Bovine virus diarrhoea virus RNA synthesis
by Peter Clayton Roberts
A thesis submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy in
Microbiology
Montana State University
© Copyright by Peter Clayton Roberts (1990)
Abstract:
The size of the genomic RNA of the pestivirus bovine virus diarrhoea virus (BVDV) is unclear. Early
estimates ranged from 8.2-10.5 kilobases (kb). More recently a size of 12.5-13 kb has been reported.
Very little is known concerning the synthesis of BVDV RNA in infected cells. The goals of the project
were: to accurately size the genomic RNAs of cytopathic (cBVDV) and noncytopathic (ncBVDV)
BVDV strains; and to compare the synthesis of BVDV RNA in cells infected with different BVDV
strains.
The BVDV genome was sized by comparison of BVDV-specific intracellular and virion RNAs to
vesicular stomatitis virus mRNAs and denatured bacteriophage λ DNA restriction fragments. This
resulted in a size estimate of 12.6 kb for the BVDV genome. No significant difference in genome size
was detected between the strains studied. For the study of RNA synthesis, total cell RNA was prepared
from infected cells at various times postinfection. The RNA was dot blotted onto duplicate
nitrocellulose filters, that were probed with either plus or minus sense 32P-labelled BVDV RNA
transcribed from a BVDV cDNA. Quantitation of the RNAs was achieved by scanning autoradiographs
with a densitometer. The concentration of BVDV RNA was calculated using BVDV-specific RNA
calibrations. The quantity of BVDV RNA varied markedly between cells infected with different strains
of the virus. There was a lag of 5-10 h before BVDV RNA was detected. With cBVDV isolates the
quantity of both plus and minus sense RNA increased until 22 h postinfection. In cells infected with
ncBVDV, plus and minus sense RNA levels varied throughout the course of the experiment. The ratio
of plus to minus sense RNA was higher in cells infected with ncBVDV as compared to cells infected
with cBVDV.
The conclusions to be drawn from this work are: 1) the BVDV genome is approximately 12.6 kb in
size; 2) the synthesis of BVDV RNA follows a similar time course to that observed in cells infected
with flaviviruses; and 3) cBVDV isolates have lost control of minus sense RNA synthesis, possibly due
to an inability to encapsidate viral plus sense RNA efficiently. BOVINE VIRUS DIARRHOEA VIRUS RNA SYNTHESIS
by
Z
Peter Clayton Roberts
A thesis submitted in partial fulfillment
of the requirements for the degree
Doctor of Philosophy
in
Microbiology
MONTANA STATE UNIVERSITY
Bozeman, Montana
May 1990
^ 5^-3 S'
ii
APPROVAL
of a thesis submitted by
Peter Clayton Roberts
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been found to be satisfactory regarding content, English usage, format, citations,
bibliographic style and consistency, and is ready for submission to the College of
Graduate Studies.
/7
Date
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Approved for the Major Department
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iii
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TABLE OF CONTENTS
Page
TABLE OF C O N T E N T S........................................................................................
v
LIST OF T A B L E S......... .......................................................................................
viii
LIST OF F IG U R E S ................................................................................. .............
ix
ABSTRACT ..............................................
xi
INTRODUCTION ..................................................................................................
I
The Pestiviruses.............................................................................................
History ...........................................................................................................
The D isease....................................................................................................
Acute In fe c tio n ....................................................................................
Haematological Changes ..................................................... .............
Immunosuppression............................................................................
TransplacentalJnfection......................................................................
Persistent Infections.............................................................................
Mucosal Disease ...........
Epidemiology ..................
Control of BVDV ...............................................................................
The Virus ............' ........................................................................................
M orphology............................................................ .................. .. . . . .
The G e n o m e ......... ..............................................................................
Structural P ro te in s........................................................! ....................
Physicochemical Properties ...............................................................
Viral Replication and M orphogenesis........................................................
The Virus R e c e p to r......... ...................................................................
Growth Phases ............................................................................
RNA Species and Synthesis...............................................................
Intracellular Polypeptides...................................................................
Genome Organisation . ......................................................................
Putative Protein F unctions................................................... . ; . . . .
Morphogenesis ......................................................: ...........................
Overview of the P ro je c t...............................................................................
2
3
6
6
8
9
11
14
15
19
20
23
23
24
27
29
30
30
31
32
32
35
37
38
39
MATERIALS AND METHODS ........................................................................
41
Biologicals and B uffers.........................................
Cells, Viruses and Plasmids ........................................................................
Preparation of BVDV A ntiserum ...............................................................
Infectious Virus Assays ...............................................................................
41
44
45
46
vi
TABLE OF CONTENTS-Continued
Page
End Point Dilution Microtitration Assay (EPDMA) ..................... 46
Plaque A ssa y ................................................................................. . . . 47
Biological Cloning of BVDV Isolates ............................' .......................... 47
Virus Growth C u rv e s.................................................................................... 48
Preparation of Cellular R N A ...................................................................... 49
Cytoplasmic RNA .............................................................
49
Nuclear Fraction R N A ........................................................................ 50
Total Cell R N A .................................................................................... 50
Virus Purification and RNA E xtraction..................................................... 51
Radiolabelling of Viral RNA ................................ ............................. . . . . 52
Preparation of X DNA Molecular Weight M a rk e rs ....................... ..
53
Agarose Gel Electrophoresis of R N A ........................................................ 53
Formamide Gels ............; ................................................................... 53
Glyoxal Gels .............................................................................................54
Northern Transfers ...................................................................................... 54
RNA Dot Blots ............................................................................................. 55
Agarose Gel Electrophoresis of D N A .................................................' . . . 55
Preparation of Plasmid D N A ...................................................................... 55
Transformation of Escherichia coli D H 5 .......................................... 55
Screening of T ransform ants............................................................... 56
Large Scale Plasmid Purification ............................................
57
Purification of the pBV4-p80 BVDV cDNA segment .................. 58
Preparation of BVDV-specific P ro b e s............................................ '.......... 58
Nick Translation of c D N A ................................................................. 58
In vitro Transcription of pBV 4-p80..............................
58
Hybridisations ............................................................................................... 59
Detection of 3’ Polyadenosine Sequences ................................................. 60
Quantitation of BVDV-specific R N A ...................................................: . 61
R E S U L T S ............'................................. ' ........................................■. •....... ...........
63
Selection of Cell L in e s ............................i ................................................... 63
Cytopathic Effect of the BVDV Strains .......................... ........................ 64
Titration of Infectious V iru s ........................................................................ 65
Raising of BVDV-specific Antiserum ................................. ■
............. 66
End Point Dilution Microtitration A ssay.......................................... 67
Comparison With Plaque Assay . ............................ ......................... 69
Biological Cloning of BVDV S tra in s.......................................................... 70
Virus Growth K in e tic s......... ............................................i ......................... 71
Purification of BVDV ................................................................................. 71
Sizing of the BVDV Genomic R N A .......................................................... 75
Northern Blot Comparisons ......................................................................■. 80
Time Course of BVDV RNA Synthesis ................................................... 81
BVDV NADL RNA Synthesis........................................................... 86
BVDV Singer RNA Synthesis .......................................................... 89
BVDV Oregon C24V RNA Synthesis.............................................. 89
V ll
TABLE OF CONTENTS-Contouerf
Page
BVDV New York-1 RNA Synthesis ................................. ................ 92
BVDV TGAC RNA S ynthesis............................................................ 94
BVDV TGAN RNA Synthesis............................................................ 94
Comparison of Plus Sense RNA Levels With Levels of Infectious
Virus .' ...■...........................................................
Comparison of the Ratio of Plus to Minus Sense R N A ................ 97
DISCUSSION .........................................................................................................
Virus Strains ..................................................................................................
Development of the Experimental System ..............................................
BVDV RNA: Size and Synthesis . . ........................................................
98
98
99
104
LITERATURE CITED ................ .....................................................................
Ill
A P P E N D IX ...........................................................................................................
137
Abbreviations
................................................... .......................■.................
137
9
viii
LIST OF TABLES
Table
'
Page
1. Members of the Pestivinis genus and their host species . . .....................
2.
3
Possible combinations of BVDV and BVDV-specific antibody in cattle
and their significance................................................................... ..
21
Size and sedimentation coefficient estimates of the genomic RNA of
BVDV .................................................................................................
25
4.
Reported sizes of the structural polypeptides of BVDV
.......................
28
5.
Molecular weights of BVDV-specific polypeptides detected in cells
infected with BVDV ..................................... .....................................
33
6.
Composition of buffers, reaction mixtures and media ............................
42
7.
BVDV strains studied .................................................................................
45
8.
Comparison of BVDV titres determined by plaque assayand EPDMA
9.
Recovery of BVDV during concentration.................................................
3.
10. Reanalysis of the size of the genomic RNA of four strains of BVDV . .
70
73
105
ix
LIST OF FIGURES
Figure
Page
1. Possible outcomes of transplacental BVDV infection of the bovine
foetus
..................................................................................................
2.
3.
13
Epidemiology of BVDV: routes and possible outcomes of infection of
c a ttle ...........................................................................................
20
Putative genomic organisation of the NADL cBVDV is o la te .................
36
4. Plasmid pB V 4-p80........................................................................................
59
5.
6.
Growth of MDBK cells in KClOO serum-free medium or DME
supplemented with either 10% equine or 10% foetal
bovine se ru m ...........................................................................................
64
Plaques of BVDV strain Oregon C24V: comparison of staining with
neutral red and crystal violet ...............................................................
66
7.
Focus of infection of the ILLC strain of BVDV on MDBK c e l ls .........
68
8.
One step multiplication curves of four BVDV strains
........................
72
9.
Comparison of sedimentation of NADL strain of BVDV on potassium
tartrate and glycerol-tartrate density gradients
........................
74
10. Sizing of the genome RNA of BVDV: comparison of viral intracellular
and virion RNA with denatured X Hindlll and Sail restriction
fragments .............................................................................................
78
11. Sizing of BVDV genomic RNA: comparison of BVDV strain Singer
mRNA with VSV m R N A .................................................................
79
12. Quantitation of BVDV intracellular R N A .....................................
84
13. Calibration curves for BVDV NADL plus and minussense RNA . . . .
85
14. Time course of RNA synthesis of BVDV NADL ...................................
87
15. Northern blots of total cell RNA from MDBK cells infected with
BVDV N A D L ......................................................................................
88
16. Time course of RNA synthesis of BVDV S in g e r............................ ' . . . .
90
X
LIST OF FIG U R E S-C onrzm ^
Figure
Page
17. Time course
of RNA synthesis of BVDV Oregon C24V ....................
91
18. Time course
of RNA synthesis of BVDV New York-1 .......................
93
19. Time course
of RNA synthesis of BVDV TGAC ................................
95
20. Time course of RNA synthesis of BVDV TGAN . . . .......................... ■.
96
ABSTRACT
The size of the genomic RNA of the pestivirus bovine virus diarrhoea virus
(BVDV) is unclear. Early estimates ranged from 8.2-10.5 kilobases (kb). More
recently a size of 12.5-13 kb has been reported. Very little is known concerning
the synthesis of BVDV RNA in infected cells. The goals of the project were: to
accurately size the genomic RNAs of cytopathic (cBVDV) and noncytopathic '
(ncBVDV) BVDV strains; and to compare the synthesis of BVDV RNA in cells
infected with different BVDV strains.
The BVDV genome was sized by comparison of BVDV-specific intracellular
and virion RNAs to vesicular stomatitis virus mRNAs and denatured bacterio­
phage X DNA restriction fragments. This resulted in a size estimate of 12.6 kb for
the BVDV genome. No significant difference in genome size was detected
between the strains studied. For the study of RNA synthesis, total cell RNA was
prepared from infected cells at various times postinfection. The RNA was dot
blotted onto duplicate nitrocellulose filters, that were probed with either plus or
minus sense 32P-Iabelled BVDV RNA transcribed from a BVDV cDNA. Quantit­
ation of the RNAs was achieved by scanning autoradiographs with a densitometer.
The concentration of BVDV RNA was calculated using BVDV-specific RNA
calibrations. The quantity of BVDV RNA varied markedly between cells infected
with different strains of the virus. There was a lag of 5-10 h before BVDV RNA
was detected. With cBVDV isolates the quantity of both plus and minus sense
RNA increased until 22 h postinfection. In cells infected with ncBVDV, plus and
minus sense RNA levels varied throughout the course of the experiment. The
ratio of plus to minus sense RNA was higher in cells infected with ncBVDV as
compared to cells infected with cBVDV.
The conclusions to be drawn from this work are: I) the BVDV genome is
approximately 12.6 kb in size; 2) the synthesis of BVDV RNA follows a similar
time course to that observed in cells infected with flaviviruses; and 3) cBVDV
isolates have lost control of minus sense RNA synthesis, possibly due to an
inability to encapsidate viral plus sense RNA efficiently.
I
INTRODUCTION
Bovine virus diarrhoea virus (BVDV) is the etiological agent of one of the
most complex disease syndromes in veterinary medicine (78). The virus appears
to be ubiquitous, since there is a high incidence of seropositive animals throughout
the world (147,197). Only recently, through a better understanding of its epidemi­
ology, has the full economic impact of BVDV become appreciated (78). The
infection of animals may not be diagnosed for several months after the introduc­
tion of the virus into a susceptible herd and losses may continue for several years
(6,79). The virus is now considered to be one of the major causes of economic
loss to the cattle industry (130).
Field isolates of BVDV are initially characterised by their cytopathic effect in
cell culture. They are usually described as either cytopathic (cBVDV) or noncytopathic (ncBVDV) (93); intermediate cytopathologies also occur (93,94,115,186).
Fernelius (93) felt that these differences were genetic in nature and labelled them
biotypes. They appear to be genetically stable traits in bovine cell lines, but can
be altered by passage in non-bovine cell lines (93) or in rabbits (94). The virus
occurs as a single serotype (55), though subtle but distinct antigenic differences
are detected between BVDV strains (40-42,55,58,62,81,92,114,115,117,136,142,
163,173,296). These antigenic differences extend to presumed identical strains
from different laboratories (42,55).
No definite correlation has been found between a specific strain of BVDV
and specific clinical symptoms, though such a correlation has been described for
the closely related Border disease virus (BDV) (215).
2
The Pestiviruses
Bovine virus diarrhoea virus is a small, spherical, enveloped virus with a
single stranded, positive polarity RNA genome. It is the type species of the genus
Pestivinis, at present classified in the family Togaviridae (299) but it is likely that
the genus will be reclassified as being in the family Flaviviridae (52,55). Other
members of the Pestivinis genus are hog cholera virus of swine (HCV) (also
referred to as classical or European swine fever virus) and BDV of sheep. There
is some evidence that a human pestivirus exists (101,221,303,305) that may cause
infantile gastroenteritis associated with respiratory inflammation (305). The
cBVDV strain Oregon C24V has been designated the type strain of the genus
( 299).
The pestiviruses are serologically related (211,216), share similarities in their
pathogenicity, especially for the foetus (125), and have a lower sedimentation
coefficient and buoyant density than members of other togavirus genera (157).
The pestiviruses are generally species specific but interspecies infections do occur
(81,273). The three pestiviruses can be differentiated by panels of monoclonal
antibodies (42,81,111,129,190,213,273,293-295,307). Monoclonal antibodies specific
for pestiviruses in general, HCV alone or BVDV alone are available; however, a
monoclonal antibody that is specific for BDV has not yet been described.
BVDV and BDV seem to be very similar and are increasingly being referred
to as the ruminant pestiviruses. They are antigenically diverse, have a wide host
range (Table I) and a similar range of biotypes (125,134,158). In contrast, HCV
has a limited host range (Table I), strains form a compact antigenic group and all
field isolates are noncytopathic (120,134). There is about 85% homology at the
amino acid level between BVDV and HCV (193). HCV also differs from the
3
Table I.
Members of the Pestivirus genus and their host species.
Virus
Host species
Bovine virus diarrhoea virus
Cattle, sheep, goats, swine, wild
ruminants, rabbits3 (5,76,78,94,119)
Border disease virus
Sheep, goats, cattle, swine (125)
Hog cholera virus
Swine (120)
a. Rabbits have not been shown to be infected outside the laboratory.
other pestiviruses in that virulent and avirulent strains occur (120). Virulent HCV
strains are antigenically different from BVDV and BDV (120); however, avirulent
HCV strains have been described that are antigenically more diverse: they appear
to be more closely related serologically to BVDV than to virulent HCV (120,158).
BVDV, BDV and avirulent HCV may prove to be the same virus, since they are
only truly differentiated on the basis of their host species.
History
It is likely that the first description of the bovine virus diarrhoea-mucosal
disease complex was that of Simonds and Brown (268) in England in 1871. Their
description probably went unnoticed by many later researchers, as they attributed
the syndrome they observed to acorn poisoning. In 1946, Olafson et al. (208)
described a highly contagious and transmissible disease of cattle in New York, of
high morbidity but low mortality. They called the disease virus diarrhoea (VD), as
no bacteria could be isolated from infectious material and diarrhoea was a
common clinical symptom (209). A similar but more severe disease syndrome,
X disease, was described by Childs (44) in Saskatchewan the same year. Both
4
acute and chronic forms of X disease were described. The low morbidity and high
mortality of X disease differentiated it from VD. In .1953, Ramsey and Chivers
(232) described a syndrome in Iowa and surrounding states they called mucosal
disease (MD). They believed that the syndrome had not previously been reported
but their description is very similar to that of Child’s X disease, a fact Ramsey
later acknowledged (cited in reference (223)). These early descriptions led to a
belief that, although they were similar in general symptoms, VD and MD were
separate disease entities.
At that time the nature of the etiological agent of VD was still in question.
In 1954, Baker et al. (4) conducted an extensive study of infectious material from
two cases of VD. The results concurred with the earlier studies of Olafson et al.
(208,209) i.e., the etiological agent of VD was a virus as no other agent was
detected. This conclusion was supported the following year by the filtration
studies of Pritchard et al. (224) and Schipper et al. (261). In 1957, Lee and
Gillespie (160) successfully propagated in vitro one of Baker’s isolates, New
York-1. The virus was found to be noncytopathic. The same year, Underdahl et
al. (282) propagated in tissue culture two cytopathic viruses isolated from the
tissues of animals from separate outbreaks of MD. Cross-neutralisation studies
showed that the two viruses were related. In addition, cattle from herds with no
history of MD were found to have serum neutralising antibody to the two viruses.
Kniazeff (cited in reference (223)) later showed that UnderdahTs viruses were
related to New York-1. In 1960, Gillespie et al. (103) described a cytopathic virus
that was isolated from the spleen of a cow reported to have VD (a later report
indicates that the cow actually had MD (197)). This isolate, Oregon C24V, has
been designated the Pestivirus type strain (299). Cross-neutralisation studies,
performed both in vivo and in vitro, showed that Oregon C24V was antigenically
5
related to New York-1. The following year the same group showed that viruses
isolated from cases of VD and MD were antigenically related (104). This finding
was supported by comparisons of VD and MD virus isolates from Europe and the
United States (152,223).
The ability to grow BVDV in tissue culture greatly facilitated studies of the
bovine virus diarrhoea-mucosal disease complex. It provided irrefutable evidence
that BVDV was the etiological agent of VD. Although virus neutralisation studies
implied that VD and MD were caused by the same virus, proof of this remained
elusive. A major problem was the inability to reproduce MD experimentally. In
their original description of MD, Ramsey and Chivers (232) described attempts to
transmit the disease to healthy calves. The only response they detected was a
transient pyrexia. Similar results were obtained in other studies (102,224,261).
Tyler and Ramsey (281) failed to detect any significant differences in the
pathologic, immunologic and clinical responses to VD and MD virus infections in
healthy calves. The shroud of mystery surrounding the pathogenesis of MD began
to be lifted in 1964 when Thomson and Savan (276) observed that only animals
without an antibody titre to BVDV succumbed to MD. In 1968, Malmquist (177)
published a landmark paper. He observed that animals suffering from MD had a
persistent BVDV viraemia but failed to develop neutralising antibodies to the
virus. He hypothesised that the affected animals had been infected in utero prior
to the attainment of immunocompetence and were subsequently born immunotolerant to the virus. Although there was circumstantial evidence that BVDV could
infect the foetus, direct proof was provided in 1969 by Ward et al. (289). They
demonstrated that BVDV could cross the placenta and that it was a teratogenic
agent. In 1974, Liess et al. (161) reproduced MD experimentally. Having also
observed that only seronegative animals died of MD, they selected four
6
seronegative cattle from herds where the majority of animals were immune to
BVDV. The four animals were challenged by intranasal inoculation with a
cBVDV strain and two subsequently died of MD. In 1978, Coria and McClurkin
(59) described an apparently healthy bull with a persistent ncBVDV viraemia,
proving the presence of persistent BVDV infections in animals other than those
with MD. In recent years, several groups have shown that MD only occurs in
persistently infected animals (25,34,99,248). The syndrome has been reproduced
by superinfection of persistently infected animals with cBVDV but not ncBVDV
(25,34). Brownlie et al. (34) proposed the hypothesis that MD is produced by
superinfection of persistently infected cattle with cBVDV. This was later modified
when it became obvious that the cytopathic virus infecting animals with MD was
always antigenically homologous to the original persisting ncBVDV (36). The
hypothesis is now that MD arises in persistently infected animals either through
mutation of ncBVDV to cBVDV or superinfection with an antigenically homolo­
gous cBVDV. Current research is aimed at elucidating the mechanism of
induction of MD.
The Disease
Acute Infection
Seronegative and immunocompetent animals postnatally infected with
BVDV undergo an acute infection. The primary site of infection is probably the
respiratory tract, in particular the bronchiolar epithelia and tonsils (10). From
there the virus is disseminated to epithelial and lymphoid tissues throughout the
animal, especially those of the alimentary tract and skin. This dissemination may
be achieved by transport of the virus in cells of the reticuloendothelial system (10).
7
Most acute infections are subclinical. They are characterised by a transient
pyrexia that is often biphasic, a profound leucopenia and a viraemia that may
persist for 2 weeks (78,223). During the period of viraemia, virus is shed at low
levels in body secretions and excretions; however, transmission to other animals is
not considered to be very efficient (78). Two weeks after infection animals start
to produce serum neutralising antibody (38). This response peaks after 10-12
weeks and is generally considered to protect the animal for life (38,78); in two
cases, however, immunity was shown to only last for 3-4 months (127,224).
Clinical acute infections are rarely encountered. They generally occur in
animals 6 months to 2 years old. Prior to six months of age, colostrum-derived
neutralising antibody protects the calf from BVDV infection (267). Severe,
clinical epizootics are diagnosed as bovine virus diarrhoea (BVD). Affected
animals may present a variety of symptoms other than those described above:
inappetence, rapid respiration, depression, oculonasal discharges and occasional
ulceration and erosion of the oral mucosa (5). Mucus is sometimes present in the
faeces, often as strands that can be nearly an inch thick and several feet long
(223). Diarrhoea, when present, appears 1-7 days after the febrile period (223).
It may be explosive in character and present continuously or intermittently
(78,223). In the later stages large quantities of bright red blood may be present in
the faeces (223). Diarrhoeic animals are often severely dehydrated and can lose
as much as 25% of their total bodyweight (223). Affected dairy cows have a
marked reduction in milk yield (6,78).
The lesions of BVD are less severe than would be expected from the
symptoms (208). There are diffuse reddened areas in the mucosa of the entire
alimentary tract but they are more prevalent in the upper regions. These areas
may develop into erosions and shallow, punched-out ulcers, especially in the oral
8
cavity, oesophagus, abomasum and caecum (208,223). In nonfatal cases the ulcers
heal very rapidly (208).
The morbidity rate in infected herds is generally close to 100% of the
susceptible animals present when based on seroconversion. The mortality rate
ranges from 0-20% (223). Death is. due to the effects of either the primary
infection, especially dehydration and emaciation, or secondary infections by
opportunistic pathogens (212).
There is some evidence that acute BVDV infection of neonatal calves can
result in a severe, sometimes fatal, enteritis (5). This has been reproduced
experimentally, with both colostrum-fed and colostrum-deprived animals (153).
The importance of the virus in neonatal calf diarrhoea is still an open question
(5). Older, colostrum-fed calves undergo acute infections, as described above
(207). Another syndrome that has been associated with neonatal infection with
BVDV is hyena disease, a disorder of skeletal development (86).
Haematological Changes
The most obvious haematological change after BVDV infection is the
transient ieucopenia. Bolin et al. (23) showed that 4 days after infection the total
white blood cell count is significantly reduced. The most marked reduction is that
of T lymphocytes. Most cell populations return to or exceed pre-exposure levels
within 3 days but lymphocyte numbers do not recover until 2 weeks postinfection.
Flow cytofluorimetric analysis showed that helper T cells, cytotoxic/suppressor T
cells, B lymphocytes and neutrophils are depleted (83). The numbers of other
lymphocytes and monocytes are not affected. There are some indications that
Ieucopenia, involving both neutropenia and lymphopenia, correlates with a more
severe clinical course than lymphopenia alone (212).
9
Several recent reports have described the association of thrombocytopenia,
with resultant haemorrhagic conditions, with severe outbreaks of BVD (57,212,
233). The thrombocytopenia has been reproduced experimentally (56,57).
Platelet destruction appears to be due to direct interaction with the virus (56). As
the incidence of thrombocytopenia is low (233) and can be reproduced experimen­
tally with specific strains of ncBVDV (56,57) it is likely that it is induced by a
limited number of BVDV strains.
Immunosuppression
Although acute infections with BVDV are generally innocuous, there is
strong evidence that they involve a profound, transient immunosuppression. The
most obvious manifestation of this is the transient leucopenia. BVDV infections
also result in the destruction of lymphoid tissues (2,10). The most severely
affected lymphoid tissues are those in the submucosa of the intestine, especially
the Peyefs patches and the area distal to the ileocaecal valve. The tonsils and
lymph nodes exhibit a loss of differentiation between the cortex and the medulla,
with general loss of lymphocytes, especially from the germinal centres (2,10).
There is often a depletion of thymocytes in the thymus (2,10). In the spleen, the
periarteriolar lymphoid sheath is severely affected (10). In both lymphoid and
nonlymphoid tissues BVDV antigen is present predominantly in cells of the
reticuloendothelial system (10).
In vitro studies have shown that the virus can replicate in several different
lymphoid cell populations: B and T lymphocytes (2,3,18,257,279); neutrophils
(257,258); and monocytes and macrophages (2,18,278,279). The virus has been
isolated from, and can replicate in, the leucocytes of actively immunised animals
(2,196,279). BVDV infection can result in the suppression of cellular functions.
10
The B lymphocyte response to pokeweed mitogen is suppressed (2,3,155). This
results in a significant reduction in plasma cell development and IgG and IgM
synthesis. Antibody secretion but not synthesis is affected in persistently infected
animals (202). The mitogenic response of T lymphocytes is depressed by BVDV
infection (2,28,100,143,154,155,201,256,257). A more marked depression of the
blastogenic response to phytohemagglutinin compared with that to ^concanavalin A
has been noted (28,257). The blastogenic response can be partially restored by
addition of isoprinosine (100). In two cases, however, BVDV infection did not
affect the mitogenic response of lymphocytes (39,269). Interleukin-2 receptor
expression may be compromised in BVDV infected lymphocytes (100). Infection
of monocytes results in a significant decrease in their random locomotion and
chemotaxis (151). Infected neutrophils exhibit a defect in the myeloperoxidase,
hydrogen peroxide, halide system and antibody dependent cell-mediated cytotox­
icity (257,258). Active BVDV infection of cells is required to produce the defects
in cellular response and function; heat-killed or ultraviolet-irradiated virus has no
effect (2,3,83,151,201,257,258).
Apart from the direct action of BVDV on lymphoid cell populations it also
impacts on the immune system in other ways. Bovine foetal lung cells infected
with BVDV release substances that suppress concanavalin A stimulation of bovine
leucocytes (180). The agent responsible has been tentatively characterised as a
prostaglandin. The serum of animals suffering from MD has been shown to
suppress the mitogenic response of lymphocytes (155,269). BVDV also affects the
production and action of interferon (IFN). Cells infected with BVDV do not
produce IFN (66) but can be induced to produce IFN by treatment with polyriboinosinic-polyribocytidylic acid (253). The production of IFN by phytohemaggluti­
nin stimulated lymphocytes infected with BVDV is reduced (234). In contrast,
11
animals infected both in utero and postnatally have been shown to produce IFN in
response to BVDV infection (234,240). BVDV-infected tissue culture cells are
refractory to IFN action (66,255); however, pretreatment of cells with IFN prior to
infection results in reduced virus yields (15,66,97). Recombinant bovine IFN-Cn1I,
IFN-y and tumour necrosis factor alpha, when added to the growth medium of
BVDV-infected cells, cause enhanced cBVDV cytopathogenesis and a cytopathic
effect in ncBVDV infected cells (15).
A major effect of BVDV-induced immunosuppression is the potentiation of
opportunistic pathogens (5). Evidence is accumulating that the virus plays a major
role in bovine respiratory tract disease (5,181,270). It is believed to act synergistically with several organisms, the most important of which is Pasteurella haemolytica (5,218,220,235,280). Though BVDV infection can impair bacterial clearance
from the lungs (220), the clearance of P. haemolytica may actually be enhanced
(280). It has been suggested that the more severe pulmonary lesions observed in
dual infections of BVDV and P. haemolytica are a product of neutrophil degranu­
lation (280). Bacterial blood clearance mechanisms are also impaired, resulting in
an endogenous bacteraemia in 85% of animals with acute BVDV infections (219).
Other pathogens that BVDV may act synergistically with are infectious bovine
rhinotracheitis virus (82,235), parainfluenza-3 virus (33), respiratory syncytial virus
(33), bovine leukosis virus (244), Coxiella burnetii (222), Leptospira interrogans
serovar hardjo (222) and salmonella (304).
Transplacental Infection
BVDV infection of pregnant, seronegative, immunocompetent cattle general­
ly results in transplacental transmission of the virus to the foetus (reviewed in
reference (285))i Such infections are the major source of economic losses due to
12
BVDV (13,121,249); 78-90% of total losses (13). Embryonic death has been
produced early in gestation after intrauterine infections with BVDV; however, this
is unlikely to be a common occurrence in the field (285). Exposure of bovine
embryos, with or without the zona pellucida, to BVDV in vitro did not result in
embryonic death (9). The virus has also been shown to impair fertilisation (109).
Placental attachment, which occurs after about 35 days of gestation, appears to be
necessary for transfer of BVDV from the dam to the foetus (150). After this early,
refractive period, the virus is thought to cross the placenta by sequential infection
of adjacent cells (285). Transplacental infections do not occur in seropositive
animals, limiting the number of foetuses at risk.
The outcome of foetal infection with BVDV is dependent primarily on the
developmental age of the foetus, though the genotype of the host and the virus
strain may also play a role (12,285). Congenital infections can result in prenatal
death, stillbirth, malformations, lesions or the birth of calves with a persistent
viraemia (Figure I) (285). Abortions are common early in gestation, though they
can occur as a consequence of foetal infection and death at any time (70,285).
Foetal mummification may also occur (38,70). During the second trimester of
pregnancy, congenital malformations are the most frequent sequelae to foetal
infection. Common lesions are cerebellar hypoplasia, dysmyelination of the
central nervous system, ocular defects such as retinopathy and cataracts, hydranencephaly, intrauterine growth retardation of the whole animal or specific organs
(especially the brain, thymus and lungs) and skeletal defects (19,285). The
outcome of BVDV teratism is the birth of calves that may be stillborn, stunted,
ataxic, blind or that manifest various neuromuscular disorders.
In sheep and cattle the generation of malformations appears to coincide with
the initial development of the immune system (164,250,251). The bovine foetus
13
Prenatal effect
Foetal death
Postnatal corollary
Abortion, stillbirth
Persistent
infections
Mucosal disease
I
Ocular
lesions
Blindness
Cerebellar
hypoplasia
Ataxia
Immune
response
...............
Organogenesis
Iiiiiii
Neutralising antibody
Birth
100
200
Gestational age (days)
Probability of occurrence: high
Figure I.
300
low |
Possible outcomes of transplacental BVDV infection of the bovine
foetus. (Adapted from reference (78)).
becomes immunologically competent to BVDV by day 100 of gestation; however,
neutralising antibody to the virus is not produced before day 180-200 of gestation
(17,30,32,262). The production of neutralising antibody coincides with elimination
of BVDV from the foetus (30). This suggests that there can be a long lag before
the virus is cleared from the foetus. During this period the immature foetal
immune system could act against virus-infected cells causing the various terato­
genic effects.
In the third trimester of pregnancy BVDV infection has little effect on the
foetus, due to the ability of the foetus to mount a normal immune response to the
virus. Abortions may still occur at this stage of gestation. One outcome of
14
infection of the foetus late in gestation is the birth of calves with the typical
lesions of BVD (289).
Persistent Infections
Transplacental infection of the foetus during the first 4 months of gestation
may result in the birth of animals persistently infected with ncBVDV (188). In
experimental studies the success rate in producing persistently infected calves may
approach 100% (70,188). Only ncBVDV can cause persistent infections (37). It
has been estimated that persistently infected animals comprise between 0.4-1.8%
of the cattle population (24,80,137,194,214).
The virus is able to persist in pestiviraemic animals through immunotolerance. The immunotolerance is a product of the infection of the foetus prior to
the development of the immune system. The immunotolerance involves not only
the antibody response but also T cell dependent mechanisms (12). It appears to
be highly specific for epitopes of the persisting virus (22). The infected animals
are able to mount normal humoral immune responses to other pathogens and
antigens (59,188,269) and to heterologous BVDV strains (22,26,28,58,59,72,85,
162,266,269,301). The detection of immune complexes containing BVDV antigens
in the kidneys of clinically normal, persistently infected animals implies that the
immunotolerance is not complete (64). The immune complexes may be formed
with non-neutralising BVDV-specific antibodies that are detected in the serum of
some persistently infected animals (22,72).
Persistently infected calves may appear perfectly normal, though various
teratological disorders are also observed (70,188). Common manifestations are
intrauterine growth retardation of the whole animal or specific organs, especially
the thymus, and defects of the central nervous system (70). In clinically healthy
15
cattle* persistently infected with BVDV microscopic lesions are only detected in
the brain and the kidneys (64). BVDV antigen can be detected in epithelial and
immune cells in most tissues (12). In the central nervous system BVDV antigen is
only observed in neurons (89). In peripheral blood leucocyte (PBL) populations,
virus is associated with lymphocytes, monocytes and an uncharacterised population
of cells (18,29). Cytofluorimetric analysis showed that the virus is associated with
an average of 4.4% of total leucocytes, 5.4% of T lymphocytes and 2.1% of B
lymphocytes (29). Significant differences are found between animals in terms of
the number of cells associated with virus; however, these differences do not
correlate with persistent infection by a specific virus strain. The use of doubleimmunolabelling procedures for phenotyping virus-infected cells showed that
5-36% of PBL are infected, again with a marked variation between animals (18).
T lymphocytes represent 40-50% of the infected cells: 60% of these are of the
cytotoxic/suppressor phenotype and the remainder are helper cells. Of the
remaining infected cells less than 4% are B lymphocytes, 17-24% are monocytes
or macrophages and the remaining 24-40% are uncharacterised cells. Infectious
virus can be isolated from all the cell populations except B lymphocytes. Total
leucocyte numbers are the same as in uninfected control animals; however, there
is a significant reduction in the number of T lymphocytes with a concomitant
elevation of the other cell populations, especially monocytes.
Mucosal Disease
The mortality rate of persistently infected cattle is extremely high, often
exceeding 50% in the first year (78). Animals die from diverse respiratory and
enteric infections (6,188), but the most important clinical cause of death is MD
(78). Mucosal disease occurs sporadically, affecting animals 6 months to 2 years
16
of age (5,33,78). Outbreaks of MD often occur concomitantly amongst animals of
a similar age in a herd, implying the presence of a common precipitating factor
(78). The number of affected animals in a herd is usually low, between 1-5%, but
they invariably die (5,38). The first clinical symptom is usually anorexia (38).
Other symptoms are pyrexia, depression, profuse watery diarrhoea, rapid respira­
tion and nasal discharges (5,33,78,223). Oral erosions and ulcerations are usually
evident, often accompanied by profuse salivation (5,33). Dermatosis is common
(5,33). Lameness occurs as a result of erosion and ulceration of the interdigital
cleft and coronary band (5,33). Death usually occurs 3 days to 3 weeks after the
first clinical symptoms are observed, though some animals die before exhibiting
any symptoms (5,33,38,78).
The characteristic lesions of MD resemble a graft versus host reaction (11).
Severe ulcerative lesions and erosions of the mucosae are found throughout the
alimentary tract (5,14,33,78,223). The ulcers are unusual as lymphoid cells initially
do not invade the area around the lesion (William Quinn, personal communica­
tion). Severe thymic atrophy is observed (2,10). T lymphocytes are depleted in
peripheral lymphoid tissues and lymphocytes are lost from germinal centres
(2,10,14). In PBL populations there are increased numbers of B lymphocytes and,
occasionally, suppressor T cells; null cells-non-characterised lymphocytes-are
I
decreased in number (155). Other leucocyte populations are not affected. Both
ncBVDV and cBVDV can be isolated from the blood and many tissues of affected
animals (6,33,35,36,50,135,166,185,203,265,266,301). The ncBVDV and cBVDV
present in an individual animal are antigenically very similar, if not identical
(36,58,135,136,265,266). There are indications that the two biotypes may have
different tissue tropisms, cBVDV being more readily isolated from the intestines
and ncBVDV more prevalent in all other tissues (35,50,166,265,301).
17
Occasionally a more protracted course of disease is observed. This was
originally termed chronic BVD but chronic MD is a more apt description (5).
Animals so afflicted become progressively more emaciated with persistent or
intermittent diarrhoea (5,14). Intractable ocular and nasal discharges are common
(5). As with acute MD, the animals invariably die; however, death may not occur.
for more than 18 months after the onset of symptoms (5).
The pathogenesis of MD is still unclear. It is generally accepted that in utero
infection resulting in persistent infection with ncBVDV is a prerequisite for MD
and that both virus biotypes are present in animals with MD (6,33,35,36,185). It
has been hypothesised that MD results from persistently infected animals
becoming superinfected with cBVDV either through mutation of the persistent
ncBVDV or superinfection with an antigenically homologous cBVDV
(36,38,58,136). In natural outbreaks of MD it appears that both processes occur
(122,277). A single animal is often observed to have MD up to a month before
other herd members (122). It seems likely that the ncBVDV mutates in this
sentinel animal. There is some evidence that the change of biotype may be the
result of recombination of the BVDV genomic RNA with cellular RNA (192).
Cytopathic BVDV is then transmitted by this animal to the rest of the herd. As
persistently infected animals in the herd are probably infected with the same
ncBVDV, they will readily contract MD. A similar theory applies to BDV in
sheep. Sheep that have recovered from Border disease are persistently infected
with BDV. In these animals an MD-Iike syndrome occurs spontaneously,
suggesting mutation of the persisting noncytopathie virus (99). The syndrome can
also be produced by superinfection with the homologous, persisting virus but not a
heterologous virus (99).
18
Experimental induction of MD tends to indicate that the process is more
complex (25,26,34,36,58,123,266,301). Challenge of persistently infected animals
with homologous cBVDV results in fatal MD after 2-3 weeks (301). However,
challenge of persistently infected cattle with a heterologous cBVDV can also result
in MD (266,301), but the onset of MD is delayed (301) and the cBVDV isolated
from the animals is always antigenically homologous to the persistent ncBVDV not
the original challenge virus (266,301). The animals usually produce neutralising
antibody against the challenge virus. A similar situation can arise after vaccination
of persistently infected animals with a modified live BVDV vaccine (26,28,85,162,269). In the case of heterologous challenge the narrowly defined immunotolerance for the persisting ncBVDV may select for antigenically homologous
cBVDV in a generally heterologous cBVDV population. The delay in onset of
MD would be due to the selected virus having to replicate to a high enough
concentration to induce disease. A homologous cBVDV challenge would require
less time to reach such a level.
Several other theories have been expounded to explain the conundrum of
MD induction. These include: hormonal changes associated with puberty (248),
the cooperation of an unrelated defective satellite agent (166) or an immune
response to an antigenically different BVDV strain (126). Although the evidence
mitigates against them, none of these alternatives can be totally discounted at
present.
The evidence implies that antigenically homologous cBVDV is required for
the development of acute MD. It has been suggested that the degree of homology
between the persistent ncBVDV and challenge cBVDV is reflected in the type of
clinical response observed (36). There are indications that infection with a
heterologous cBVDV induces chronic MD (38). In one case, though, cytopathic
19
virus could not be isolated from the affected animal (28). It was suggested that
the animal was superinfected with ncBVDV. At present there is no direct
evidence that, superinfection with ncBVDV can cause chronic MD. Experimental
challenge of persistently infected animals with ncBVDV produces no untoward
reactions (25,203).
Epidemiology
Persistently infected cattle are the root of the epidemiology of the BVD-MD
complex (Figure 2). They are the major mechanism by which the virus is
maintained in the cattle population (78). They continually shed a large amount of
virus that is readily transmitted to susceptible animals. The most devastating
economic effects of BVDV occur when a persistently infected cow or heifer is
introduced into a susceptible breeding herd (78,248). The herd is likely to
experience any or all of the possible sequelae of acute BVDV infections. All the
reproductive losses and abnormalities associated with BVDV infection may occur,
including the birth of another generation of persistently infected animals.
Persistently infected animals can be bred. In the case of persistently infected
heifers, the offspring are also persistently infected (188,272). Such vertical
transmission can result in several generations of persistently infected animals
(166,249,272). The persistently infected bull is less important in the epidemiology
of BVD V. They produce semen that is often of poor quality and that is contami­
nated with BVDV (7,187,239,297). The presence of virus.in the semen may
occasionally result.in vertical transmission in cattle and in sheep (98,195). It may
also be a problem if the bull is mated with a seronegative cow; the virus seems to
prevent conception in seronegative animals (187,302), though not in every case
(195,297).
20
P ersistently infected
animal
Immmunosuppression
Seronegative
im m unocom petent
animal
Transplacental
infection
Acute infection
Teratogenic
effects
Seroconversion
Foetal death
Stillbirth
Figure 2. Epidemiology of BVDV: routes and possible outcomes of infection of
cattle
It is not clear what role the non-bovine host species of BVDV play in the
epidemiology of the virus (78).
Control of BVDV
It is unlikely that BVDV could be eradicated, as was achieved with HCV in
the U.S.A. and Britain, due to its general innocuity and ubiquity in domestic and
wild ruminants (249). The aim of any control strategy, therefore, should be to
21
prevent transplacental infections, the means by which the virus is maintained in
the cattle population and the major source of economic losses (13,121,249). To
this end, it is necessary to address the immune and virus status of animals in a
herd (78,249). Table 2 summarises the four possible combinations of immunity
and viraemia with respect to BVDV.
Table 2.
Possible combinations of BVDV and BVDV-specific antibody in cattle
and their significance.
Virus
Antibody
-
-
"+■
-
+
-
+
+
Significance
Fully susceptible to acute infection
Immune
Persistently infected.
Early acute infection, prior to seroconversion. ,
Persistently infected but with low level of antibody.
Acute infection undergoing seroconversion.
Persistently infected cattle need to be identified, as do susceptible animals
(78). Recently, several enzyme-linked immunosorbent assays (ELISA) have been
described for the detection of antibodies to BVDV in serum (20,47,138,140,145,
146,148,300). These can be used to detect animals with low or no immunity to
BVDV, that are then screened for persistent viraemia. Viraemic animals can be
managed in two ways: they can be culled or they can be retained to act as
vaccinator animals until they attain a suitable weight for slaughter (78). Persis­
tently infected cattle should not be bred. Seronegative, nonviraenhc animals
should be vaccinated or exposed to a persistently infected animal and not bred
until they have seroconverted.
Vaccination of animals presents its own problems. The vaccines that are
available are so unsafe that none has been licensed for use in Britain (78).
22
Modified live virus vaccines can induce all the clinical manifestations of BVDV
infections, including those of the foetus (165,249). They are also immunosup­
pressive (165,249,257). The criterion by which they are termed ‘attenuated’ seems
rather dubious as they seem to do little except pre-empt a natural infection (249).
Recently, a temperature-sensitive BVDV vaccine strain has been described that
appears to obviate these problems (167,168). Killed virus vaccines are also
available, though these are also of dubious quality as they are inefficacious in
some cases (249). To produce a protective immune response a large antigenic
dose has to be administered which makes these vaccines rather expensive to
produce. Their advantage over the live vaccines is the lack of adverse clinical
reactions but the duration of immunity may be shortlived (249). Recently
detergent solubilised BVDV has been shown to be as good an immunogen as a
killed virus vaccine (198). It is not clear, at present, whether a single vaccine
strain will provide adequate protection against all field viruses. The evidence from
the studies with persistently infected animals would argue that a multivalent
vaccine will be required. Studies of BDV in sheep Corroborate this (286).
Another potential problem is contamination of live virus vaccines with
nonattenuated BVDV (249). In the past, vaccine preparations of other bovine
viruses have been contaminated with ncBVDV, with serious consequences. The
source of the contamination is probably the bovine serum used to supplement the
growth medium of the cells the vaccine viruses are propagated in. It has been
shown that every lot of commercially produced fetal bovine serum is contaminated
with ncBVDV (139). Therefore, great care needs to be exercised in the
production of live virus vaccines. In the future recombinant DNA techniques will
probably be utilised to produce cheaper, safer and more efficacious vaccines (121).
23
The Virus
Morphology
The first electron micrographs of negatively stained BVDV virions were
prepared by Kniazeff (cited in reference (223)). The micrographs revealed
particles 35-55 nm in diameter. Later studies of both negatively stained virus and
sections through virus pellets furnished a large range of sizes for BVDV virions
(reviewed in references (134,200)). Most estimated the virus particles to be
between 40 nm and 60 nm in diameter, though sizes greater than 100 nm were
reported. Similar results were obtained in studies of BVDV infected cells, with
the majority of virion size estimates ranging from 45-55 nm (16,43,110,174),
though sizes in excess of 100 nm were also reported (241,288). The larger virions
may result from the packaging of more than one virus nucleocapsid in a single
envelope (288).
The virus envelope is generally described as smooth, though projections are
observed on some particles (46,171,241). This correlates with studies of HCV
where glycoprotein spikes are observed on some particles (84). These spikes are
lost upon osmotic shock. The envelope of both BVDV and HCV has a character­
istic "rosary" substructure composed of electron-lucent beads about 6 nm in
diameter, only visible after removal of surface material (131).
Within the virus envelope is the virus core or nucleocapsid, with a diameter
of 20-30 nm (43,110,118,131,170). The core exhibits far less size variation than the
virus particles, implying that the virus envelope is responsible for the size hetero­
geneity of the virions (131). The core has a central component with a diameter of
13 nm (131). The core appears to be polygonal, suggesting cubic symmetry
24
(131,170). Ringlike subunits are present on the surface of some cores (131,170)
and similar structures are seen in disintegrating virus particles (241).
The Genome
Early studies of BVDV showed that virus replication is not inhibited by
halogenated deoxyuridines (inhibitors of DNA synthesis) indicating that the virus
has an RNA genome (reviewed in reference (134)). Supporting evidence was
provided by later studies that showed that viral replication is not inhibited by
actinomycin D (AMD). Diderholm and Dinter (65) showed that the genome is
infectious; furthermore, they showed that infectivity is lost after treatment with
RNase but not DNase. The infectious nature of the genome was confirmed by
Moennig (cited in reference (134)) and Horzinek (132). These results proved that
the genome of BVDV is single-stranded, plus-sense RNA.
A wide range of sizes have been reported for the genome of BVDV, derived
from both sedimentation on sucrose gradients and gel electrophoresis (Table 3).
In most cases only one RNA species was detected; however, in two cases several
minor RNA species were also detected (31,225). Recent data indicate that the
genome is 12.5-13 kilobases (kb) long (54,237). The genomic RNA of HCV
appears to be slightly smaller (199,259), approximately 12.3 kb (193). The base
composition of the genomic RNA of BVDV is 31.7% A, 22.2% U, 25.7% G and
20.4% C (55). The genomic RNA is not polyadenylated (228,237).
The genomic RNA of the cBVDV strains Osloss and NADL has recently
been molecularly cloned and sequenced (54,237). The Osloss sequence is 12,490
bases long and has a novel genetic organisation: two open reading frames (ORF)
were found, one apparently encoding the structural and the other the nonstructural proteins (236,238). In contrast to this, the NADL sequence is 12,573
25
Table 3.
Size and sedimentation coefficient estimates of the genomic RNA of
BVDV.
Reference
Size
daltons
Moennig (cited in reference (133))
kilobases
Sedimentation
coefficient
37-40S
3.00xl06
Horzinek (132)
Pritchett, Manning and Zee (225)
38S
38S
31S=
24S=
3.22xl06
2.09xl06
1.22xl06
38S
Zeegers and Horzinek (306)
Felmingham and Brown (88)
40S
3.60xl06
38-39S
35S<,46Sc
27Snc,47Snc
Brinton (31)
Purchio, Larson and Collett (228)
2.90xl06
Renard et al. (237)
Collett et al. (54)
8.2
33S
12.5
12-13
c. Minor RNA species in virions of cBVDV isolates
nc. Minor RNA species in virions of ncBVDV isolates
nucleotides long and only has one ORF. This encodes 3,988 amino acids,
equivalent to 449 kilodaltons (kDa) of protein. Collett et al. (55) compared the
sequences of the two viruses. They found that there is 74% homology at the
nucleotide level. They also found that insertion of two nucleotides (or the
deletion of one) at Osloss nucleotide 4,241 results in a single ORF, with the
previously noncoding region now coding for amino acids in register with, and
homologous to, those of NADL. This indicates that the double ORF of the Osloss
sequence is a cloning or sequencing artifact.
The NADL sequence has been intensively analysed (54). At the 5’ end of
the genome there is a stretch of 385 nucleotides before the initiation codon.
There are two short ORFs present in this sequence but it is not known if they are
26
expressed. At the 3’ end of the genome there are 223 nucleotides present after
the termination codon at nucleotide 12,350. The sequence GTATA appears in
repeats in both the 5’ and 3’ noncoding regions. The 3’ noncoding region also
contains a longer repeated sequence involving two closely spaced 50 nucleotide
stretches in which 30 nucleotides are identical. It is not known what functional
significance these sequences may have. It is not known if the entire genome of
BVDV has been cloned, as the ends of the genomic RNA have not been
sequenced for comparison with the cDNA sequence.
The genome of the Alfort strain of HCV has also been cloned (193). The
sequence of 12,284 nucleotides bares many similarities to that of BVDV- There is
a nucleotide homology of 66% but an amino acid homology of 85% with respect
to the NADL sequence. The HCV sequence contains an ORF of 3,898 codons.
There are 363 nucleotides preceding the initiation codon at the 5’ end of the
sequence. Short ORFs are present in this region. At the 3’ end 224 nucleotides
are present after the termination codon. The HCV ORF contains 90 codons less
than that of NADL. The 90 amino acid difference is accounted for by an
insertion at amino acid 1,536 of HCV. Comparison with the Osloss sequence
shows that there is an insertion of 76 amino acids at this point (192). Surprisingly,
the insertion in Osloss was found to be homologous to the sequence of animal
ubiquitin (192), but no homology has been found between the NADL insertion
and any other nucleotide sequence. This indicates that the BVDV genome RNA
can undergo recombination with cellular RNA.
27
Structural Proteins
Frost and Liess (96) were the first to investigate the structural proteins of
BVDV, observing three proteins on polyacrylamide gels. Several groups have
since studied the structural proteins of the virus. There is little correlation
between reports, either in the number of polypeptides or their relative molecular
weights (Mr) (Table 4). As the same strain was studied in several cases, the
discrepancies are not a reflection of differences between strains.
The only common polypeptide detected is a 50-57 kDa glycoprotein. It is
referred to as gp53 in the proposed nomenclature for BVDV polypeptides (55).
(The proposed nomenclature of Collett et al. (55) (Table 5) will be used, as far as
possible, in this manuscript.) This glycoprotein is present in the envelope of the
virus. The neutralising monoclonal antibodies to BVDV that have been produced
all react with this glycoprotein (21,58,71,172,173,290); however, they do not all
bind to the same epitope, as shown by their varying reactivities with different virus
strains (21,58,71). It appears that 10 epitopes (55) in at least three domains are
involved in virus neutralisation (21). Eight epitopes were clustered in a single
domain (55). One domain is highly conserved between BVDV strains and is
strongly associated with neutralisation. The other two domains are weakly
associated with neutralisation; one appears to be virus-specific and the other
appears to be generally conserved among BVDV strains. Similar results are found
with HCV (291,292). However, HCV monoclonal antibodies, specific for different
domains, can act synergistically to enhance neutralisation (291) but this has not
been observed with BVDV monoclonal antibodies (55). A domain of the glyco­
protein of the Singer cBVDV strain containing a neutralisation epitope has been
expressed in a vaccinia virus vector (77). Neutralising antibody specific for BVDV
is produced in mice immunised with the recombinant virus.
28
Table 4.
Ia
Reported sizes of the structural polypeptides of BVDV.
II
Ill
IV
NADLb NADL OC24Vc
NADL
V
VI
VII
VIII
IX
Singer
NADL
Osloss
NADL
Singer
gp55
gp53
gp52-56
gp48
gp48
140d
138
117
93-110
115
115
80
80
100 •
gp75
66
70
50-59
25
gp57
gp54
gp44
gp45
34
35
gp54
gp55
32
26
28
gp25
23
20
16
a. Reference.
I
Pritchett and Zee (226)
II
Felmingham and Brown (88)
III
Matthaeus (182)
IV
Akkina (I). Strain studied not known but probably NADL
V
Coria, Schmerr and McClurkin (61)
VI
Purchio, Larson and Collett (229)
VII
Renard et al. (238)
VTTT
Collett, Larson, Belzer and Retzel (53)
IX
Etchison (unpublished data)
b. BVDV strain
c. Oregon C24V
d. Mr polypeptide
gp. glycoprotein
29
Etchison (James Etchison, personal communication) found that gp48, which
is often detected in purified virus preparations, is not enriched in peaks of virus
infectivity on rate-zonal and isopycnic density gradients. However, four other
polypeptides are enriched (Table 4, column IX). This tends to imply that gp48 is
not an integral part of the virus structure.
The only other structural polypeptide that has been identified is the capsid
protein. Matthaeus (182) suggested that the 34 kDa polypeptide he detected
served this role. The recent data of Collett et al. (53) and Etchison (James
Etchison, personal communication) suggest that it is somewhat smaller, 16-20 kDa.
In the proposed nomenclature this is referred to as p20 (55).
Matthaeus (184) compared the cross-reactivity of the three structural
polypeptides of BVDV he detected (Table 4, column III) with antisera against
BVDV or HCV. The most marked difference was that HCV antibodies failed to
immunoprecipitate the 34 kDa polypeptide, Matthaeus’s. putative capsid protein.
Of the two glycoproteins he detected, gp57 reacted more strongly with BVDV
antibodies than did gp44, while the opposite was true with HCV antibodies. He
suggested that the 34 kDa polypeptide is species-specific and the glycoproteins are
group-specific within the Pestivinis genus.
Physicochemical Properties
The physicochemical properties of BVDV have been reviewed by Brinton
(31) and Horzinek (134). Infectivity is lost after exposure of the virus to ultra­
violet radiation or treatment with ether, chloroform, deoxycholate, saponin,
nonidet P40, Tween 80, sodium dodecyl sulphate, formaldehyde, B-propiolactone
and trypsin. The virus is relatively stable in a pH range of 5.1-93, with a
maximum stability at pH 7.4 (116). The virus also has reasonable thermal
30
stability, losing only 0.2-1.0 Iog10 ID50 units after 24 h at 37°C (134). Tempera­
tures above 50°C result in more rapid inactivation.
The reported values for the buoyant density of BVDV, ranging from
1.09-1.17 g/ml, reflect the size heterogeneity of the virus particles (134). Laude
and Gelfi (157) observed that the buoyant density of pestiviruses also varies
according to the cell line in which they are propagated. Two peaks of infectivity
are often observed when BVDV is sedimented to equilibrium on caesium chloride
or potassium tartrate gradients but a single peak, at a density of 1.15-1.16 g/ml, is
generally observed with sucrose gradients (134). The sedimentation coefficient of
the virus has been calculated (157). Analysis by rate zonal centrifugation on
7-35% sucrose gradients yields a value of ISSt IIS 20jwand analysis on isokinetic
gradients yields a value of 139+12S20w.
Viral Replication and Morphogenesis
The Virus Receptor
The initial event in virus replication is the binding to cellular receptors. The
cell receptor for BVDV has not been identified. However, a monoclonal
antibody, raised against a cellular component, has been described that specifically
blocks binding of cBVDV to bovine cells (55). The binding of HCV and BDV is
not impaired. This implies that there is a specific receptor for BVDV on bovine
cells. As the infectivity of BVDV was not totally blocked, the virus may also be
taken up by a passive mechanism. Another monoclonal antibody, that completely
blocks the binding of bovine enterovirus 3 to its receptor, also interferes with
cBVDV infectivity (274), implying that the receptor for BVDV and’bovine
enterovirus 3 are closely associated.
31
Growth Phases
Early studies of virus growth kinetics in cell culture were reviewed by
Horzinek (134). Major differences are found, apparently dependent on the cell
lines and virus strains used. Adsorption appears to be a slow process and is
maximal after 60-100 min. Different adsorption rates are observed with different
cell lines: bovine spleen cells have been shown to adsorb BVDV 13 times faster
than bovine kidney cells (260). In general the growth kinetics are as follows: the
latent phase lasts for 4-12 h, exponential growth occurs until 12-36 h postinfection
(pi) and a plateau phase follows this (134). The duration of each phase appears
to be dependent more on the cell line and virus involved than on the multiplicity
of infection (moi). Titres of released virus are generally higher than those of cellassociated virus (95,115,205,287).
Studies of infected cells by immunofluorescence have shown the presence of
viral antigen in the nucleus early in infection (91). Viral antigen is then observed
perinuclearly and later becomes dispersed throughout the cytoplasm. The
fluorescence disappears late in infection (91,175).
Noncytopathic BVDV causes persistent infections of susceptible cells in
culture (60). Persistently infected cells continuously produce virus. The cells
cannot be freed of the infection by passaging in medium containing antibody to
the virus (60) or by treatment with IFN or tumour necrosis factor alpha (15). The
persistent infections are not due to the presence of defective interfering particles
(60), although these are detected in cBVDV cultures (67,87).
BVDV replication is inhibited by proflavine sulphate and acriflavine dihydro­
chloride (68). The nature of the inhibition is not known but it has been suggested
that it is a product of the high degree of secondary structure of BVDV genomic
RNA (55). Resistant virus strains have been described (69,247). Nuttall (205)
32
observed that cBVDV yield is reduced in the presence of low concentrations of
AMD but ncBVDV yield is enhanced under the same conditions.
RNA Species and Synthesis
Two RNA species are detected in cells infected with BVDV (88,228): one is
the same size as the viral genomic RNA and the other is twice the genomic
RNA’s mass. The larger species is not degraded by low concentrations of RNase,
implying that it is the replicative form (RF) of RNA; the degradation of this RNA
at higher concentrations of RNase indicates that it is not a true duplex molecule
(228). Denaturation results in only genome-size RNA being detected (228). In no
case has a subgenomic messenger RNA been detected.
Little is known of the kinetics of RNA synthesis. Nuttall (cited in reference
(134)) detected [3Hjuridine incorporation 7.5 h pi; maximum incorporation was
observed after 10.5 h. Moorman and Hulst (199) found that the time course of
HCV RNA synthesis paralleled that of virus growth.
Intracellular Polypeptides
As with the viral structural proteins, the numbers and sizes of virus-specific
polypeptides detected in infected cells varies between reports (Table 5).
There is a marked heterogeneity in the sizes of some polypeptides between
both biotypes and strains when directly compared (1,73,217). The most marked
variation is found in gp53, the prominent Mr 52-64 kDa glycoprotein that is often
resolved as a doublet; this is also the case with HCV (292). This heterogeneity is
due to differential glycosylation of a precursor polypeptide (1,53,74,292). The
precursor polypeptide is detected in cells labelled in the presence of tunicamycin
or after endoglycosidase F digestion of the glycoprotein. It has been estimated to
be 51-52 kDa (1,74) or 42 kDa (53) in size. Analysis of the nucleotide sequence
Table 5.
Molecular weights of BVDV-specific polypeptides detected in cells infected with BVDV.
Ia
NADLb
Singer
OC24Vc
III
• II
NY-Id
NADL
Singer
165e
cBVDV
165
ncBVDV
IV
V
VI
VII
VIII
NADL
NADL
NADL
Singer
Proposed
names
165
gpl65
I
145
135
115
115
115
115
118
118 .
118
120
120
133
135
125
105
pl25
80
85
p80
75
77
gpll6
90
80
80
80
80
80
gP?5
gp62
gp75
87
80
gp75
gp69
gp65
gp62
gp62
58
54
gp54
gp45
gp54
35
35
gp56-58
gp56-58
gp55-64
gp57
gp53-55
gp53
gp52-56
gp45
gp55
45
gp48
gp48
gp48
gp49
gp47
gp48
gp40
35
38
gp37
37
37
37
38
gp54
gp53 ..
gp48
37
36
32
33
25
gp23
32
gp26
19
a. Reference
I
Akkina (I)
IV Pocock et al. (217)
VII Etchison (unpublished data)
b. BVDV strain
c. Oregon C24V
II
Purchio et al. (229)
V
Magar et al. (172)
VIII Collett et al. (55)
d. New York-1
e. Mr polypeptide
III
VI
gp25
23
20
16
Donis and Dubovi (73-75)
Collett et al. (53)
gp. glycoprotein
gp25
p20
w
w
34
of both BVDV and HCV shows that there are four potential N-glycosylation sites
on the precursor (53,292). In cells infected with HCV, gp53 is found connected to
a glycoprotein of 31 kDa (292).
Monoclonal antibodies have been produced that recognise several BVDV
polypeptides. Neutralising monoclonal antibodies recognise only gp53 (21,58,71,173,290). The non-neutralising monoclonal antibodies that have been produced
recognise gp48 (58,71), gp53 (58,71), a 20 kDa polypeptide (145), a 79 kDa and
73 kDa polypeptide (213), and a 73 kDa polypeptide with minor specificity for
several polypeptides from 28-51 kDa (213).
One consistent difference has been detected between cytopathic and
noncytopathic strains (1,12,27,73,172,217): a prominent virus-specific polypeptide
of Mr 80-87 kDa, that is detected in cells infected with cBVDV strains, is not
detected in cells infected with ncBVDV strains. However, in ncBVDV-infected
cells a polypeptide of Mr 105-120 kDa, that is also detected in cells infected with
cBVDV, is present at an increased level. These two polypeptides are designated
p80 and pl25 (55). Peptide analysis has shown that the two polypeptides are
related (1,229). This suggests that the lack of p80 in ncBVDV-infected cells is
due to a defect in processing of pl25. Collett et al. (53) showed that p80 of the
cBVDV strain NADL is derived from a 125 kDa precursor, the other product
being a 54 kDa protein. In addition to the above processing difference, a
polypeptide, or doublet, of Mr 90 kDa has been detected in cells infected with
ncBVDV but not cBVDV (73).
Donis and Dubovi (72) studied the antibodies present in animals naturally or
experimentally infected with BVDV. They found strong immune responses to the
following major polypeptides detected in infected cells: the gp48, gp53, pl25 and
p80 in cBVDV-infected animals, pl25 in ncBVDV-infected cells and, occasionally,
35
a 37 kDa polypeptide. Bolin (22) studied the specific immune response of
persistently infected cattle to a live cBVDV vaccine. He found a highly strainspecific neutralising antibody response to gp53. He also found a less specific non­
neutralising antibody response to p!25, p80, gp53 and gp48. In normal calves
vaccinated with modified live cBVDV vaccine, Bolin and Ridpath (27) detected
antibodies to p!25, p80, gp53, gp48, gp25 and a 39 kDa polypeptide. Some calves
also produced antibody to gp62 and a 37 kDa polypeptide.
Genome Organisation
Collett et al. (53) generated a panel of sequence specific antisera raised
against short segments along the genome of the NADL cBVDV strain expressed
in Escherichia coli. These were used to immunoprecipitate authentic BVDV
polypeptides from lysates of infected cells. From this data they were able to
identify precursor-product relationships between various polypeptides. This
information, coupled with the nucleotide sequence, was used to propose a
tentative scheme for the organisation of the BVDV genome (Figure 3). Polypep­
tides were not detected for two hydrophobic regions of the translated genome
sequence, representing 30 kDa and 38 kDa of protein. Peptide sequencing of the
polypeptides needs to be performed in order to accurately locate the genes on the
genome and to substantiate precursor-product relationships.
The genetic organisation of the genome is very similar to that of flaviviruses,
the structural genes being located at the 5’ end of the genome. There is a striking
similarity between the hydrophobicity profiles of BVDV and flavivirus polyproteins
(52,53,55). The BVDV ORF is probably translated as a single polyprotein, which
is cotranslationally and posttranslationally processed (55).
36
NADL genomic RNA open reading frame (11,964 nucleotides)
5’
3'
p20"
gp ll6
pl25b
pl33
gp48d gp25
< Structural proteins > <
Vegetative functions
ggg
immunoprecipitated polypeptides
I lll
regions with no detected intracellular protein product
>
a. Capsid protein
b. Polypeptide that is not cleaved during ncBVDV replication
Envelope glycoprotein possessing epitopes recognised by
neutralising monoclonal antibodies
d. Possibly not a structural polypeptide
Figure 3.
Putative genomic organisation of the NADL cBVDV isolate. (Adapted
from reference (53)).
Unfortunately, in vitro translation studies do not illuminate this process
(230). Genomic BVDV RN A, after denaturation with methyl mercury, directs the
synthesis of a range of polypeptides of Mr 50-150 kDa. Immunoprecipitation of
the products with BVDV-specific antiserum does not reveal any products that
comigrate with authentic viral proteins. When free polyribosomes are used to
program translation, two polypeptides are synthesised that comigrate with
37
authentic BVDV 80 kDa and 115 kDa polypeptides. Membrane-bound polysomes
do not direct the synthesis of any BVDV-specific polypeptides.
Putative Protein Functions
It has been hypothesised that p80 and pl25 may play a role in RNA replica­
tion or translation (73). Some evidence for a role of this polypeptide in viral
RNA metabolism comes from earlier studies on a soluble antigen present in
cBVDV infected cell cultures (183,184,283,284). This antigen is 82-85 kDa in size
(183,284) and is detected both as a monomer and as a dimer (284). It is not a
structural protein of BVDV (183) but is antigenically related to a nonstructural
polypeptide of HCV (183,284). It is the only BVDV polypeptide that has been
shown to have enzymatic properties: it reduces nucleotide tri- and diphosphates to
their respective monophosphates and may be a protease (183,284).
Recent computer analysis of the amino acid sequence of NADL has
identified domains on p!25 that are also present on flavivirus NS3 (8,52,55,106).
They contain highly conserved amino acid residues typical, in an N-proximal
domain, of chymotrypsin-like serine proteases (8,106) and, in a C-proximal
domain, of RNA helicases (106). The amino acids are conserved not only in
pestiviruses and flaviviruses but also alphaviruses (106). It is thought that cleavage
of pl25 occurs between the two domains (106).
Some amino acid residues are also conserved between BVDV p!33/p58:p75
and flavivirus NS5 (52). A conserved Gly-Asp-Asp sequence in this protein is
typical of RNA dependent RNA polymerases, implying that p!33/p58:p75 is the
BVDV replicase (52,55).
38
Morphogenesis
Very little is known about the morphogenesis of BVDV. Chasey and Roedef
(43) and Gray and Nettleton (HO) observed modifications of the endoplasmic
reticulum (ER) in cBVDV infected cells. The modifications are tubular structures
65-83 nm in diameter and of variable length. One end is sealed and the other
terminated in the rough or smooth ER. Mature virions are observed within the
tubules and larger vesicles. No virions are observed free within the cytoplasm or
budding through intracellular membranes or-plasma membranes of the infected
cells. Mahnel (174) found that cells persistently infected with ncBVDV contain
smaller and fewer vesicles than cells infected with cBVDV. Again, no virions are
observed in the cytoplasm or budding through membranes.
Ward and Kaeberle (288) used immunoperoxidase to stain infected cells.
Electron micrographs showed the presence of viral antigen around the periphery
of vacuoles. Early in infection such vacuoles have well defined limiting
membranes and contain intensely staining particles, thought to be viral nucleocapsids. Later in infection, when mature virus is present in the vacuoles, no
limiting membranes are detected.
Based on their electron microscopic observations of infected cells, Bielefeldt
Ohmann and Bloch (16) suggested the following sequence of events occurred in
BVDV morphogenesis. Initially virus proteins accumulate in matrices of fibrillar
material. These become more condensed and are internalised within membrane
vesicles formed from coalescing smooth bi- and multilaminated membranes.
Virion maturation takes place within these vesicles through the interaction of virus
nucleocapsids and membrane units. Virus release is achieved either through
disintegration of the cell or exocytosis.
39
Overview of the Project
Although the genomic RNA of two strains of BVDV has been molecularly
cloned, there are still major gaps in our knowledge of BVDV RNA. The actual
size of the genome is still unclear and it is likely that the size varies between
strains. More importantly, no refereed reports concerning the size of the
ncBVDV genome have been published. Another major gap involves the synthesis
of BVDV RNA in infected cells. The ultimate aim of this project was to accurate­
ly measure the genome of both cBVDV and ncBVDV strains and to define the
time course of RNA synthesis in infected cells.
Before these questions could be addressed, it was necessary to define a
suitable system for studying BVDV. A major problem encountered in working
with BVDV is the contamination of cell lines with adventitious virus from bovine
serum. Cell lines have to be screened for the presence of the virus and then kept
free from contamination. I felt that it would be wise to find a substitute for
bovine serum for the growth of the cells. Another important consideration was
that, as the virus does not replicate to high titres, a cell line should be chosen that
would maximise virus yield. I also deemed it necessary to develop a reliable assay
for the titration of ncBVDV.
There is a lack of standard procedures for the purification of BVDV. The
purification of the virus is complicated by the heterogeneous size and density of
the virus, and the low yields of virus from cells. The next part of my research
involved a series of experiments to develop a system for the purification of BVDV
while limiting the loss of infectious virus.
For the study of the size of the BVDV genomic RNA, the main requirement
was the use of defined molecular weight markers for the calibration of agarose
40
gels. Initially denatured DNA restriction fragments were used. Later studies
involved using VSV mRNAs. The results were checked using northern blots.
During the genome sizing studies, I noticed that there were distinct
differences between the BVDV strains in terms of their intracellular viral RNA
levels. This led to speculation that the genomic RNA of ncBVDV strains was
more efficiently packaged into virions. I addressed this question by quantitating
the levels of BVDV-specific plus sense and minus sense intracellular RNAs. The
results of this study led to a hypothesis that cBVDV strains have lost control of
minus sense RNA synthesis, due to a defect in their ability to target plus sense
RNA for encapsidation.
/
41
MATERIALS AND METHODS
Biologicals and Buffers
Reagent grade chemicals were obtained from J. T. Baker Chemical Com­
pany, Fisher Scientific, Sigma Chemical Company or Eastman Kodak Company.
Radioisotopes were obtained from NEN Research Products, DuPont Company.
Cell culture medium was obtained from KC Biologicals, Sigma Chemical Company
or Irvine Scientific. Equine serum was obtained from Hyclone Laboratories, Inc.
or Sigma Chemical Company. Fluorescein conjugated rabbit anti-bovine gamma­
globulin was purchased from Antibodies Inc. or Sigma Chemical Company. Calf
intestinal alkaline phosphatase (CIP), R Q l DNase, RNasin, T4 polynucleotide
kinase, Riboprobe Gemini System II kit and restriction endonuclease Sail were
purchased from Promega. Bacteriophage X DNA, restriction endonuclease
H indlll and vanadyl ribonucleoside complexes (VRC) were purchased from
Bethesda Research Laboratories. Proteinase K and restriction endonucleases Bgll,
Bglll, Hpal, Kpnl, Pvul and Xbal were obtained from Boehringer Mannheim
Biochemicals. Percoll, Sephacryl S-IOOO and dephosphorylated oligodeoxythymidylic acid12.18 were obtained from Pharmacia.
The composition of buffers, reaction mixtures and media formulations are
listed in Table 6.
42
Table 6.
Composition of buffers, reaction mixtures and media.
Buffer
Composition
CaCl2 solution
60 mM CaCl2, 15% vol/vol glycerol, 10 mM PIPES
(pH 7.0)
CIP buffer
50 mM Tris-HCl (pH 9.0), I mM MgCl2,
0.1 mM ZnCl2, I mM spermidine
ChloroformAAA
96% vol/vol chloroform, 4% vol/vol isopentanol
Denhardt’s solution
0.02% wt/vol ficoll, 0.02% wt/vol PVP,
0.02% wt/vol BSA
DME-%
DME supplemented with n% vol/vol equine serum
DNA-TAE buffer
40 mM Tris-acetate (pH 7.7), 20 mM sodium
acetate, 2 mM EDTA
Glyoxal solution
I M deionised glyoxal, 50% vol/vol DMSO,
10 mM NaPO4 (pH 6.65)
GTE
50 mM glucose, 25 mM Tris-HCl (pH 8.0),
10 mM EDTA
Hybridisation solution
50% vol/vol deionised formamide, 5X SSPE,
10 jug/ml sheared salmon sperm DNA, 500 /rg/ml
yeast RNA, IX Denhardt’s solution, 10% wt/vol
dextran sulphate
LB
1% wt/vol tryptone, 0.5% wt/vol yeast extract,
0.5% wt/vol NaCl, I mM NaOH
LB agar
LB, 1.5% wt/vol agar
LB+Amp
LB, 100 Mg/ml ampicillin
LSB
0.2 M NaCl, 20 mM Tris-HCl (pH 7.4),
I mM EDTA
Lysis buffer
0.14 M NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl
(pH 8.6), 0.5% NP-40, 10 mM VRC
MTH
0.5 M MOPS, 0.5 M TES, 0.5 M HEPES (pH 7.2)
NET
0.1 M NaCl, 10 mM Tris-HCl (pH 7.4),
I mM EDTA
43
Table 6, continued.
Buffer
Composition
NT buffer
50 mM Tris-HCl (pH 7.5), 50 mM NaCl,
10 mM MgCl2, 7 mM 2-mercaptoethanol,
50 jug/ml BSA
PBS
0.8% wt/vol NaCl, 0.02% wt/vol KC1,
0.115% wt/vol Na2HPO4, 0.02% wt/vol KH2PO4,
0.01% wt/vol CaCl2, 0.01% wt/vol MgCl2-SH2O
Phenol solution
63% vol/vol phenol, 37% vol/vol LSB,
7 mM 8-hydroxyquinoline
PK buffer
0.15 M NaCl, 0.1 M Tris-HCl (pH 7.5),
12.5 mM EDTA, 1% wt/vol SDS
PNE
20 mM PIPES (pH 6.5), 0.15 M NaCl,
I mM EDTA
PNK buffer
50 mM Tris-HCl (pH 7.6), 10 mM MgCl2,
5 mM DTT, 0.1 mM spermidine, 0.1 mM EDTA
Prehybridisation solution
50% vol/vol deionised formamide, 5X SSPE,
10 jug/ml sheared salmon sperm DNA, 500 //g/ml
yeast RN A, 5X Denhardt’s solution
RNA-TAE buffer
40 mM Tris-acetate TpH 7.0), 20 mM sodium
acetate, 2 mM EDTA, 50% vol/vol deionised
formamide
SOC medium
0.5% wt/vol yeast extract, 2% wt/vol tryptone,
10 mM NaCl, 2.5 mM KC1, 10 mM MgCl2,
10 mM MgSO4, 20 mM glucose
Solution D
4 M guanidinium thiocyanate, 25 mM sodium
citrate (pH 7.0), 0.5% wt/vol sarcosyl,
0.1 M 2-mercaptoethanol
SSC
0.15 M NaCl, 15 mM sodium citrate (pH 7.0)
SSPE
0.18 M NaCl, 10 mM NaPO4 (pH 7.7),
I mM EDTA
TE
20 mM Tris-HCl (pH 7.4), 2 mM EDTA
TE8
10 mM Tris-HCl (pH 8.0), I mM EDTA
TE+S
20 mM Tris-HCl (pH 7.4), 2 mM EDTA,
0.1% wt/vol SDS
44
Table 6, continued.
Buffer
TNES
Composition '
10 mM Tris-HQ (pH 7.4), 0.1 M NaCl,
2 mM EDTA, 0.2% wt/vol SDS
Cells. Viruses and Plasmids
The Madin-Darby bovine kidney (MDBK) cell line (169) was obtained from
the American Type Culture Collection (ATCC, CCL 22), at passage 113, The
bovine turbinate (BT) cell line (189), at passage 8, was the kind gift of Dr. Arlan
McClurkin of the National Animal Disease Center (NADC), Ames, Iowa. The
bovine lung cell line (BLF) was the kind gift of Dr. David Reed of Iowa State
University. The cell lines were maintained in DME-10. They were screened for
BVDV contamination by direct immunofluorescence, using fluorescent BVDVspecific antiserum obtained from the National Veterinary Services Diagnostic
Laboratory, Ames, Iowa.
All BVDV strains were the kind gift of Dr. Arlan McClurkin and Dr. Steven
Bolin of the NADC. These are listed in Table 7. Virus stocks were prepared by
infecting MDBK cells either as monolayers or cells in suspension. After allowing
the virus to adsorb for I h a t 37°C, the inoculum was aspirated and replaced with
fresh DME-2. The cells were incubated at 37°C until either extensive cell lysis
was observed, in the case of most cytopathic strains, or for 48 to 72. h, in the case
of noncytopathic strains and Oregon C24V. The tissue culture fluids (tcf) were
harvested, clarified by low speed centrifugation and stored at -70°C.
The Mudd-Summers strain of the Indiana serotype of vesicular stomatitis
virus (VSV) was provided by Dr. James Etchison.
45
Table 7.
BVDV strains studied.
Strain
Biotype
New York-1
nc1
Reference
Baker et al. (4)
Lee and Gillespie (160)
Oregon C24V
C2
Gillespie et al. (103)
NADL
C
Gutekunst and Malmquist
(H 3)
Singer
C
McClurkin et al. (189)
TGAC3 -
C
Bolin et al. (23,25)
TGAN
nc
1. noncytopathic
2. cytopathic
3. Originally thought to be a cytopathic isolate, TGA (23). Later discovered to be
a mixture of two biotypes that were each biologically cloned (25).
The plasmid pBV4-p80, provided as plasmid DNA, was the kind gift of Dr.
Marc Collett. This is a pGEM-4 plasmid with a 2,306 base insert of BVDV strain
NADL cDNA. The sequence is that between nucleotides 5,644 and 7,949 (54),
representing the p80 polypeptide gene, inserted in the Smal site of the vector.
Preparation of BVDV Antiserum
A black angus heifer, seronegative and virus negative for BVDV, was
infected with BVDV strain New York-1 by intranasal instillation of 20 ml of
infected cell culture fluid, with a titre of LSxlO8 infectious units (IU) of virus
per ml. Eight weeks later, the animal was boosted with 4 ml of a 1:1 emulsion of
46
New York-l-infected cell culture fluid and incomplete Freund adjuvant, adminis­
tered intramuscularly. Serum was prepared from blood collected 2 wk later.
Infectious Virus Assays
End Point Dilution Microtitration Assay CEPDMA')
. The assay was adapted from that developed by Robb and Martin for titrating
simian virus 40 (SV40) (243) and later adapted for the titration of murine coronaviruses (242). Ten fold serial dilutions of BVDV were prepared in a suspension of
IxlO5 MDBK cells per ml in DME-2. The suspension was then dispensed into.
Terasaki plates (Nunc), 10 £d of suspension being dispensed into each well.
Alternatively, uninfected cells were dispensed into the wells and infected the next
day with serial dilutions of virus in DME-2. To prevent dehydration during
incubation, 0.5 ml of DME-2 was distributed around the inside edge of the plates.
The plates were incubated for 72 h at 37°C. The medium was aspirated, the
plates rinsed twice with cold PBS, once with cold absolute methanol and flooded
with cold absolute methanol. Five minutes later, the methanol was removed and
the plates allowed to dry at room temperature. The plates were used immediately
or stored at -70°C.
The Terasaki plates were rehydrated by rinsing once with PBS. The primary
antiserum was diluted in PBS containing 1% wt/vol porcine gelatin and 10 gl
dispensed into each well of the Terasaki plate. The plates were incubated for 2 h
at 37°C, in a humid atmosphere. After incubation the plates were rinsed five
times with PBS containing 0.05% vol/vol Tween 80. The secondary antiserum,
fluorescent rabbit anti-bovine gammaglobulins, was diluted and dispensed, and the
plates incubated, as for the primary antiserum. The plates were washed twice
u
47
with PBS containing 0.05% vol/vol Tween 80 and twice with distilled water (5 min
per wash).
Prior to observing the cells for fluorescence, the wells were filled with PBS.
An Olympus inverted microscope, with an epifluorescence attachment, was used to
observe wells for BVDV-specific fluorescence. The virus litre was calculated from
the number of uninfected wells using the Poisson distribution. A Turbo Pascal
(Borland) computer program was written to perform these calculations.
Plaque Assay
Six well plates (Nunc) were seeded with 4x105 MDBK cells per well in
DME-IO and incubated overnight. The next day the medium was aspirated and
0.2 ml of 10 fold serial dilutions of virus in DME-2 were dispensed onto the
monolayers. Virus was allowed to adsorb for I h, with regular agitation. The
monolayers were overlaid with 3 ml 0.7% wt/vol SeaPlaque agarose (FMC) in
DME-2. The agarose was allowed to gel at room temperature and the plates
incubated inverted at 37°C. Three to 5 days later, the cells were fixed with 0.5 ml
2% vol/vol glutaraldehyde, the agarose removed, the monolayers stained with
0.4% wt/vol crystal violet in 20% vol/vol ethanol and the plaques counted.
Alternatively, the cells were stained with 3 ml 0.01% wt/vol neutral red in DME-2
for at least 6 h at 37°C in the dark, fixed with 0.5 ml 2% vol/vol glutaraldehyde,
the agarose removed and the plaques counted.
. Biological Cloning of BVDV Isolates
The BVDV strains New York-1, Singer, NADL and Oregon C24V were all
cloned by limiting dilution. Initially, stocks of each were serially diluted in a
suspension of SxlO5 MDBK cells per ml in the following ranges: Singer and
48
NADL, IO'3 to IO"6; Oregon C24V, IO'4 to IO"7; and New York-1, IO'2 to IO'5.
Thirty, 10 /xl aliquots of each dilution were dispensed into the wells of Terasaki
plates. These were incubated for 48 h at 37°C. The tcfs from each well were
transfered to the equivalent well of a fresh Terasaki plate, which was stored at
4°C. The original Terasaki plate was processed for immunofluorescence, as for an
EPDMA. The tcf from an infected well, selected at random from the lowest
dilution exhibiting less than 100% infection, was diluted 10"2 to IO'4, dispensed and
incubated as described above. The remaining tcf were stored at -70°C. This
process was repeated six times in all, tcf being harvested 30 to 48 h after each
infection. After the last cloning, five or six infected tcf were selected at random
and amplified in a 96 well plate and then a six well plate. The final tcfs were
dispensed as I ml aliquots and stored at -70°C.
Virus Growth Curves
Twenty, six well plates were seeded with 3x105 MDBK cells per well, five
wells per plate. Next day, one well of each plate was infected at a moi of one
with BVDV strains Singer, NADL, Oregon C24V or New York-1 in a volume of
0.2 ml. The fifth well of each tray was mock infected with 0.2 ml of DME-2. The
virus was allowed to adsorb for I h and 2.8 ml of DME-2 was added to each well.
■:
At specific times pi the tcf was harvested from the wells of one plate and 3 ml of
fresh DME-2 was dispensed into each well. The tcfs and the plate were placed at
-V0°C. The plates were thawed, the cells scraped off and the cell suspension
refrozen. The tcfs were also thawed and refrozen. The released and cell-associ­
ated virus titres of Singer, NADL and Oregon C24V were measured by plaque
assay and those of New York-1 by EPDMA.
49
Preparation of Cellular RNA
Cytoplasmic RNA
Cytoplasmic RNA was usually extracted using a method adapted from that
described by Maniatis et al. (178). Cell monolayers were rinsed four times with
ice-cold PBS and the cells scraped off into I ml of PBS. The cells were pelleted
by centrifugation in a microfuge for 3 min and lysed in ice-cold lysis buffer (50
per 35 mm dish or equivalent). The nuclei were removed by centrifugation
through a 24% wt/vol sucrose pad. The upper layer was collected, adjusted to IX
PK buffer and digested with proteinase K at 37°C for 30 min. The mixture was
phenol extracted (mixing with an equal volume of phenol solution, then 0.5 vol­
umes of chloroform/IAA and the aqueous phase was recovered by centrifugation),
chloroform/IAA extracted and the RNA precipitated by adding 2.5 volumes
ethanol and incubating at -20°C for at least 2 h. The RNA was recovered by
centrifugation and the pellet washed with 75% vol/vol ethanol, 0.1 M sodium
acetate (pH 5.2) and dried in vacuo. The RNA was dissolved in TE+S.
Occasionally, a second method was used for preparing cytoplasmic RNA.
Cell monolayers were rinsed once with ice-cold NET. The cells were lysed by
adding I ml of NET plus 1% vol/vol NP-40 and incubating the dishes on ice for
5 min. The lysate was placed in a 15 ml Corex tube, the volume adjusted to 2 ml
with NET and 0.2 ml of 10% wt/vol SDS, 0.2 ml of 4 M NaCl and 0.1 ml of
0.2 M EDTA were added. Proteinase K was added to approximately 40 jiig/ml
and the mixture was incubated at 50°C for 15 min. It was phenol extracted twice
and chloroform/IAA extracted twice. Sodium chloride was added to a final
concentration of 1.4 M and the RNA was ethanol precipitated. The RNA was
50
recovered by centrifugation, the pellet washed with 70% vol/vol ethanol, dried in
vacuo and dissolved in TE+S.
Nuclear Fraction RNA
Nuclear fraction RNA was prepared by resuspending the pelleted nuclei,
from the cytoplasmic RNA extraction procedure above, in lysis buffer. The DNA
was sheared by forcing the suspension through a 20-gauge needle. The nucleic
acids were extracted as described above for cytoplasmic RN A. The DNA was
removed by digestion with R Q l DNase.
Total Cell RNA
The acid guanidinium thiocyanate-phenol-chloroform extraction method of
Chomczynski and Sacchi (45) was used. Cells were solubilised in 2 ml solution D
per 10 cm diameter dish. To this was added 0.2 ml 2 M sodium acetate (pH 4.0),
2 ml phenol and 0.4 ml chloroform/IAA, with thorough mixing between additions.
The mixture was vortexed vigorously for 10 s and incubated on ice for 15 min.
The aqueous and organic phases were separated by centrifugation at 10,000 g for
20 min at 4°C. An equal volume of isopropanol was added to the aqueous phase
and the RNA precipitated at -20°C for at least I h. The RNA was recovered by
centrifugation, redissolved in 400 /ri solution D and reprecipitated in an equal
volume of isopropanol at -20°C. The RNA was recovered by centrifugation,
washed with 75% vol/vol ethanol, 0.1 M sodium acetate (pH 5.2), dried in vacuo
and dissolved in TE+S.
51
Virus Purification and RNA Extraction
Tissue culture fluids from infected cells were initially clarified by centrifuga­
tion at 4000 g and 4°C for 20 min. The tcf was adjusted to pH 6.5 by addition of
0.1 volumes of 10X PNE. Large volumes were concentrated by tangential flow
ultrafiltration, using a Minitan unit (Millipore) with 100,000 molecular weight limit
filters. Virus was recovered from the Minitan retentate or unconcentrated tcf by
one of three methods.
1) Virus was pelleted by centrifugation at 115,000 gmax for 3 h at 4°C. If the virus
was to be further purified, the pellets were resuspended in PNE. If the viral RNA
was to be extracted directly, the pellets were either resuspended in lysis buffer,
and processed as described for cytoplasmic RN A, or solubilised in solution D, and
processed as described for total cell RNA.
2) For rough virus purification, retentate or tcf was underlaid with 20% wt/wt
glycerol in PNE, which was underlaid with 80% wt/wt glycerol in PNE. These
discontinuous gradients were centrifuged at 275,000 gmax for 1.5 h. For extraction
of viral RNA, the interface between the glycerol layers was collected, diluted with
one volume of 2X PK buffer and processed as described for cytoplasmic RNA.
3) For very pure virus, retentate or tcf was layered on top of a 20-0% wt/wt
glycerol, 0-40% wt/wt potassium tartrate isopycnic gradient (108). Both compo­
nents were prepared in PNE. The gradients were centrifuged at 90,000 gav for
16 h at 4°C. The gradients were fractionated and fractions with a density between
1.11 and 1.19 g/ml pooled, diluted with PNE and virus recovered by centrifugation
as described under I) above.
If desired, viral RNA was purified by sedimentation on 10-30% wt/wt sucrose
gradients, prepared in TNES. The viral RNA was denatured by incubation in
52
50% vol/vol DMSO at 70°C for 5 min and diluted to 25% vol/vol DMSO with
TNES before layering on the gradient. Gradients were centrifuged for 4.5 h at
275,000 gmax and 20°C and fractionated. For detecting BVDV vRNA an aliquot of
each fraction was either TCA precipitated, if the RNA was radiolabelled, or dot
blotted and hybridised with a BVDV-specific probe.
Radiolabelling of Viral RNA
[32Pj-Iabelled RNA was prepared essentially as described previously (246).
Briefly, monolayers of MDBK or BT cells were infected with virus in DME-2 or
mock infected with DME-2 alone. Virus was allowed to adsorb for I h at 37°C.
The inoculum was aspirated and fresh DME-2 added. This was replaced 4 h prior
to the addition of label with phosphate free DME-2, supplemented with dialysed
equine serum and buffered with MTH. Immediately before adding label the
medium was replaced with fresh phosphate free DME-2, to which was added
[32P]-orthophosphate to a final concentration of 150 /xCi/ml. When desired, AMD
was added to the DME-2 and the cells incubated for 10 min before adding label.
Cytoplasmic and virion RNA were extracted, after the desired labelling period had
elapsed, as described above.
Labelling of RNA with [3Hj-Uridine was performed basically as for [32Pjlabelling except deficient medium was not used. After infection, the cells were
maintained in DME-2. Labelling was carried out in fresh DME-2 to which
[5,6-3Hj-uridine was added to a final concentration of 50 juCi/ml. When required,
AMD was added to the medium as described above.
53
Preparation of A DNA Molecular Weight Markers
Lambda DNA was digested with the restriction endonucleases LTmdIII or
Sail, according to the. suppliers instructions. To label the 5’ termini of the
fragments the forward reaction was used (178). Five picomoles of 5’ restriction
fragment ends (10
of H indlll digested DNA; 26.7 ng of Sail digested DNA)
were dephosphorylated using CIP in 50 /d of CIP buffer. The DNA was phenol
extracted, purified by Sephadex G-50 chromatography and ethanol precipitated.
The DNA was recovered by centrifugation, the pellet dried and dissolved in water.
The 5’ ends were labelled using 20 units of T4 polynucleotide kinase in 50 jul of
PNK buffer containing 150 ^Ci [y-32P]dATP (GammaPrep-A, Promega). The
DNA was purified as described above.
Agarose Gel Electrophoresis of RNA
Formamide Gels
These were prepared as previously described (246). Twenty centimetre,
1% wt/vol agarose gels were prepared in RNA-TAE buffer. Prior to electro­
phoresis, 8 jjA of RNA dissolved in TE+S was mixed with 12 jul of deionised
formamide buffered with 2 mM phosphate (pH 7.0) and incubated at 65°C for
I min. Twenty microlitres of a 1:1 mixture of glycerol and deionised formamide,
containing 0.2% wt/vol agarose beads and bromophenol blue, were added and the
sample was loaded on the gel. Electrophoresis was overnight at 50 Vi If desired,
gels were stained with ethidium bromide (10 jUg/ml in RNA-TAE buffer minus
formamide) for 30 min, destained for I h i n water and photographed. For
autoradiography, gels were fixed in 10% vol/vol formaldehyde for 30 min, washed
in water for 30 min, blotted onto Whatman DE81 paper and autoradiographed.
54
Glyoxal Gels
These were prepared according to the protocol of McMaster and Carmichael
(191). In general, 0.7% wt/vol agarose gels (20 cm x 22 cm) were prepared in
10 mM sodium phosphate (pH 6.65). The RNA samples were denatured at 50°C
for I h i n glyoxal solution. The samples were chilled on ice and 0.1 volumes of
50% vol/vol glycerol, saturated with bromophenol blue, were added. Electro­
phoresis was at 100 V with buffer recirculation. Gels were stained either for
10 min with acridine orange (33 Mg/ml in 10 mM phosphate buffer) and destained
in three changes of 10 mM phosphate buffer in an enameled metal pan (191) or
for 20 min in 50 mM NaOH, 0.5 jug/ml ethidium bromide then for 40 min in
0.1 M tris-HCl (pH 7.4), 0.5 Aig/ml ethidium bromide. The gels stained with
acridine orange were photographed with the aid of a red 25A filter (Kodak). For
autoradiography, gels were either fixed and blotted as described above for
formamide gels or fixed in 20% vol/vol methanol in 10 mM sodium phosphate and
dried down onto 3MM paper (Whatman). For fluorography the gels were soaked
for 10 min in Fluoro-Hance (Research Products International Corp.), blotted or
dried down as described above and exposed to preflashed X-ray film (156).
Northern Transfers
RNA was transfered from glyoxal gels to nitrocellulose (Schleicher and
Schuell, BA85) essentially as described by Thomas (275). Transfers were
performed in IOX SSPE overnight. Filters were dried and baked for 2 h at SO0C
in vacuo. The filters were treated with 20 mM Tris-HCl (pH 8.0) for 5 min at
65°C to reverse glyoxylation. Filters were stored dry at room temperature.
55
RNA Dot Blots
Samples for RNA dot blots were either denatured with glyoxal solution, as
described above for glyoxal gels, or with 7.5% vol/vol formaldehyde (63). The
RNA was adjusted to a final concentration of 15X SSPE and, if desired, serially
diluted in 15X SSPE. Nitrocellulose was prewet in water, equilibrated in
IOX SSPE and placed on top of two sheets of 3MM paper soaked in IOX SSPE ,.
in a Biorad manifold. Wells were rinsed with 0.5 ml IOX SSPE, the samples
applied to the filter and the wells rinsed with a further 0.5 ml of 10X SSPE. The
filters were dried and baked, as described above for northern transfers.
Agarose Gel Electrophoresis of DNA
Electrophoresis of DNA was performed in minigels, either after denaturation
with glyoxal as described above or as previously described (204). In the latter
case, 0.8% wt/vol agarose minigels were prepared in DNA-TAE buffer. One
microgram of DNA dissolved in 15 /d of TE8 was mixed with 5 /d of 0.2% wt/vol
. agarose beads in 10% glycerol, 10 mM Tris-HCl (pH 7.5), 10 mM EDTA satu­
rated with bromophenol blue. To this was added 2.5 //I of 80% vol/vol glycerol
saturated with bromophenol blue. Electrophoresis was at 50 V for 2 h. The gels
were stained with ethidium bromide and photographed.
Preparation of Plasmid DNA
Transformation of Escherichia coli DH5
Transformation of Escherichia coli (E. coli) DH5 cells was performed using
the basic calcium chloride protocol (263). To prepare competent cells, 200 ml
culture of E. coli DH5 in LB was incubated at 37°C, until the culture reached an
56
OD590 of 0.375. The culture was chilled on ice and the cells collected by centrifu­
gation at 1,000 g for 7 min. They were gently washed with ice cold CaCl2 solution,
resuspended in 40 ml of CaCl2 solution and incubated on ice for 30 min. The
cells were collected as above, resuspended in 8 ml CaCl2 solution and left on ice
overnight.
The pBV4-p80 plasmid DNA preparation provided by Mark Collett was
diluted in CaCl2 solution to give a final concentration of I Mg/^l- To 10 /il of this
DNA solution was added 100 /d of the competent E. coli DH5 suspension. After
incubation on ice for 10 min, the cells were heat-shocked at 42°C for 2 min, I ml
of LB added and the suspension incubated for I h at 37°C. The cells were diluted
in SOC medium and spread on LB+Amp agar plates, which were incubated
overnight at 37°C.
Screening of Transformants
Transformants were screened by alkaline lysis plasmid minipreps (159).
Individual colonies from the LB+Amp plates were inoculated into 10 ml of
LB+Amp and incubated overnight at 37°C. A 1.5 ml aliquot of each culture was
pelleted by spinning in a microfuge for 20 s. The cells were resuspended in 100 ^l
TE8 and incubated at room temperature for 5 min. Cells were lysed by adding
200 ii\ 0.2 M NaOH, 1% wt/vol SDS and incubating on ice for 5 min. After
adding 150 ^l 3 M potassium acetate the lysate was vortexed, incubated on ice for
5 min and debris removed by centrifugation. Two volumes of 95% ethanol were
added, followed by incubation at room temperature for 2 min. Nucleic acids were
pelleted, washed with 70% vol/vol ethanol and dried. After redissolving in 20 /rl
TE8, RNA was removed by digestion with 10 ng DNase-free RNase. Plasmid
DNA was analysed by electrophoresis in agarose gels.
57
Large Scale Plasmid Purification
Large scale plasmid purification involved the use of the alkaline lysis method
for producing crude cell lysates, followed by purification on CsCl gradients (128).
One litre of LB+Amp was inoculated with a 10 ml overnight culture of E. coli
DH5 transformed with pBV4-p80 and incubated overnight at 37°C. Cells were
recovered by centrifugation at 5,000 g for 10 min, resuspended in 40 ml GTE and
lysed by mixing gently with 80 ml 0.2 M NaOH, 1% wt/vol SDS, followed by
incubation for 10 min at room temperature. Potassium acetate (pH 4.8) was
gently mixed in to a final concentration of I M. After incubating at room tem per­
ature for a further 5 min, debris was removed by centrifugation at 5,000 g for
10 min. The supernatant was carefully decanted off, I volume of isopropanol
added and the nucleic acids allowed to precipitate at room temperature for
45 min. Nucleic acids were pelleted, lyophilised until nearly dry and redissolved in
8 ml TE8.
To purify pBV4-p80 plasmid DNA, the nucleic acids were sedimented on
CsCl isopycnic gradients (128). The nucleic acid solution was adjusted to a final
volume of 10 ml in TE8, 0.4 mg/ml ethidium bromide, in which was dissolved 9.5 g
CsCl. This solution was dispensed into two 12 ml centrifuge tubes and overlaid
with mineral oil. The tubes were centrifuged for 45 h in an SW 41 Ti rotor at
210,000 g and 20°C. Plasmid bands were visualised by U.V. light and the bottom
band collected by side-puncture of the tube. Ethidium bromide was removed by
repeated extractions with water-saturated n-butanol. The DNA was desalted by
Sephadex G-50 chromatography.
The plasmid DNA was characterised by digestion with restriction endo­
nucleases.
58
Purification of the pBV4-p80 BVDV cDNA segment
The BVDV cDNA segment was excised from pBV4-p80 by digestion with
restriction endonucleases Kpnl, Xbal and Bgll (Figure 4). For purifying the
BVDV cDNA insert, lorn melting temperature agarose was used (264). The
fragments were electrophoresed on a 1% wt/vol low melting agarose gel, prepared
in DNA-TAE buffer. The largest DNA band was excised, the agarose melted at
65°C, diluted in LSB and the DNA recovered using an Elutip-d column
(Schleicher and Schuell).
Preparation of BVDV-specific Probes
Nick Translation of cDNA
This was performed as previously described (204). Briefly, 0.5 /zg cDNA was
incubated with I ng DNase I, 6 U DNA polymerase I, 125 pCi a-32P-dCTP and
2 pM dNTPs in 50 pi of NT buffer at 15°C for 2 h. Unincorporated nucleotides
were removed by Sephadex G-50 chromatography.
In vitro Transcription of pBV4-p80
For SP6 RNA polymerase transcription, to produce BVDV-specific plus
sense RNA probes, pBV4-p80 plasmid DNA was linearised by digestion with
Xbal. For TV RNA polymerase transcription, to produce BVDV-specific minus'
sense RNA probes, the plasmid DNA was digested either with Kpnl, followed by
digestion with 5 U//zg Klenow fragment of DNA polymerase I to remove 3’
overhanging ends, or with Hpal.
In vitro transcription was performed using a Riboprobe Gemini System II kit.
Reactions were performed with 0.5 /zg linearised pBV4-p80 DNA and 50 pCi
O--32P-CTP, in a volume of 20 /zl, according to the manufacturer’s instructions.
59
KPn ' Wpa I
BVDV
cDNA
Figure 4.
Plasmid pBV4-p80. The map shows the restriction sites used in extrac­
tion of the BVDV cDNA and in in vitro transcription. SP6 transcrip­
tion gives viral-sense RNA; T l transcription, viral complementary
RNA.
Template DNA was removed by digesting with I U RQ l DNase at 37°C for
15 min.
Hybridisations
Northern and dot blot hybridisations with cDNA probes were performed as
described previously (204). Nitrocellulose filters were prehybridised in prehybridi­
sation buffer (0.05 ml per cm2 of filter area) for 3 to 6 h at 42°C, in heat-sealed
60
polythene bags. The prehybridisation solution, was replaced with hybridisation
solution (0.01 ml per cm2 of filter area) containing IxlO6 cpm/ml of 32P-Iabelled
cDNA probe. Hybridisations were incubated at 42°C overnight. The next day, the
filters were washed twice in 2X SSPE, 0.1% wt/vol SDS for 10 min at room
temperature; twice in 0.1X SSPE, 0.1% wt/vol SDS at room temperature; and in
0.1X SSPE, 0.1% wt/vol SDS at 65°C for I h. The damp filters were wrapped in
plastic film and autoradiographed.
Hybridisations with RNA probes were performed as described for cDNA
probes with the following exceptions. Prehybridisations and hybridisations were
performed at 55 to 60°C. The filters were washed twice with IX SSPE for I h at
65°C and with 0.1X SSPE for I h at 60°C.
If filters were to be reprobed, the old probe was stripped off by rinsing them
with boiling water.
Detection of 3’ Polvadenosine Sequences
Half a microgram of dephosphorylated oligo (dT)12„18.was end labelled using
T4 polynucleotide kinase as described above. Labelled oligonucleotides were
recovered by affinity chromatography on DEAE Trisacryl (LKB). The oligonuc­
leotides were bound in TE8 and eluted in TE8, I M NaCl. Filters to be probed
were prehybridised at 25°C for I h in 20 ml of 6X SSPE, 0.1% SDS, 5X Denhardt’s solution. The filters were hybridised overnight at 25°C with IO6 cpm/ml of
[32P]-labelled oligo (dT) in 10 ml of 6X SSPE, 0.1% SDS, IX Denhardfs solution,
200 ng/ml tRNA. The filters were washed three times with 3X SSPE, 0.1% SDS
at room temperature and then for 10 min in IX SSPE, 0.1% SDS at 25°C. The
filters were blotted dry, wrapped in plastic film and autoradiographed.
61
Quantitation of BVDV-specific RNA
Plus and minus sense RNA was transcribed from pBV4-p80, as described for
preparation of RNA probes, except reactions were carried out in a volume of
100 /il with 2 fig of linearised DNA and 0.5 mM CTP for 2 h. The RNA was
glyoxalated, as described above, and diluted to give a range of concentrations of
0.316 to 1000 pg in 50 /d 12X SSPE. Total cell RNA was glyoxalated and diluted
to a final concentration of 5, 0.5 and 0.05 Hg in 50 ^l of 12X SSPE.
Duplicate
dot blots were then prepared, as described above. Filters were hybridised at SB0C
overnight with either IxlO6 cpm/ml of minus sense RNA probe or 2xl06 cpm/ml
plus sense RNA probe. After washing, the filters were dried and both filters
autoradiographed on the same sheet of preflashed X-ray film.
For calculating the RNA concentration, the autoradiographs were scanned
with a Hoefer GS-300 scanning densitometer and the peaks integrated using the
Hoefer GS-350 Data System software supplied with the instrument. Linear
regression analysis was performed on the integrated values for the in vitro tran­
scribed, serially diluted RNA samples, using Sigmaplot 4.0 (Jandel Scientific) to
provide calibration curves for BVDV-specific RN A. Only curves with an R value
greater than 0.98 were used. The BVDV-specific RNA content in the cellular
RNA samples was calculated by the standard equation,
y - mx + b
These were corrected for nonspecific binding by subtracting the value for mockinfected cell RNA. Values which did not exceed the maximum value for the
calibration curve were corrected to picograms of BVDV RNA per microgram of
total cell RNA using the following equation,
62
pg BVDV-specific RNA
i
Atg RNA in sample
N
length of BVDV genome (12.6 kb)
:------------
length of BVDV sequences in probe
The mean BVDV-specific RNA concentration was then calculated for each time
point from the values for individual dilutions and. exposures.
63
RESULTS
Selection of Cell Lines
A major complication of studying BVDV in cell culture is that ncBVDV
contaminates many, if not all, lots of commercially prepared bovine serum
(139,206,252). This can result in infection of susceptible cells, grown in medium
containing contaminated serum. Any cell line, to be used in the study of BVDV,
has to be free of the virus and maintained in this pristine state. Another problem,
encountered in the use of bovine serum, is the presence of BVDV neutralising
antibody.
Three cell lines were considered for use in these studies: MDBK, BT and
BLF cells. All three cell lines were shown to be free of BVDV by direct immuno­
fluorescence. All three cell lines supported the growth of BVDV.
To obviate the potential problems of maintaining the cells in bovine serum
supplemented medium, the growth of the different cell lines in KClOO serum-free
medium (the kind gift of Paul Baker) and in medium supplemented with equine
serum was studied. Only MDBK cells grew in KClOO medium and they grew very
slowly. None of the cell lines were adversely affected by growth in equine serum
(Figure 5).
I decided to use MDBK cells for my research, as they grew much faster than
the other two cell lines and, being a permanent cell line, were not likely to
undergo crisis at some point in the future. Some experiments were also per­
formed using BT cells.
64
96
108
120
Tim e (h)
Figure 5. Growth of MDBK cells in KClOO serum-free medium (■) or DME
supplemented with either 10% equine ( • ) or 10% foetal bovine ( A)
serum.
Cvtopathic Effect of the BVDV Strains
The project initially involved the comparison of four strains of BVDV.
These were the noncytopathic strain New York-1 and the cytopathic strains
Singer, NADL and Oregon C24V. Due to the different cytopathology of Oregon
C24V, it is classified as being a different biotype from the other two cBVDV
65
strains (93). The Singer and NADL strains were highly cytopathic. They
produced extensive vacuolation in infected cells by 24 h pi. Total destruction of
the cell monolayers occured by 36-48 h pi. Oregon C24V was less cytopathic.
Vacuolation of infected cells was observed, though this occurred later than with
Singer or NADL. The cell monolayer was rarely extensively damaged, lysis of
cells appearing to be due to coalition of the vacuoles, rather than the cellular
degeneration observed with the other cytopathic strains. In the case of cells
infected with the noncytopathic strain, New York-1, no cellular damage was
observed.
The nature of the cytopathologies of the four strains seemed to influence
the amount of viral antigen present in the cells. When infected cells were studied
by immunofluorescence, Singer and NADL-infected. cells fluoresced brightly;
Oregon C24V-infected cells fluoresced less brightly; and New York-l-infected cells
fluoresced poorly (data not shown).
Titration of Infectious Virus
The variation in cpe between the strains influenced the method by which
they were titrated. Singer and NADL were titrated using standard plaque assays,
in which the cell monolayers were stained either with neutral red or with crystal
violet. Oregon C24V was also titrated by plaque assay but plaques were only
readily detected by neutral red staining (Figure 6). Plaques were occasionally
observed after prolonged staining of New York-l-infected cell monolayers with
neutral red (data not shown) but this was not reproducible, so a different assay
method was sought. Several alternative assays have been developed to titrate
ncBVDV strains but these were indirect, time consuming, statistically inaccurate or
not comparable with plaque assays (90,95,105,140,141,149,176,210,231,271). To
66
Figure 6.
Plaques of BVDV strain Oregon C24V: comparison of staining with
(A) neutral red and (B) crystal violet. The monolayers were stained 4
days after infection.
obviate these drawbacks, an end point dilution assay, originally developed for
titrating SV40 (243), was adapted for the titration of BVDV (245). As this assay
involved indirect immunofluorescence for detecting BVDV-infected cells, BVDVspecific antiserum had to be produced.
Raising of BVDV-specific Antiserum
When initially setting up the EPDMA, bovine hyperimmune serum raised
against the NADL strain of BVDV (the kind gift of Steven Bolin), was used. This
worked well for assaying cBVDV strains but the assay of New York-1 proved
more difficult. The level of virus-specific fluorescence observed was often barely
above background, increasing the chance of scoring false positives or negatives.
Fernelius (90), showed that fluorescent antiserum raised against ncBVDV is better
for detecting both biotypes than that raised against cBVDV. I decided that
antiserum raised against ncBVDV strain New York-1 should be used in the assay.
67
• The stock animals of the Veterinary Research Laboratory were screened to
identify cattle seronegative for BVDV by a plaque reduction assay. These animals
were then screened for persistent BVDV infection, by co-cultivation of PBL with
MDBK cells. The MDBK cells were subsequently assayed for the presence of
BVDV by indirect immunofluorescence. Three seronegative and BVDV-free
animals were identified. One was selected at random and immunised with tcf
containing live New York-1 virus.
The specificity of the antiserum for BVDV was titrated by comparing the
immunofluorescence observed on BVDV-infected and uninfected MDBK cells.
With uninfected cells, a 1:12 dilution of the antiserum resulted in the elimination
of nonspecific fluorescence, while BVDV-infected cells were readily detected. The
cells infected with BVDV had a distinctive fluorescence that was confined to their
cytoplasm (Figure 7); uninfected cells either did not fluoresce or had a low level
of fluorescence over the entire cell.
End Point Dilution Microtitration Assay
BVDV-specific fluorescence could be detected as early as 12 h pi in MDBK
cells (data not shown). With care, plates could be scored after 24 h but the
accuracy of the microtitration assay was enhanced by incubating the cells either
48 h, for titration of cBVDV strains, or 72 h, for titration of ncBVDV strains.
This allowed the virus to propagate, amplifying the viral antigens in infected wells,
resulting in more rapid and accurate scoring of the plates. The wells infected with
higher virus dilutions had discrete foci of fluorescence that were easy to score.
With lower virus dilutions the fluorescence was less distinct, due, presumably, to
the adsorption of the primary BVDV-specific antiserum over the entire monolayer. This meant that the secondary antiserum was also widely distributed,
68
Figure 7. Focus of infection of the ILLC strain of BVDV on MDBK cells. The
cells were fixed 72 h pi. Virus-infected cells were detected by indirect
immunofluorescence. X150.
resulting in an apparent reduction in fluorescence, causing the wells to appear
uninfected. For this reason, the plates infected with higher virus dilutions were
scored first.
The virus titre was calculated from the Poisson distribution using the
following equation;
P(O) = e m
where P(0) is the number of uninfected wells and m is the multiplicity of infection
per well (IU per 10 /d). For an example of how the virus titre is calculated,
consider-a Terasaki plate where 24 of 36 wells were uninfected at a dilution of
10‘6. This gives a value of P(O) of 0.67 (24/36) so m equals 0.41 (-In P(O)). To
69
find the titre per millilitre of the original virus stock, m is divided by the product
of the volume of the well (0.01 ml) and the dilution (IO"6). The result is a titre of
4. IxlO7 lU/ml.
. Large statistical errors occur when litres are calculated from plates where
either very few or most of the wells are infected (243). The accuracy of the assay
is also dependent on the number of wells used per dilution. In general, half the
wells of a Terasaki plate, 30 or 36 wells depending on whether it was a 60 dr 72
well plate, were used per dilution.
Comparison With Plaque Assay
To assess the accuracy of the microtitration assay as compared with a plaque
assay, ten strains of BVDV (6 cBVDV and 4 ncBVDV) were titrated using the
two methods. Four of the strains compared were the standard laboratory strains
studied throughout this project, the other six were paired strains isolated from
animals with naturally acquired MD (Steven Bolin, personal communication).
Plaque assays were carried out in duplicate plates. After 4 days, one plate was
stained with neutral red and the other with crystal violet. The titre was taken as
the average of the duplicate plates, except for Oregon C24V where only the plate
stained with neutral red was considered.
The litres determined by the individual assays were in very close agreement
(Table 8). Although the data presented were from a single experiment, similar
results were consistently produced. The detection of plaques of the ncBVDV
SSDN strain, indicated that this putatively noncytopathic strain was contaminated
with cBVDV. This was not surprising as this strain, and the cBVDV SSDC strain,
were recovered from an animal suffering from chronic BVD (Steven Bolin,
70
personal communication); the two BVDV biotypes present had obviously not been
totally separated from each other in the ncBVDV stock.
Table 8.
Comparison of BVDV titres determined by plaque assay and EPDMA.
Strain
Virus titre
Plaque assay
EPDMA
(PFU/ml)
(lU/ml)
Singer
NADL
LQxlO7
7. IxlO6
LlxlO7
6.9xl06
Oregon C24V
5.3x107
5.4xl07
a
2.5xl07
TGAC
5.6x10s
4.9xl05
TGAN
a
3.6xl06
ILLC
2.2xl06
3.6xl06
ILLN
a
4.9x106
SSDC
7.9xl06
9.4xl06
SSDN
6.0xl05
l.OxlO6
New York-1
a. Plaques not detected; noncytopathic.
Biological Cloning of BVDV Strains
For consistency, I decided to biologically clone the four strains by limiting
dilution. The viruses were subjected to six rounds of selection, two wells of the
Terasaki plate being selected at each round for subcloning.
At one point, subcloned NADL failed to yield any virus. MDBK cells were
infected with the remaining seven infected tcf s from the previous round of NADL
cloning. Two of these yielded virus, which was further subcloned.
71
, Of the final five or six clones of each strain, one was selected at random for
further study. These were Singer E6, NADL B7, Oregon C24V D9 and New
York-1 CS.
Virus Growth Kinetics
The first step, in developing a protocol for the purification of BVDV, was to
ascertain the time postinfection that maximum amounts of virus were present. The
growth curves of the four strains, in terms of both released and cell-associated
virus, are shown in Figure 8. The high levels of free virus initially observed were
probably residual virus from the inoculum. The level of free virus was always
greater than that of cell-associated virus. The litres of Oregon. C24V were
somewhat lower, probably because the virus samples were frozen and thawed
three times, whereas the other virus samples were only frozen and thawed twice.
Each strain had a latent phase for cell-associated virus of approximately 9 h. In
the case of strain New York-Ii the latent phase for released virus and for cellassociated virus appeared to be the same. However, the three cBVDV strains all
had a longer latent phase for released virus of 12-15 h. The rate of cBVDV
production appeared to be higher than that of ncBVDV during the logarithmic
phase. The peak litre of Singer and NADL occurred at approximately 30 h, that
of Oregon C24V at 36 h and that of New York-1 at 42 h. Virus was collected at
these times for subsequent virus purifications.
Purification of BVDV
Concentration of BVDV from large volumes of tcf was readily achieved by
the use of a Minitan Ultrafiltration System, with 100,000 molecular weight limit
filters. The retentate from the Minitan could be further concentrated using a
72
S in g er
O regon C24V
0
12 2 4 36 48 60 72 8 4 0
NADL
New Y o r k - 1
12 2 4 36 4 8 60 72 8 4
Time p o s ti n f e c ti o n (h)
Figure 8. One step multiplication curves of four BVDV strains. Titres are
PFU/ml except New York-1 which is lU/ml. ( • ) free virus, (O) cellassociated virus.
73
CX-30 filter unit (Millipore). This resulted in the rapid concentration of virus,
with negligible loss of viability. Table 9 shows the recovery of BVDV strain
Oregon C24V during concentration by ultrafiltration.
Table 9.
Recovery of BVDV during concentration.
Sample
Volume
(ml)
Virus titre
(RJ/ml)
Total virus in
sample (IU)
% virus
recovery
Medium
1,100
2.3x10?
2.5xl010
100
Minitan fil­
trate
1,120
I.'2xl04
IJxlO 7
0.05
Minitan retentate
41
6.3xl08
2.6xl010
104
CX-30 retentate
9
3.4xl09
3.IxlO10
124
The purification of the virus was complicated by its heterogeneous size and
density. Several approaches were assessed: sedimentation on Percoll, potassium
tartrate or glycerol-tartrate density gradients; and Sephacryl S-1000 column
chromatography. Percoll gradients and Sephacryl S-1000 column chromatography
failed to resolve the virus as a discrete peak (data not .shown). Figure 9 shows a
comparison of BVDV sedimentation on a 10-40% wt/wt potassium tartrate
gradient and a 30-0% wt/wt glycerol, 0-50% wt/wt potassium tartrate gradient.
The glycerol-tartrate gradient resolved the virus as a single broad peak. The
potassium tartrate gradient resolved the virus as an initial peak in the same
density range as the peak on the glycerol-tartrate gradient, but most of the virus
was recovered from the bottom of the gradient. Potassium tartrate isopycnic
density gradients generally resolved the virus as two peaks; a broad peak at a
density of 1.13-1.15 g/ml and a sharp peak at a density of 1.22 g/ml (data not
shown). Glycerol-tartrate gradients resolved the virus as one broad peak at a
74
D ensity (g /m l)
Figure 9. Comparison of sedimentation of NADL strain of BVDV on potassium
tartrate (O) and glycerol-tartrate (#)density gradients.
density of 1.13-1.17 g/ml. A gradient of 20-0% wt/wt glycerol, 0-40% wt/wt
potassium tartrate gave the best resolution of virus (data not shown). These
conditions were used in all subsequent virus purifications. The virus appeared as
a diffuse band at a density of 1.11-1.19 g/ml. This band contained 90 to 95% of
the virus loaded on the gradient (data not shown). For recovery of virus, gradient
fractions in this density range were pooled, diluted and centrifuged. The virus
pellets were often quite loose, so the supernatant had to be carefully decanted.
75
Sizing of the BVDV Genomic RNA
A time course study of BVDV-specific RNA synthesis in Singer-infected
MDBK cells, indicated that BVDV-specific cytoplasmic RNA concentration
peaked at approximately 18 h pi (data not shown). Cytoplasmic RNA was
labelled with 32P-Orthophosphate in the presence of AMD. Surprisingly, in
addition to BVDV-specific RNA, two lower molecular weight bands were seen.
These were not subgenomic BVDV mRNAs, as they were also present in the
mock-infected control RNA samples. The two bands disappeared after RNase
treatment, as did the Singer mRNA band (data not shown). They were not
present in intracellular RNA from BT cells, indicating that they were cell-specific.
Recently, the same bands were described in MDBK cells from the same source
(259). As I felt that these bands might interfere with the identification of BVDVspecific RNAs, subsequent studies were carried out in BT cells.
For comparing intracellular BVDV-specific RNAs, BT cells were pulsed for
6 h with 32P-orthophosphate, in the presence and absence of AMD, prior to RNA
extraction. Cytoplasmic RNA was extracted at the assumed peak of RNA
concentration and peak of virus production for each strain: Singer and NADL
18 h and 30 h pi; Oregon C24V 22 h and 36 h pi; and New York-1 24 h and 42 h
pi, respectively. The RNA was electrophoresed on an agarose-formamide gel.
The autoradiograph of the gel showed that a high molecular weight RNA species
was present in cells infected with Singer, NADL and Oregon C24V but not in the
mock-infected control or New York-l-infected cell RNA samples (data not
shown). This RNA was more prominent at the time of peak RNA synthesis than
at the time of peak virus production. It was assumed that this was cBVDV
genomic RNA. There was no evidence of any BVDV subgenomic mRNA species.
76
To find out how large the cBVDV genomic RNA band was, the RNA
samples were glyoxalated and electrophoresed on a 0.8% wt/vol agarose gel with
glyoxalated lambda H indlll fragments as molecular weight markers. The molecu­
lar weight markers were visualised by acridine orange staining. Comparison of the
migration of the markers and the genomic RNA band, using the Gel program of
Fristensky, indicated, that the BVDV genome was 16.3 kb in size. There was no
detectable difference in migration of the genomic RNA band of the three cBVDV
strains (data not shown).
The inability to detect BVDV genomic RNA in cells infected with New
York-1 was puzzling. A series of experiments were performed to address this
question. Genome size RNA was only found in New York-1 virions. It was not
detected in the cytoplasm or nucleus of New York-1 infected MDBK or BT cells,
at the level of sensitivity of in vivo 32P-Iabelling. However, genomic RNA could be
detected in cytoplasmic RN A, by probing northern transfers of infected cell RNA
with BVDV-specific probes (see below). Comparison with X H indlll fragments
indicated that the New York-1 virus genome was 14.8 kb in size, somewhat
smaller than that of the cBVDV strains (data not shown).
To see if the genomic RNA of the cBVDV strains and New York-1 were
actually different sizes, the cytoplasmic and .virion RNAs of the four strains were
again compared. Bovine turbinate cells were labelled for 12 h prior to RNA
extraction. Cytoplasmic RNA was extracted from Singer and NADL-infected cells
at 18 h pi, from Oregon C24V-infected cells at 21 h pi and from New York-Iinfected cells at 25 h pi. Virions were pelleted directly from tcf and RNA was
extracted from the pellets. The RNA was electrophoresed on a Wo wt/vol
agarose-formamide gel. The autoradiograph of the gel showed that the different
cytoplasmic RNA preparations were not equivalent in terms of the quantity of
77
label present in ribosomal RNA (rRNA), implying that the total amount of RNA
was different. As far as possible, the different cytoplasmic RNA preparations
were balanced on the basis of 18S rRNA labelling and the virion RNAs on the
basis of 32P content. The RNA samples were glyoxalated and electrophoresed on
a 0.7% wt/vol agarose gel, with end labelled, glyoxalated A HindlW and A Sail
fragments as molecular weight markers. Lambda Sail fragments were included, as
one fragment is 15,258 bases, approximately the same size as the BVDV genomic
RNA. Although some minor differences were noted between the migration of
genomic RNA bands of the four strains, these were not deemed to be significant
(Figure 10). The genomic RNA was estimated to be 13.4 in size. As the
molecular weight standards were DNA fragments, the estimate had to be correct­
ed for the difference in mass of nucleotides in RNA and DNA. This resulted in
an adjusted size estimate of 12.8 kb. Major differences were seen in the ratio of
label incorporation into the cytoplasmic BVDV RNA of the different strains, as
compared with rRNA. In decreasing order of intracellular BVDV RNA content
were Singer, NADL, Oregon C24V and New York-1. As before, no BVDV RNA
was detectable in the cytoplasmic RNA sample from New York-l-infected cells.
As a final check of the size estimate for the BVDV genome, VSV mRNAs
were used as molecular weight standards. Cytoplasmic RNA, labelled with 32P in
the presence of AMD for 3 h, was extracted from VSV-infected MDBK cells 5 h
pi. Cytoplasmic RNA was extracted from Singer-infected BT cells 18 h pi, after a
4 h 32P pulse in the presence of AMD- Singer virion RNA was also prepared.
The RNAs were glyoxalated and electrophoresed on a 0.7% .wt/volume agarose
gel. The resultant autoradiograph yielded a size estimate of 12.4 kb for the
BVDV genome (Figure 11).
78
>
T
S
Figure 10. Sizing of the genome RNA of BVDV: comparison of viral intracellular
(C) and virion (V) RNA with denatured X HindlW (H) and 5fl/I (S)
restriction fragments. MI, mock-infected cell RNA.
12.4 kb
11162
.... #
W
1672
1333
830
Figure 11. Sizing of BVDV genomic RNA: comparison of BVDV strain Singer
mRNA with VSV mRNA. RNA was labelled in the presence of AMD.
MI, mock-infected cell RNA. RNA size is expressed in terms of
nucleotides.
80
The final size estimate of the BVDV genomic RNA was the average of the
estimates from the DNA and RNA markers: 12.6 kb.
Northern Blot Comparisons
The difficulty encountered in comparing the intracellular RNA species of the
four strains, demanded that a more sensitive approach be used. Northern
transfers of RNA from glyoxal gels to nitrocellulose were performed. The RNA
samples included cytoplasmic and total cell RNA, from BVDV infected and
uninfected BT and MDBK cells, and virion RNA. All 10 of the BVDV strains
available were represented.
Initially, the filters were probed with random primed cDNAs, reverse
transcribed from Oregon C24V and Singer vRNAs. These gave rather unsatisfac­
tory results.
The next probe used was the BVDV cDNA from the plasmid pBV4-p80.
Computer-assisted analysis of the nucleotide sequence of the BVDV cDNA insert,
showed that it did not contain restriction sites for the restriction endonucleases
Kpnl and Xbal. Restriction sites for these two enzymes flank the restriction site of
Sm al in the multiple cloning site of pGEM-4, where the cDNA had been inserted.
Digestion of pBV4-p80 with Kpnl and Xbal yielded a fragment containing the
whole BVDV sequence plus only 11 nucleotides from the vector. However, the
remaining vector was also a single fragment, of a size that could cause problems in
gel purification of the BVDV cDNA fragment. I decided that the vector fragment ’
should be digested further. The restriction endonuclease Bgll was chosen, as this
would cut the pGEM-4 fragment approximately in half and the restriction site was
not present in the BVDV cDNA. Triple digestions of pBV4-p80 were followed by
purification of the Kpnl-Xbal fragment by low melting agarose gel electrophoresis.
81
The fragment was nick translated and used to probe northern transfers. The
genomic RNA of all the strains was detected on one or more filters (data not
shown). The results obtained supported the conclusions from the in vivo 32P-Iabelling studies: no significant difference in the size of the genomic RNAs of the
strains and no subgenomic mRNAs were detected.
The final probe used was a 32P end labelled oligo (dT)12.18, to see if a
3’ polyadenosine sequence was present on any of the genomic RNAs. With low
stringency hybridisations, at 19°C to detect sequences longer than 12 bases, there
appeared to be specific hybridisation to Oregon C24V virion RNA and possibly to
TGAC virion RNA (data not shown). However, this was accompanied with high
nonspecific background. Increasing the stringency of hybridisation, so that only
15-mers or longer sequences would be detected, resulted in no specific hybridisa­
tion to BVDV RN A, though nonspecific binding was markedly reduced. At both
high and low stringency, there was considerable hybridisation to cellular mRNAs.
This shows that the genomic RNA of all the strains was not 3’ polyadenylated but
that Oregon C24V and TGAC may have short stretches of adenosine residues.
Time Course of BVDV RNA Synthesis
The differences in cytoplasmic BVDV RNA levels observed between strains,
that did not appear to be associated with lower virus yields, indicated that there
might be differences between the strains in terms of the control of RNA synthesis
or RNA packaging. To address this question, six BVDV strains were compared
on the basis of the time course of plus and minus sense RNA synthesis, in infected
MDBK cells.
For quantifying the RN A, in vitro transcribed BVDV-specific plus and minus
sense RNA was prepared, using pBV4-p80 as a template. For SP6 RNA
82
polymerase transcription, to produce plus sense BVDV-specific RNA, the plasmid
was digested with restriction endonuclease Xbal. Gel electrophoresis of the RNA
transcripts showed that they were homogeneous; a single RNA species, that
migrated to the same position as denatured pBV4-p80 Kpnl-Xbal fragment DNA
(data not shown). Transcription with T7 RNA polymerase proved to be more of a
problem. Using pBV4-p80 digested with Kpnl as the template resulted in a
heterogeneous RNA product: a smear of RNA, the same size as or larger than the
SP6 transcript (data not shown). This was due to the 3’ protruding ends produced
by digestion with Kpnl (227). To overcome this problem, the 3’-5’ exonuclease
activity of the Klenow DNA polymerase was used to blunt end the template prior
to transcription. This greatly reduced the heterogeneity of the RNA product,
resulting in a major band the same size as the SP6 transcript (data not shown).
The smear was still evident after gel electrophoresis though.
For the preparation of total infected cell RNA,
two sets of 10 cm diameter
dishes were seeded with, MDBK cells 10 h apart. They were also infected 10 h
apart, at an moi of 4. Virus was allowed to adsorb for I h. The inoculum was
rinsed off, the monolayer washed twice with DME-O and 5 ml of DME-2 placed in
each dish. One hour after the second set of dishes had been infected, and every
2 h thereafter, total cell RNA was extracted from one dish of each set. This gave
a range of samples from I h to 24 h pi.
An initial attempt at quantifying the BVDV-specific RNA in total cell RNA
with dot blots was somewhat flawed. A basic problem was that the samples were
on two filters. Even though both filters were calibrated in the same way, they did
not seem to match. This may have been due to differences in hybridisation
conditions. Low stringency hybridisation conditions were used, which resulted in
high levels of hybridisation to mock-infected cell RNA. Formaldehyde was used
83
to denature the RNA prior to blotting, which appeared to inhibit the degree of
hybridisation. To quantify the hybridisations, the filters were cut up and the
individual dots were scintillation counted. This was inherently inaccurate, as
distortion of the filter during the hybridisation process made it difficult to accu­
rately locate the dots. The calibration curves appeared to lose linearity above
I ng of target RNA.
I was also concerned by the heterogeneity of the RNA used for the minus
sense RNA calibrations and probes, derived from Kpnl digested pBV4-p80
templates. I decided that it would be better to linearise pBV4-p80 with a different
restriction endonuclease, that did not produce 3’ overhanging ends. No suitable
restriction site was available in the multiple cloning site ,of pGEM-4. Analysis of
the BVDV cDNA sequence showed that a SgZII site was located at the very end
of the cDNA sequence. Attempted digestion of pBV4-p80 with SgZII showed that
this restriction site was not present. A Hpal restriction site, 149 nucleotides into
the BVDV cDNA sequence, was present, so this enzyme was used for linearising
pBV4-p80. This template was used for providing the minus sense RNA for
calibrations and probes for plus sense RNA.
The dot blot and hybrid detection protocols were modified in the light of the
problems encountered in the initial study. The concentration of RNA in the total
cell RNA preparations was remeasured. The RNA samples were glyoxalated to
denature them. The range of RNA concentrations were reduced for both the
cellular RNA and the in vitro transcribed RNA. All samples were applied to a
single pair of duplicate filters. Hybridisation conditions were more stringent and
carefully controlled and plus and minus sense RNA hybridisations were performed
at the same time to match conditions for each. The filters were autoradiographed
on the same sheet of preflashed X-ray film and the hybrids were quantitated by
84
3#
<
z
DC
O
3)
SP6
♦
Ml
I
Time pi Ihl
3
5
7
9
11
#
#
5
12
#
1 0 0 0 pg
0.5
0.0 5
0.05
IOOOpg
t
0
05
#
5
#
13 14
Ml
#
0.5
•
0 .05
1
*
0
# # # #
#
16 18
2 0 22 2 4
3
7
5
9
0.316p g
11 12
O 0 05
0.5
5
0.316 pg
• # » # # # #
13
14
16 18 2 0
2 2 24
Figure 12. Quantitation of BVDV intracellular RNA. The illustration shows an
autoradiograph of dot blots probed for BVDV NADL intracellular
RNA. SP6 is the plus sense RNA calibration, T7 is the minus sense
RNA calibration.
85
scanning the autoradiographs with a Hoefer GS-300 densitometer. These modifi­
cations resulted in the production of much cleaner data. A typical autoradiograph
is shown in Figure 12, with the calibration curves for plus and minus sense RNA
derived from it shown in Figure 13. The system was able to detect as little as I pg
of BVDV RNA.
15000
12500
10000
7500
5000
2500
B VD V R N A (p g )
Figure 13. Calibration curves for BVDV NADL plus ( • ) and minus (O) sense
RNA, derived from Figure 12 as described in the text. Above 100 pg
of RNA the curves were no longer linear.
As a further check, 5 jug of each RNA preparation was electrophoresed on a
0.8% wt/vol agarose gel. The ribosomal RNAs were visualised by UV shadowing
to check the integrity of the RNA. In no case was any degradation observed and
the concentrations were consistent between samples. The RNA was then transfered to nitrocellulose by northern transfer.
86
A cursory analysis of the RNA synthesis curves, generated from the dot blot
autoradiographs, showed that there was a marked difference in the levels of
intracellular BVDV RN A, of both senses, between strains (Figures 14,16-20).
BVDV NADL RNA Synthesis
As NADL was the BVDV strain the cDNA was cloned from, I shall describe
the results with this strain first. Both plus and minus sense RNA were first
detectable, at very low levels at 7.2 h pi and 9 h pi, respectively, but were not
really evident until 12.25 h pi (Figure 14). There appeared to be an initial peak
of RNA synthesis at 14 h pi but synthesis did not really increase markedly until
after .16 h pi. Plus sense RNA synthesis increased rapidly, reaching a peak of 7 ng
per jug of total cell RNA at 22 h pi. Minus sense RNA increased rapidly between
16 h and 18 h pi, remained fairly constant until 22 h pi, at a concentration of
1.5-1.75 ng per jitg of total cell RN A, after which time it decreased.
Probing of the northern blot with plus sense RNA supported the conclusions
from the dot blots. Hybridisation to minus sense RNA was visible at 9 h pi
(Figure 15). This hybridisation was not to genome size RNA but to an apparently
higher molecular weight smear of RNA, which, at some time points, appeared to
be a discrete band. Hybridisation to genome size minus sense RNA was detected
later in infection but not to the same extent as to the high molecular weight
species. Probing with minus sense RNA revealed a similar picture, except a
greater proportion of hybridisation was to genome size RNA (Figure 15). Plus
sense RNA was detectable at 7.2 h pi.
87
log virus litr e (lU /m l)
Time post infection (h)
Figure 14. Time course of RNA synthesis of BVDV NADL The RNA concentra­
tion and virus titres were measured as described in M aterials an d
M eth ods.
88
Time pi |h)
Ml
1
3
5
7
+
Figure 15. Northern blots of total cell RNA from MDBK cells infected with
BVDV NADL The filter was first probed for minus sense RNA (-)
with an SP6 polymerase transcript of pBV4-p80. After autoradio­
graphy, the probe was stripped off with boiling water and the filter
reprobed for plus sense RNA (+ ) with a T7 polymerase transcript of
pBV4-p80. The 12.6 kb bands represent BVDV genome size RN A.
RI and RF represent putative replicative intermediate and replicative
form RNA respectively. MI is mock-infected cell RNA.
89
BVDV Singer RNA Synthesis
Plus sense RNA was first detected at 7 h pi and the concentration increased
rapidly until it exceeded the calibration range, at approximately 16 h pi (Figure 16)
Although not shown, the RNA concentration peaked at between 18 h and 20 h pi,
at a concentration of approximately HO ng per ng of total cell RNA i.e. 11% of
total cellular RN A. Minus sense RNA followed a similar time course to that of
NADL minus sense R N A It was first detected at 12 h pi and increased to a
plateau, of approximately 10 ng per /xg of total cell RN A, at 18-24 h pi.
The northern blot, probed with plus sense RNA, showed a similar pattern
of hybridisation to that seen with NADL (data not shown). Most probe hybridised
to the high molecular weight RNA species. This hybridisation was first evident at
5 h pi, indicating a shorter lag phase than was evident from the graph. Very little
minus sense RNA was present in the genome size band at any time point com­
pared to the high molecular weight smear. Probing with minus sense RNA,
showed that plus sense BVDV RNA was detectable at 1.7 h pi. This was prob­
ably the infecting virus genome. Plus sense RNA began to increase at 7 h pi. As
with NADL, the probe hybridised not only to a discrete band, the Singer genome,
but also to higher molecular weight RNAs and to a smear of RNA beneath the
genome RNA band.
BVDV Oregon C24V RNA Synthesis
. Plus sense RNA was first detected at 11 h pi (Figure 17). Like NADL, there
was an initial small peak. The amount of plus sense RNA rapidly increased after
13 h pi, peaking at 22 h pi, at a concentration of approximately 2.25 ng per jug of
total cell RNA. Minus sense RNA followed a very similar time course. It was
first detectable at 11 h pi. It increased steadily from there, peaking at 22 h pi, at
90
•
+RNA
D
-RNA
I Virus li t r e
log viru s litr e (lU /m l)
ng BVDV RNA per /xg cell RNA
I
O
4
8
12
16
20
24
Time post infection (h)
Figure 16. Time course of RNA synthesis of BVDV Singer. The RNA concentra­
tion and virus litres were measured as described in M aterials an d
M eth ods.
91
•
+RNA
O -RNA
I Virus l i t r e
log virus litr e (lU /m l)
ng BVDV RNA per yu-g ce ll RNA
I
O
4
8
12
16
20
24
Time post infection (h)
Figure 17. Time course of RNA synthesis of BVDV Oregon C24V. The RNA
concentrations and virus litres were measured as described in M aterials
a n d M eth ods.
92
a concentration of approximately 0.5 ng per ng of total cell RN A.
The northern blot exhibited a similar distribution of target RNA to that of
NAJDL and Singer. Minus sense RNA was detected mainly in a high molecular
weight smear. It was first detectable at 7.25 h pi. Only at 22 h pi was any
detected in a discrete genome size band (data not shown). As with Singer, plus
sense BVDV RNA was detectable in a genome size band at 1.4 h pi but only
started to increase after 7.25 h pi.
BVDV New York-1 RNA Synthesis
The time course of New York-1 RNA synthesis was rather different to that
of the cBVDV strains (Figure 18). Plus sense RNA was detectable at 1.6 h pi. It
increased rapidly between 9 h and 14 h pi, at which time it became relatively
constant, at a concentration of 1.7 ng per
of total cell RN A. There was
another rapid increase between 20 h and 22 h pi, to a peak of 2.4 ng per /ig of
total cell RNA. Minus sense RNA synthesis was also somewhat different. It was
first detectable at 11 h pi. As with the plus sense RNA, there was a biphasic time
course with peaks at 14 h and 20 h pi, of 0.09 ng and 0.15 ng per fig of total cell
RNA, respectively.
Minus sense RNA was detected on the northern blot at 5 h pi (data not
shown). As with the other strains, this was in a high molecular weight RNA
smear. However, compared with the other strains, more was present in a discrete
genomic size band later in infection. Surprisingly, plus sense RNA was detected at
1.6 h pi but was not detectable again until 9 h pi. This is also reflected in the plus
sense RNA plot from the dot blots. It is unclear what caused this. Most of the
plus sense RNA on the northern blot was detected in a discrete genome size
band.
93
•
+RNA
D
-RNA
I Virus lit r e
log virus litr e (lU /m l)
ng BVDV RNA per yu-g cell RNA
I
O
4
8
12
16
20
24
Time post infection (h)
Figure 18. Time course of RNA synthesis of BVDV New York-1. The RNA
concentrations and virus litres were measured as described under
M aterials a n d M eth ods.
94
BVDV TGAC RNA Synthesis
Plus sense RNA was not detectable until 12.75 h pi (Figure 19). After this it
increased rapidly to a biphasic peak between 18 h and 24 h pi, of 1.1-1.2 ng per
Hg total cell RN A. Minus sense RNA was detectable at 9.8 h pi. It increased
steadily after this until 22 h pi and increased sharply between then and 24 h pi.
The northern blot supported the findings of the dot blots (data not shown).
Minus sense RNA was detected at 9.8 h pi and was detected mainly in a high
molecular weight smear. Plus sense RNA was detectable at 12.2 h pi and was
mainly present in a discrete genome size band.
BVDV TGAN RNA Synthesis
TGAN had a similar biphasic time course to New York-1 (Figure 20). Plus
sense RNA was first detected at 12 h pi, after which it increased rapidly to an
initial peak of 1.15 ng per [Ag total cell RNA at 14 h pi. There was a second peak
of approximately 1.4 ng per
[Ag
of total cell RNA at 22 h pi. Though not evident
from the graph, minus sense RNA was detected at 12 h pi, increased to an initial
peak at 12.7 h pi, with a second peak at 18 h pi and was rapidly increasing at the
termination of the experiment. The two peaks were at a concentration of
0.012 ng per
[Ag
of total cell RN A.
The northern blot showed that minus sense RNA was present at 8 h pi, in a
high molecular weight RNA smear (data not shown). Probing with minus sense
RNA showed that plus sense RNA was detectable at 12 h pi, in a discrete genome
size band. The concentration of the plus sense RNA was very low.
•
+RNA
a
-RNA
I
I Virus lit r e
log virus litr e (lU /m l)
0 .7 5
ng BVDV RNA per
Hg cell RNA
95
0
4
8
12
16
20
24
Time post infection (h)
Figure 19. Time course of RNA synthesis of BVDV TGAC. The RNA concentra­
tions and virus litres were measured as described in M aterials an d
M eth ods.
96
•
+RNA
D
-RNA
I Virus lit r e
log virus litr e (lU /m l)
ng BVDV RNA per /Ltg ce ll RNA
I
O
4
8
12
16
20
24
Time post infection (h)
Figure 20. Time course of RNA synthesis of BVDV TGAN. The RNA concentra­
tions and virus titres were measured as described in M aterials an d
M eth ods.
97
Comparison of Plus Sense RNA Levels
With Levels of Infectious Virus
The level of virus production was measured at approximately 6 h intervals
during the course of the experiment. The virus titres compared to plus sense
RNA synthesis can be seen in Figures 14,16-20. No relationship was observed
between the production of plus sense RNA and production of infectious virus. In
fact, the strains that produced the lowest cellular levels of plus sense RNA
generally had the highest virus titres.
Comparison of the Ratio of Plus to
Minus Sense RNA
Though the ratio of minus sense to plus sense RNA of each strain was
rather variable, general trends were observed (data not shown). The ratio
observed for cBVDV strains was generally less varied throughout the course of
infection. The ratios of NADL and Oregon C24V remained quite constant, while
those of Singer and TGAC started off quite high but fell steadily throughout the
course of infection. With the ncBVDV strains, there was a much greater degree
of variation. Two major peaks were observed in each case, which occurred
immediately after the peaks in minus sense RNA levels. The average ratios of the
two ncBVDV strains were also higher than those of the cBVDV strains.
Although not as consistent as the the data from the second set of quantita­
tions, the general trends evident in the quantitations from the original study
supported the conclusions concerning the RNA ratios.
98
DISCUSSION
Virus Strains
The four BVDV strains I studied in the bulk of this work-Singer, NADL,
Oregon C24V and New York-1—could be considered the standard BVDV strains.
They represent all the important traits of BVDV cytopathology. Most of the
important studies of BVDV at the molecular level have involved one or more of
these strains. The strains TGAC and TGAN were included in later studies
because of the emerging data on the pathogenesis of MD. The two strains were
originally isolated from a cow with MD and have since been used to reproduce
the disease experimentally. Due to their known ability to cause severe disease,
they act as a useful yardstick for the relevance of the findings with the four
original strains, which had been maintained in cell culture for many years. The six
virus strains were all obtained from the same source. This is an important point
in light of the differences found in ostensibly identical strains from different
laboratories (42).
I have used the term strain to represent virus from established laboratory
stocks. I would term BVDV recovered from a field case an isolate. Some
workers in pestivirology refer to all BVD viruses as isolates, as the viruses cannot
be truly differentiated on a serological basis, so they cannot be truly called strains.
99
Development of the Experimental System
When I started this research project, three things were evident: there was no
standard system for growing the virus; there was no standard system for titrating
the virus; and there was no standard system for purifying the virus. To quote
Horzinek (134):
...the dilemma encountered by all workers in this field: the lack of suit­
able systems for in vitro virus growth and titration. Without exaggera­
tion it can be stated that this is the reason why non-arbo togaviruses
rank among the least known animal viruses, especially with regard to
their molecular structure and biology.
Most studies of BVDV are carried out in primary bovine cell lines. The
inherent variation between cell preparations and likely problems in maintaining a
system free of BVDV, rendered the use of primary cells impractical. An estab­
lished cell line was required. Routine isolations of BVDV, for diagnostic pur­
poses, are carried out with BT cells (William Quinn, personal communication). As
this is a secondary cell line, that undergoes crisis at approximately passage 30
(Arlan McClurkin, personal communication), an early passage of the cell line was
procured. These cells grew rather slowly and did not achieve very high cell
densities. The BLF cell was also a secondary cell line. It grew faster than BT
cells and to higher cell densities. The cell line had only recently been established,
so its long term stability in culture was unknown. The MDBK cell line was a
permanent cell line of known growth characteristics. It grew faster than the other
two cell lines and to a higher cell density. In this cell system the litres of the
BVDV strains Singer, Oregon C24V and New York-1 were consistently between
3x107 and IxlO8 lU/ml or PFU/ml. This is an unusually high titre for BVDV
(134,237). Strain NADL was consistently of lower titre, approximately
3x10* PFU/ml. Although this cell and virus combination had been used in the past
100
(179), it had probably fallen from favour as the MDBK cell line available from the
ATCC for many years was persistently infected with ncBVDV. Fortunately, a
stock of the cells had been discovered that was free of BVDV and this was the
cell line that I used, MDBK (NBL-1).
The evidence of a MDBK cell contaminant that I found during the 32P-Iabelling studies was puzzling. The two prominent RNA bands observed migrated at
the same rate as 16S and 23S RN A, the size of prokaryotic rRNA. This suggested
a contaminant but no evidence for the presence of mycoplasma or bacteria was
found. As the putative contaminant did not appear to affect BVDV replication
!
and BVDV-specific probes did not hybridise to the RNA its presence was ignored.
When first obtained, the cell lines were maintained in medium supplemented
with foetal bovine serum. The serum was treated with /3-propiolactone, which
destroyed any virus in the serum; however, it would not remove BVDV neutral­
ising antibody from the serum. An alternative to bovine serum was equine serum.
Horses are not infected by BVDV '(124), so there was no possibility that equine
serum would contain BVDV or neutralising antibody to it. The cell lines all grew
well in medium supplemented with equine serum, so it was used for all subsequent
cell maintenance. The use of serum-free medium was also studied but it impaired
the growth of the cells.
The development of the EPDMA was an important aspect of my initial
studies of BVDV. As I previously stated, several different assays were available
for the titration of ncBVDV but these were indirect, time consuming, relatively
inaccurate or not comparable with plaque assays (90,95,105,141,176,210,271).
Several of the assays are dependent on the indirect effects of ncBVDV on the
replication of challenge viruses to indicate dilution end points. Two, the interfer­
ence assay of Gillespie et al (105), and the reverse plaque assay of Itoh et al.
101
(141), rely on the ability of ncBVDV to inhibit cBVDV cpe. The interference
assay suffers from ill-defined end points, leading to statistical inaccuracies. The
END assay depends on the enhancement of Newcastle disease virus cytopathogenicity (210) and has been shown to be less sensitive than the other assays for
ncBVDV (141). The PINBA assay of Maisonnave and Rossi (176) relies on
challenge with VSV. The accuracy of this assay varies between cell lines, some
cells infected with ncBVDV being resistant to challenge with VSV (254).
Compared to the direct assays described for titrating ncBVDV, EPDMA is
generally more accurate statistically, due to the larger number of replicates
involved (90,95,140,149). Two plaque assays have been described for the direct
titration of ncBVDV. Straver (271) described the visualisation of plaques by
prolonged staining of monolayers with neutral red. I have seen this phenomenon
but it is not very reproducible and the staining period is extremely long. Rae et al.
(231) recently described a fluorescent focus forming assay for ncBVDV. This is a
plaque assay in which plaques are visualised by indirect immunofluorescence. It is
likely that this assay is as accurate as EPDMA.
In addition to the advantages of EPDMA for titrating, ncBVDV, there were
also certain advantages to using it over plaque assays for cBVDV. It took less
time to set up, required less materials and could be read in two or three days as
compared to four or five days for a plaque assay.
The ability to titrate both biotypes of BVDV with the same assay was a
distinct advantage. As plaque assays and EPDMA gave equivalent titres for
cBVDV strains, IU and PFU could be assumed to be equivalent. This agreed
with the findings of Robb and Martin (243). I felt that it was reasonable to
assume that titration of ncBVDV strains by EPDMA was equally accurate. The
protocol also proved extremely useful in the biological cloning of the different
102
BVDV strains. The only disadvantages of the assay were due to low specificity or
titre of the antiserum used. This made it difficult to identify ncBVDV infected
wells. The use of the antiserum raised against ncBVDV strain New York-1
alleviated these problems. The use of a BVDV-specific monoclonal antibody
would probably enhance the assay considerably.
The growth curves of BVDV have been observed to be cell line and virus
strain specific (134). The latent phase of 9 h was comparable to the 4-10 h that
has been generally observed. The growth curves after this are rather complicated.
There are indications that they do not represent a single growth cycle. There is a
distinct shoulder on most of the curves at 18-24 h pi. This may represent the end
of the logarithmic phase of growth of the inoculum. The curve then increases
again in all cases. This may represent a second cycle of virus growth, due to virus
released in the initial growth phase infecting cells not infected by the inoculum.
As an moi of I was used for the initial infection, 36.8% of the cells were not
infected, according to the Poisson distribution. If one assumes that the cellassociated virus was more indicative of actual virus growth and the free virus was
more a measure of virus release, then the logarithmic phase of Singer and NADL
lasted from 9-18 h pi and of Oregon C24V and New York-1 from 9-21 h pi. The
peak of virus release occurred after this, between 30 h and 42 h pi; again, compa­
rable to previous reports (134). The plateau that was reached at this point bore
indications that cycles of virus growth were still occurring. However, monolayers
infected with Singer and NADL were destroyed by 48 h pi. This implies that
there is little virus growth occurring during this phase. Overall, there appears to
be little difference between the strains. The highly cytopathic strains appear to
have a shorter logarithmic phase. As is generally found with BVDV (134), the
four strains had 1-1.5 Iog10 more released virus than cell-associated virus.
103
The concentration and purification protocol that was defined gave very high
yields of virus. Ultrafiltration has been used by several groups for concentration
of BVDV (61,171,237). The use of glycerol-tartrate gradients has been reported
independently for the purification of BVDV (229). The two methods have not
been used in concert before. Although some loss of virus occurred during
ultrafiltration, this was a negligible amount. Other groups have reported much
greater losses, from 50% (171) to 85% (61), compared with 0.05% in the present
study. Further losses also occurred from sedimentation on glycerol-tartrate
gradients but these were much less than those incurred from the other purification
methods studied. Why glycerol-tartrate gradients resolve BVDV as a single peak,
while potassium tartrate gradients resolve the virus in two peaks, is not clear. The
glycerol may help maintain the integrity of the viral envelope, the major viral
component responsible for its buoyant density. The major drawbacks to glyceroltartrate gradients were that the virus was resolved in a broad peak and had to be
separated from the potassium tartrate after sedimentation. This was achieved by
diluting fractions containing virus and collecting the virus by centrifugation.
However, this resulted in large losses in the yield of infectious virus (data not
shown). A better approach might have been to desalt the virus by gel filtration,
followed by concentration using a CX-30 filter unit.
Though the degree of purity of the virus was not studied in terms of the
ratio of infectious virus to total protein content, the recovery of infectious virus
was far better than achieved by other methods (61,134,171,229,237).
Overall, the system developed for the growth, titration and purification of
BVDV would appear to be equal or superior to those previously described.
104
BVDV RN A: Size and Synthesis
The size estimate for the genomic RNA of BVDV was comparable to the
actual length of the BVDV NADL cDNA sequence of Collett et al. (54): 12.6 kb
compared with 12,573 bases. In the light of the difference of 42 nucleotides
between the ORF of BVDV strains Osloss and NADL (55), the autoradiograph of
the size comparison of the four BVDV strains was reanalysed. Some difference in
size had previously been observed but this was dismissed as being due to distortion
of the gel or the difference in the width of the band, due to differences in radio­
activity. The reanalysis results are shown in Table 10. The comparison shows that
NADL has the longest genome and, as expected, New York-1 has the shortest.
Interestingly the estimate for the size of the genome of New York-1 is very similar
to the length of the molecularly cloned cDNA of HCV. The genomes of Singer
and Oregon C24V are approximately the same size. The mean size of the
genomes is the same as the original size estimate, showing the accuracy of the
original estimate. Due to the degree of error incurred in measuring the distance
of migration of bands on autoradiographs, the actual differences in genome size
are probably not as great as estimated.
The time course of BVDV RNA synthesis appears to follow the same time
course as virus growth, as has also been observed for HCV (199). This implies
that the time course of RNA synthesis will be cell line and virus strain dependent.
The former was not investigated but the latter certainly proved to be the case.
Great variation was found not .only in the time course but also the quantity of
RNA synthesis between the different strains. The early events in RNA synthesis
were best defined by the time course of BVDV Singer. A lag phase of 5 h
occurred before minus sense RNA was detected. Plus sense RNA production was
105
Table 10. Reanalysis of the size of the genomic RNA of four strains of BVDV
Strain
NADL
Singer
Oregon C24V
New York-1
Mean size
Genomic RNA (kb)
. 13.2
12.9
12.8
12.5
12.8
net size of genome (kb)a
13.0
12.6
12.5
12.2
12.6
a. net genome size was calculated in each case from the difference between the
two Singer strain estimates
detected 2 h later. Both plus and minus sense RNA synthesis increased exponen­
tially after this. A similar time course for protein synthesis has been described in
Singer-infected cells (75). Though not evident from the data in most cases,
probably due to the lower BVDV RNA concentrations, a similar time course
probably applied to the other strains. However, the NADL strain had a distinct
4 h lag compared to the other strains. The latent phase before RNA synthesis
was detected has also been observed in cells infected with flaviviruses (298).
The two biotypes seemed to differ in the time course of RNA synthesis
during the eclipse phase. In the case of cytopathic strains, plus and minus sense
RNA production increases to a peak at 20-22 h pi. The increased minus sense
RNA synthesis, observed between 22 h and 24 h pi in cells infected with the
Singer and TGAC strains, may indicate the start of a second round of virus
production, as was observed in the virus growth curves. The most remarkable
difference between the four cytopathic strains is the level of RNA synthesis. The
Singer strain produced 1.5 times more plus sense RNA than NADL, 40 times
more than Oregon C24V and 80 times more than TGAC. Surprisingly, this was
not reflected in the production of infectious virus, TGAC-infected cells producing
106
as much as Singer infected cells. However, the total number of virus particles
produced was not assessed.
The noncytopathic strains appeared to exert more control over RNA
synthesis. The initial exponential increase in RNA levels only lasted until 14 h pi.
Minus sense RNA synthesis increased again from 16-20 h pi with a concomitant
increase in plus sense RNA levels until 22 h pi. As with the cytopathic strains,
there was no relationship between plus sense RNA levels and the yield of infec­
tious virus.
Apart from the difference in the time course of RNA synthesis, ncBVDV
strains differed from cBVDV strains in the ratio of plus to minus sense RN A. In
cells infected with ncBVDV only 1-3% of total BVDV RNA was minus sense
RNA; whereas, in cBVDV infected cells minus sense RNA represented 5-20% of
total BVDV RNA. The ratio fluctuated markedly during ncBVDV infection but
remained relatively constant during cBVDV infection. What could cause this
major difference in ratios? Minus sense RNA has a single role in virus replica­
tion; the template for plus sense RNA transcription. Conversely, plus sense RNA
has three roles: mRNA, template for minus sense RNA transcription and genome
of progeny virus. These roles are not necessarily mutually exclusive. In flavivirus
replication, encapsidation is thought to be controlled by binding of the C protein,
equivalent to the BVDV p20 capsid protein, to the plus sense RNA (298). This
would also prevent its use as a template for minus sense RNA transcription. It is
likely that a similar process occurs in BVDV replication. The homology in
functional domains of flavivirus NS3 and BVDV p!25 may hold the key. In
flavivirus infected cells, NS3 is thought to be part of the replication complex with
NS5, the viral RNA dependent RNA polymerase (298). In cBVDV infected cells,
p!25 is cleaved. This cleavage apparently occurs between a protease and a RNA
107
helicase domain (106). The RNA helicase is thought to be involved in the
dissociation of daughter plus sense RNA strands from the minus sense RNA
template in the replication intermediates of RNA viruses (107). The disassociation of the two domains may render newly synthesised plus sense RNA less
accessible to p20, resulting in the use of the RNA as a template for minus sense
RNA transcription. This would have two results, an increase in minus sense RNA
levels, with a subsequent increase in plus sense RNA synthesis, and a reduction in
the amount of progeny virus. This was indicated by the lower ratio of plus to
minus sense RNA in cBVDV infected cells I detected. The increased plus sense
RNA synthesis would increase the amount of BVDV mRNA, reflected in the
higher level of BVDV antigen detected in infected cells by immunofluorescence.
As p 125 is also detected in cBVDV infected cells, albeit at much lower levels than,
the cleavage products, encapsidation of plus sense RNA would occur in replication
complexes containing the uncleaved protein. The increased levels of BVDV
proteins would offset the lower net encapsidation rate, by increasing the number
of replication complexes and the concentration of structural proteins required for
virion assembly. The amount of virus produced would depend on the ratio of
cleaved pl25 to native pl25 in the replication complexes. In ncBVDV infected
cells all the replication complexes would contain p!25. The degree of minus sense
RNA transcription would be controlled by the availability of p20. Initially, most
plus sense RNA would be mRNA or template for minus sense RNA, as the levels
of p20 would be low. As the level of BVDV mRNA increased, so would the level
of BVDV proteins, due to the translation of the mRNA. This would increase the
level of p20, leading to more encapsidation, with a reduction in minus sense RNA
synthesis. The p20 levels would then fall due to removal by encapsidation and a
108
fall in BVDV mRNA levels. A dynamic equilibrium would then be set up, as I
observed in the case of ncBVDV infected cells.
One intriguing implication of the low level of pl25 in cBVDV-infected cells,
compared to the level of p80, is that cBVDV is replication defective. As with
other replication defective viruses, it requires a helper virus for its replication, the
helper virus being ncBVDV. A low level of ncBVDV is required to allow encapsidation of the cBVDV genome. It has been hypothesised that MD is actually the
result of a mixed infection, in which cBVDV is present the whole time. Mucosal
disease results when the cBVDV is able to replicate to a higher level than
ncBVDV in certain tissues (12).
The low ratio of plus to minus sense RNA in cells infected with cBVDV may
be responsible for the cpe observed. The low ratio implies the presence of higher
levels of double stranded RNA (dsRNA) in cBVDV-infected cells than in
ncBVDV-infected cells. It is known that dsRNA is cytotoxic (144). It is also
known that dsRNA is a potent inducer of IFN-a and IFN-/? (144). These two
cytokines can also induce cytotoxic effects in cells (144). The reaction of cBVDVinfected cells to the direct and indirect cytotoxic effects of dsRNA could result in
the cpe observed. The low ratio of plus to minus sense RNA observed in Oregon
C24V-infected cells, without appreciable cpe in most of the cells, implied that the
virus-cell interactions resulting in cpe were not solely due to the presence of
dsRNA.
The northern blots probed for plus sense and minus sense RNA produced
some interesting results. The most obvious observation was that denaturation of
the BVDV RNA was not complete. The RNA of BVDV has a high degree of
very stable secondary structure ((55,134), personal observation). The distribution
of BVDV RNA on the autoradiographs bares a striking similarity to the
109
distribution of flavivirus 44S genomic RNA, RI and RF (48,49,51). The RI and
RF of flaviviruses contain both plus sense and minus sense RNA, as found on the
northern blots in this case.
The incomplete denaturation observed on the northern blots raises a
question of how quantitative the dot blot assay was. I feel that denaturation was
not a problem as the baking of the filters will have denatured the RNA and the
high temperature of hybridisation in the presence of formamide will also cause the
relaxation of double stranded regions. There is also the question of probe
specificity. Again, I don’t feel that this is a problem as the probe used was from
the most highly conserved region of the genome (55), showing 96-99% homology
between NADL and Osloss.
The proof of my hypothesis, that the cleavage of pl25 leads to a loss of
control of minus sense RNA synthesis due to a failure to target plus sense RNA
for encapsidation, can be approached in several ways. The involvement of p20 in
control of RNA synthesis could be easily assessed by measuring intracellular levels
of the protein during virus replication. An antigen capture assay, using monospecific antisera or a monoclonal antibody specific for p20, could be used. If p20
were involved in controlling minus sense RNA synthesis, then the intracellular
concentration of the protein should fluctuate out of phase with minus sense RNA
concentration i.e. p20 levels should be high when minus sense RNA levels are low
and vice versa. For assessing how cleavage of p!25 affects RNA synthesis directly,
some very complex tools will be required that are not presently available in
pestivirology. One approach would be the in vitro assembly of BVDV replication
complexes into which native and cleaved p!25 could be selectively incorporated.
Although flavivirus replication complexes have been studied in vitro (49,112), I feel
that it is unlikely that such studies can be performed on BVDV in the near future,
no
due to the dearth of information on the components of BVDV replication
complexes, assuming they exist at all. Another approach would be to produce an
infectious cDNA clone of a ncBVDV strain. Site directed mutagenesis could then
be performed on the p!25 gene and the effects on RNA synthesis assessed. The
p20 gene could also be mutagenised to reduce its binding efficiency to genomic
RNA to see if this would result in the same pattern of RNA synthesis as I
observed in cBVDV-infected cells. It will probably be extremely difficult to
produce an infectious cDNA clone of a ncBVDV strain, so such experiments are
not likely to be attempted in the near future.
I ll
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APPENDIX
Abbreviations
AMD
actinomycin D
Amp
ampicillin
ATCC
American Type Culture Collection
BDV
Border disease virus
BLF
bovine lung fibroblast (cell line)
BSA
bovine serum albumin
BT
bovine turbinate (cell line)
BVD
bovine virus diarrhoea
BVDV
bovine virus diarrhoea virus
cBVDV
cytopathic BVDV
cDNA
complementary DNA
cpe
cytopathic effect
CIP
calf intestinal alkaline phosphatase
CTP
cytidine 5’-triphosphate
dATP
2’-deoxyadenosine 5’-triphosphate
dCTP
2’-deoxycytidine 5’-triphosphate
DME
Dulbecco’s modified Eagle’s medium
DMSO
dimethyl sulphoxide
DNase
deoxyribonuclease
dNTP
deoxynucleotide triphosphate
dsRNA
double stranded RNA
138
DTT
dithiothreitol
EDTA
ethylenediaminetetraacetic acid
ELISA
enzyme linked immunosorbent assay
EPDMA
end point dilution microtitration assay
ER
endoplasmic reticulum
gP
glycoprotein
HCV
hog cholera/European swine fever virus
HEPES
N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid
IAA
isopentanol
ID50
50% infectious dose
IFN
interferon
IU
infectious unit (equivalent to PFU)
kb
kilobase
kDa
kilodalton
LB
Luria broth
LSB
low salt buffer
MD
mucosal disease
MDBK
Madin-Darby bovine kidney (cell line)
moi
multiplicity of infection
MOPS
3-[N-morpholino]propanesulphomc acid
Mr
relative molecular weight
mRNA
messenger RNA
NADC
National Animal Disease Center
ncBVDV
noncytopathic BVDV
NP-40
nonidet P-40
NTP
nucleotide triphosphate
139
ORF
open reading frame
PBL
peripheral blood leucocyte
PBS
Dulbecco’s phosphate buffered saline
PFU
plaque forming unit
Pi
postinfection
PIPES
piperazine-N,N’-bis[2-ethanesulphonic acid]
PK
proteinase K
PNK
polynucleotide kinase
PVP
polyvinylpyrrolidone
RF
replicative form RNA
RI
replicative intermediate RNA
RNase
ribonuclease
rRNA
ribosomal RNA
S
Svedberg unit (IO"13 sec)
SDS
sodium dodecyl sulphate
SV40
simian virus 40
TCA
trichloroacetic acid
tcf
tissue culture fluid
TES
N-tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid
Tris
tris[hydroxymethyl]aminomethane
tRNA
transfer RNA
VD
virus diarrhoea
VRC
vanadyl ribonucleoside complexes
vRNA
viral genomic RNA
VSV
vesicular stomatitis virus
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