Molecular subtyping and quantification of Feline
Immunodeficiency Virus from domestic cats in
New Zealand
Babette Ravensbergen 3155935
Date: 21 January 2011
Supervisor at Utrecht University: Dr H. Egberink
Supervisor at Massey University: Dr. N. Cave
Dr. M. Dunowska
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Preface:
At the beginning of this research period two different projects in the field of FIV research
were defined. It was the intention that one of the projects was done by myself and the other by
Cindy Reijne (doing a research internship at the Massy University during the same period).
The first subject was to determine whether cells exposed to soluble FIV in vitro undergo
apoptosis at a comparable rate to those exposed to cells infected with FIV. The plan was to
use activated peripheral blood mononuclear cells (PBMC) from healthy cats which would be
exposed to FIV in vitro using one of two methods. The first utilizes a feline T-lymphoblastic
cell line (MYA-1) that has been infected in vitro with FIV, co-culture them with the healthy
PBMC’s and run a flow cytometric assay to determine the level of apoptosis after 24hrs. The
second experiment would have been the exposure of healthy PBMC to FIV using viral stock,
rather than infected cells and determine the percentage of apoptotic cells by flow cytometry.
The gain was to determine the degree of apoptosis in cells following exposure to FIV
compared for each of the two methods described.
The second subject was to isolate and quantify FIV derived from the plasma and PBMC’s of
naturally infected cells. The plan for this research was to infect MYA-1 cells in vitro, using
PBMC and plasma from naturally FIV infected cats and assay the cell supernatant at defined
time points, to quantify the level of virus produced by the cells. For quantification a real time
RT PCR would be used.
Our supervisor’s goal was to grow the MYA-1 cells before we came to New Zealand so we
could start with our research immediately, unfortunately the IL-2 that was needed to grow the
cells wasn’t delivered by the time we arrived. That’s why we started with learning the
techniques on healthy PBMC’s instead of MYA-1 cells and we started with two other little
study’s, one to determine why CPT tubes don’t work on cat blood and another study about
NCD, were they want to determine the best suitable method to extract NCD RNA for real
time RT PCR.
Since there would not be enough time to wait for the IL-2 and thus not enough results on FIV
for both of us to write about, we decided to change the research projects. Cindy went further
with the research about NCD and I went further with the research about FIV. However since
there was still no IL-2 available we were forced to use PBMC’s instead of MYA-1 cells and
do only virussubtyping, virusisolation, and virusquantification instead of both subjects.
Although it was hard sometimes to accept that research takes a lot of time and that things
don’t always go the way you want it to go, I learned a lot of techniques and had a great time
doing research at Massey University.
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Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Contents:
Summery
Page 4
Introduction
Page 5-9
-
Etiology
Epidemiology
Pathogenesis
Clinical Findings
Diagnosis
Management
Vaccination
Page 5
Page 5
Page 6-7
Page 7
Page 8
Page 8
Page 8-9
Material & methods
- DNA extraction
- PCR method
- Gel electrophoresis
- DNA quantification
- Sequencing
- Virus quantification
 Separation procedure
 Cell counting
 Cell culture and stimulation
 Viral RNA extraction
 Virus quantification
Page 9-15
Page 10
Page 10-11
Page 11
Page 11
Page 11
Page 11-15
Page 11-12
Page 12
Page 12-13
Page 13-14
Page 14-15
Results
- Gel electrophoresis
- DNA quantification
- Sequencing
- Virusquantification
Page 15-22
Page 15-17
Page 17
Page 17-18
Page 18-22
Discussion
Page 22-25
Acknowledgements
Page 25
Sources and manufactures
Page 26
References
Page 26-28
3
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Summery:
Feline immunodeficiency virus (FIV) affects domesticated housecats all over the world, with
a significant diversity among the five different subtypes (A, B, C, D & E). FIV has been
identified as an endemic disease with a prevalence that varies among geographic locations. In
New Zealand the prevalence ranges from 6,8% in clinically healthy cats to 20,9-27,3% in cats
with clinical symptoms. There has not been a lot of research on subtype C and because of the
high level of diversity even within subtypes, it is very plausible that studies on overseas
isolates may not accurately represent the situation in New Zealand. This research determined
whether naturally infected New Zealand cats that tested positive for FIV ELISA were really
infected with FIV and subtyped the sequence. In total 20 cats who tested positive on FIV with
the ELISA were used in this research. Blood from these cats was taken and examined by a
Gag PCR and an Env PCR reaction. The Gag PCR failed, so further research was done only
on the Env gene. From the 20 samples that were collected, 17 samples were tested positive on
the FIV Env PCR. Among the 17 samples analysed from PCR FIV positive cats, subtype C
was present in 5 samples and subtype A was prevalent in 1 sample. Although not enough
samples were subtyped to make a final conclusion, these present study results add to the
finding of other studies that the predominant FIV subtype in New Zealand appears to be
subtype C and A. Also determination of the optimal time from infection to harvest of the
supernatant with the most produced virus by FIV infected peripheral blood mononuclear cells
(PBMC’s) was performed. Healthy PBMC’s were cultured together with FIV infected
PBMC’s, a media change was performed every 3-4 days and supernatant was frozen. RNA
quantification of the supernatant was performed using a spectrophotometer. In 5 of the 7
samples the RNA concentration increased on day 18 and decreased after that. FIV infected
PBMC cultivation without donor PBMC’s indicated that the highest level of RNA was 17
days after incubation. RNA quantification was also performed using different real time RTPCR. It was found that one step real time RT-PCR wasn’t working on the RNA samples, but
two step real time RT-PCR did. No time was left to get the real time RT PCR working for the
viral RNA cell culture supernatants, so determination of the optimal time from infection to
harvest of the supernatant with the most produced virus by FIV infected PBMC’s wasn’t
performed.
4
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Molecular subtyping and quantification of Feline
Immunodeficiency Virus from domestic cats in New Zealand
Introduction:
Etiology:
The first discovery of FIV was in 1986 in a group of cats with a high prevalence of
opportunistic infections and degenerative conditions. Since that time the disease has been
identified as an endemic disease in domestic cat populations worldwide [1].
Feline immunodeficiency virus (FIV) is part of the family of Retroviridae which affects
domesticated housecats and causes a disease analogous to AIDS in humans. Retroviridae
carry a special reverse transcriptase enzyme, which can reverse RNA into DNA. Further
classification of FIV is in the genus of lentivirusses [2]. The FIV genome consists of 6
different genes made up out of 9400 nucleotides. Of these 6 genes, the envelope gene (Env) is
the most important because they determine cell tropism and influence pathogenicity. For
infecting a host cell the binding of FIV Env proteins with host cell ligands is an important
step, in addition in case of an immune response the Env proteins are important targets [3].
Epidemiology:
Based on nucleotide sequence differences coding for the V3 to V5 region of the envelope
gene, FIV is divided into five different subtypes, A, B, C, D and E . Between the five
subtypes genetic variation exist and recombinant viruses generated from subtype A/B, B/D,
and A/C envelope gene sequences are found in natural populations [4, 5]. Cats that are
naturally infected can also harbour multiple subtypes of FIV [6, 7]. Some studies even found
sequences of FIV infected cats that did not group with any known subtype, these may belong
to a novel subtype [7, 8]. The diversity in nucleotide sequence between the subtypes varies
between 17.8 % and 38 %, whereas within a subtype a diversity up to 15% is found. When
another genetic region like the Gag gene is evaluated, comparable phylogenetic clades and
subtypes are gained. Nonetheless the high diversity of the Env gene makes it the best suitable
region for subtyping [9].
Around the world there is a significant diversity among the subtypes of FIV (Fig. 1). In the
United states mostly predominates subtype A and B and in Australia and Africa subtype A is
mostly found. Cats from Europe are infected with subtypes A, B, C and D, where in the
Northern countries (like Germany and The Netherlands) mostly subtype A and in the
Southern countries (like Italy) mostly subtype B is found. However multiple seroprevalence
studies have demonstrated that cats are able to carry more than one subtype [3].
In addition FIV has been identified as an endemic disease with a prevalence that varies among
geographic locations. In the United States the prevalence varies from 4% to 24 % in clinically
ill cats and cats at high exposure risk [3, 10]. In New Zealand the prevalence ranges from 6,
8% in healthy cats to 20,9-27,3% in cats with clinically symptoms. Around the world the
prevalence of FIV ranges from 1-12 % in healthy cats to 15-44% in sick cats [11]. The
variation of the prevalence of FIV is mostly attributed to the different ways the cats are kept.
The transmission of FIV is mostly by bite and fight wounds, were the virus is present in saliva
or blood. Therefore the prevalence is higher in countries with a high number of stray cats and
in regions were cats are able to run more freely outdoors. Transmission is also possible trough
milk or in utero. The transmission after oral, intrarectal and intravaginal inoculation is
reported in experimental settings but there is no evidence that this plays an important role in
maintaining natural infections [3].
5
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Fig. 1 Worldwide distribution of FIV
subtypes based on serosurvey and
sequence analysis. [10]
Pathogenesis:
After a cat is infected with FIV, macrophages rich tissue clears out the virus and the virus will
replicate in the thymus, spleen, lymph nodes and other macrophages and lymphoid rich tissue.
Within a few weeks there is a viremia peak. FIV also spreads to mononuclear cells that are
located in the bone marrow, lung, intestinal tract, brain and kidneys. Eventually there is a
progressive disruption of the normal immune function. The first and permanently
immunologic abnormalities that occur is a decrease in the amount of CD4+ T cells, due to a
decrease production in bone marrow or thymic, lysic of the infected cells induces by FIV,
destruction of FIV infected cells by the immune system or by apoptosis [3]. The degree of
CD4, CD8 and B cells apoptosis inversely correlates with CD4 amount and the CD4/CD8
ratio, which leads to immunodeficiency [12]. Other immunologic abnormalities that occur in
FIV infected cats are lymphocytes that lose their ability to multiply when stimulated by B-and
T cell mitogens, changed production of cytokines and hypergammaglobulinemia which leads
to increased circulating immune complexes [3].
To determine the pathogenicity of the different FIV- subtypes, previous studies investigated
the correlation and intensity of in vitro induces apoptosis and the decline in the number of
peripheral blood CD4 T lymphocytes [13]. The mechanism responsible for the T cell
depletion in FIV infected cats is not totally clear, but there are parallel findings with HIV-1.
In HIV-1 infected cells most of the apoptosis occurs in bystander cells that are uninfected and
lie nearby infected cells. Research in HIV shown that several viral proteins, mostly the Env
protein, which consist of a transmembrane protein (gp41) and a surface protein (gp120),
induces apoptosis in CD4 T cells. CD4 T cells express the primary receptor, CD134 which is
a member of the tumor necrosis factor receptor superfamily. When the surface protein
attaches to the CD134 on the CD4 T cells, the conformation of the surface protein changes.
Because of the conformation change the surface protein can now attach to the co-receptor,
CXCR4 (Fig. 2.). The fusion, which is necessary for viral entry, is mediated by the
transmembrane protein. The membrane expressed Env glycoprotein also mediates a fusion
between infected cells and non-infected bystander cells, witch express the CD134 and
CXCR4. The interaction between membrane expressed Env protein and the bystander cell
will cause apoptosis, cytolysis or reduced proliferation and arrest followed by apoptosis [14,
15].
6
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Fig. 2 Binding of the surface
protein with CD134 leads to a
conformation change, so binding
of CXCR4 can be achieved [15]
To know in which way the mechanism of apoptosis is comparable to HIV-1 apoptosis
mechanism, Garg et al. did several experiments. First they determined whether T cell
activation is necessary for induction of apoptosis in FIV infected cells by co-culturing Envexpressing cells with either resting cells or cells stimulated with ConA. and found that resting
unstimulated cells did not show any significant apoptosis when co-cultured with Envexpressing cells compared to activated cells. They concluded that mitogenic activation is
required for induction of apoptosis by FIV Env and that this correlates with the expression of
more surface co-receptors CXCR4, but not more of the primary receptor CD134 in activated
cells, so CD134 binding may not be an absolute requirement for this process. However the
binding of only CXCR4 is not enough for Env-induced apoptosis, it also requires the gp41
function. Garg et al. suggested that the fusogenic potential or membrane disruption capacity
of gp41 is necessary for FIV Env mediated apoptosis, but syncytia formation between viral
and cellular membranes per se is not sufficient. Destabilization due to Env glycoprotein
interaction with bystander target cell membranes subsequently also leads to apoptosis of
target cells [14].
Clinical findings:
Because there is a diversity in subtypes and even diversity within a subtype, FIV represents a
group of viruses with that have the potential to differ in fundamental properties such as
pathogenicity. In general in a FIV positive cat there is a loss off CD4+ T cells and the
CD4/CD8 ratio will change, which leads to immunodeficiency. Clinically this
immunodeficiency status will lead to symptoms that in general can be classified in four
phases. The primary phase is the acute phase characterized by increasing viral load,
neutropenia, lymphadenopathy, fever, conjunctivitis, gingivitis and diarrhoea. This phase can
take weeks or months. The second phase is the asymptomatic phase, in this phase cats show a
long period (up to 5 years or more) of clinically normal appearance, there is a high antiviral
immune response, lower viral titters and the amount of CD4 T cells will decrease. During the
third phase, which can last between 6 months and several years, the cat shows vague signs,
include recurrent fever, leucopenia, anaemia and reduced appetite. The last phase, were the
animal enters the acquired immune deficient syndrome stage, can take months to years and
shows signs of secondary infections, weight loss, myeloproliferative disorders, tumors and
neurological signs. In the later stages of the disease the amount of viral load will increase due
to a continues drop off CD4 T cells and the cat will eventually die of a secondary infection
[15]. The clinical findings follow after infection are not only due to the immunodeficiency
status, but are the result of an interaction of many factors such as FIV isolates properties, age
at the time of infection, the amount of virus the cat is infected with, the way the cat is infected
(parenteral, mucosol or other) and whether the cat is infected with cell-free or cell-associated
virus [3].
7
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Diagnosis
As the symptoms of FIV infection vary and are mostly caused by secondary infections, the
diagnosis can’t be made based on the clinical symptoms alone. In practice the diagnosis is
often made by the detection of FIV-antibodies in the blood for example by using an ELISA or
immunochromatography. Because FIV produces a persistent infection from witch cats don’t
recover there is a direct correlation between the presence of antibodies and a FIV infection.
The ELISA’s that are used in veterinary practice usually search for antibodies against the
capsid protein p24. Because the specificity of these tests is less than 100% these test are not
so useful in areas with a low FIV prevalence. In these areas the false-positive serological
reactions may be higher than the true prevalence. So any positive results in a low-risk
population will have to be confirmed by more specific tests [16, 4]. In contrast, negative
results obtained in a cat population with a low prevalence are generally accurate. There can
still be false-negative results due to early infection, terminal stages of the disease (due to
immunodeficiency) or when sequestration of anti-FIV antibodies with virus in immune
complexes occurs [4].
A problem in diagnosing FIV infection is that currently there is FIV vaccine available to
practitioners. The used ELISA test does not distinguish between vaccinated or infected cats.
Vaccinated cats are tested positive for antibodies by ELISA, which makes this diagnostic test
useless in cats after vaccination. The American Association of Feline Practitioners and
Academy of Feline Medicine both recommend testing for FIV antibody before vaccination.
[6, 17]. There is a study that tested a discriminant ELISA to distinguish between infected and
uninfected vaccinated cats. In this study all FIV negative cats (73 unvaccinated and 89
vaccinated cats) were also tested by the ELISA as uninfected (specificity of 100%). Of the
samples collected from the 102 cats that were infected with FIV, 99 samples tested FIV
positive by the discriminant ELISA (sensitivity 97.1%). A practical strategy for the diagnosis
of FIV infection would be to use existing commercial FIV antibody assays as screening tests.
Negative results with commercial assays are highly reliable predictors for lack of infection.
Positive results can be confirmed with the discriminant ELISA. If the discriminant ELISA is
negative, the cat is probably vaccinated against FIV but not infected. Positive results are
likely to represent infection [18]. This ELISA is currently not commercially available and
more research on discriminant ELISA’s should be done.
Management
Many cats that are naturally infected with FIV don’t have severe clinical signs caused directly
by the FIV infection. With the right care FIV infected cats can live many years. Nonetheless
there has been a lot of investigation to find a treatment for FIV. Not only to treat FIV infected
cats but also for the potential benefits for HIV infections in humans [6].
Cats that are infected with FIV should be kept indoors to minimize the risk of secondary
infections and avoid potential transmission of the virus to other cats. It is important that the
cat is protected against other infections since these can cause clinical symptoms but may also
lead to progression of the FIV infection. Healthy seropositive cats should be neutered to
reduce the aggressive behavior against other cats and by that way reduce the risk of
transmission [4].
Vaccination
Because of the multiple subtypes and the diversity of strains within subtypes it is hard to
create an effective FIV vaccine. In 2002 a commercial FIV vaccine (Fel-O-Vax FIV) became
8
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
available in the USA, this FIV vaccine is now also available in Australia, New Zealand and
Japan, but is not licensed in Europe [4, 19]. This dual-subtype FIV vaccine contains
inactivated whole viruses of subtype A (FIVPet) and subtype D (FIVShi) strains [19]. This dualsubtype vaccine induces both humoral and cell-mediated immunity and was found to be
effective against homologous FIV strains and also against a subtype B strain (FIVBang) [20,
21]. In many of these challenges the used viruses may not have been representative of field
isolates. There has also been a research with UK field isolates in which the vaccine did not
protect the cats against the FIV infection [22]. More research to test the efficiency of the virus
against field isolates should be done to determine whether the vaccine is good way to protect
domestic cats against FIV.
Most research has previously focused on the most commonly isolated FIV strains worldwide
namely A and B, but recent studies have demonstrated a predominance of FIV subtype A and
C in New Zealand [12, 8]. Currently there is a long term study to test the hypothesis that
variation in pathogenicity exists among FIV subtypes in New Zealand. This is done by
looking for differences in the subtype distribution of FIV between diseased and asymptomatic
infected cats. In particular this is done to determine if the commonly diagnosed isolate of FIV
subtype C in New Zealand causes disease to the same extent as New Zealand isolates of other
subtypes. If all New Zealand FIV isolates are comparable in their ability to cause disease, the
subtype distribution among these two populations would be expected to be similar. However,
if one subtype is less pathogenic, it would be expected that this subtype will have a lower
prevalence in the diseased population compared to the asymptomatic population.
In addition to the above, Massey University wants to do more research on FIV subtype C to
determine if information gained overseas can be extrapolated to New Zealand isolates.
Especially as to the commercial vaccine Fel-O-VaxR FIV used in other countries is useful in
New Zealand. There has not been a lot of research on subtype C and because of the high level
of diversity even within subtypes, it is very plausible that studies on overseas isolates may not
accurately represent the situation in New Zealand. Because the vaccine contains only subtype
A and subtype D it is not sure whether the vaccine will be effective in protecting cats against
the FIV subtypes in New Zealand. More studies using New Zealand isolates of FIV are
required to determine if information gained overseas can be extrapolated to New Zealand
isolates and to find out if the vaccine is useful in the prevention of FIV in cat populations in
New Zealand.
The objective of this research is to isolate, subtype and quantify Feline Immunodeficiency
Virus derived from plasma and peripheral blood mononuclear cells (PBMC) of naturally
infected cats in New Zealand. These different subtypes will be used for studies in the future.
In this study it was determined whether cats positive for FIV ELISA are really infected with
FIV and the subtypes were sequenced. Also determination of the optimal time from infection
to harvest of the supernatant with the most produced virus by FIV infected PBMC’s was
performed. All the information gained by this research will be used to produce viral stock
from a range of strains of FIV from New Zealand.
Materials and methods:
Animals:
To recruit cats that are naturally infected with FIV a letter was send to veterinary clinics in
New Zealand, asking them if they could contact Massey University when they had a cat that
9
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
was tested positive on FIV by the snap-ELISA test. Blood samples from these FIV ELISA
positive cats were taken at Massey University Palmerston North by a veterinarian from the
jugulair vein. To determine whether they were really infected with FIV or that they were false
positive a nested polymerase chain reaction (PCR) on FIV Env gene and FIV gag gene was
done. Samples were stored by -70°C, before DNA extraction and PCR tests were done.
For Virus quantification blood is derived from the jugular vein from a healthy cat and from
PCR FIV positive cats.
DNA extraction:
To isolate DNA from cat blood a DNA kit (High pure PCR template preparation kit) was
used. According to the manufacturer's instructions the protocol for animal blood was used,
were DNA was extracted from 200 µL of blood:
For the isolation of nucleic acids, add 200 µL of binding buffer and 40 µL Proteinase K to the
blood sample and mix immediately. Incubate at 70°C for 10 minutes, add 100 µL Isopropanol
and mix well. Insert a High Filter tube in a collection tube and add the sample into the filter
tube. Centrifuge the entire tube (the filter and collection tube) at 8000 g for 1 minute. For
washing and elution of the sample, discard the collection tube after centrifugation and
combine it with a new collection tube. Add 500 µL inhibitor removal buffer to the upper
reservoir of the filter tube and centrifuge at 8000 g for 1 minute. Discard the collection tube
after centrifugation and combine it with a new collection tube. Add 500 µL washing buffer to
the upper reservoir of the filter tube and centrifuge at 8000 g for 1 minute. Repeat this last
washing step one time. After the three washing steps, discard the flow trough liquid and
centrifuge the sample for another 10 seconds at full speed and discard the collection tube.
To elute the DNA insert the filter tube in a clean microcentrifuge tube, add 200 µL
prewarmed (70°C) elution buffer and centrifuge at 8000 g for 1 minute. The microcentrifuge
tube now contains the eluted DNA, which can be used for PCR [a].
PCR method:
Polymerase chain reaction was performed with 1,0 μL of template DNA, 5,0 µL Faststart
Mastermix (which contains buffer, enzyme and nucleotides), 0,1 μL primer LTR-1-foreward
and 0,1 μl GAG-1-reverse (round 1) and 0,1 μL primers LTR-2-forward and a GAG-1reverse (round 2) which amplified a region in the FIV Gag gene. For the FIV Env gene other
primers were used; forward 0,1 μL VE1S and reverse 0,1 μL VE1R (round one) was used and
forward 0,1 μL VE2S and reverse 0,1 μL VE2R (round two) was used (Table 1). The primers
used for this PCR were gained from the research of Hayward and Rodrigo [23]. Two rounds
were used to gain the wanted basepare length. The primers start at different locations on the
Env or Gag gene and produce a particularly product which after round one contains 1230 and
1287 basepares. The primers of round two start at a location within the round one PCR
products, thus after round two the PCR product of the Env gene contains 858 basepares,
which is more specific to determine FIV (Table.1).
Table 1: env and gag primer details used in the nested PCR [23]
Gene region and PCR round
Primer name
Location (bp)
env round 1
VE1S
7134-7154’
VE1R
8364-8345
env round 2
VE2S
7326-7345
VE2R
8184-8165
gag round 1
LTR1
122-141”
Gag548
1409-1389
gag round 2
LTR2
285-309
Gag548
1409-1389
PCR product length (bp)
1230
858
1287
1127
‘ using TM2 as reference sequence
“ using Petaluma as reference sequence
10
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
For the Gag-gene amplifications consisted of initial incubation at 95°C for 10 min, followed
by 35 cycles of 95°C for 15 s; 60°C for 15 s, and 72°C for 1 min; with a final extension at
72°C for 7 min. After the first PCR round a second PCR round was performed using the same
amounts of reagents only using different primers and as template 1,0 µl of the samples from
the first PCR round was added. These cups were also put in the PCR machine running them at
the same temperature and cycles of the first round. After these two rounds the PCR products
of the Gag-gene consists of 858 basepares. During each amplification, controls consisting of
samples lacking DNA (negative control) and samples with DNA derived from a FIV infected
cat (positive control) were included.
For the Env-gene amplifications consisted of initial incubation at 95°C for 10 min, followed
by 35 cycles of 95°C for 15 s; 50°C for 15 s, and 72°C for 1 min; with a final extension at
72°C for 7 min. After two rounds the PCR products of the Env gene consist of 1127
basepares.
Both Env protein and the Gag protein were determined because dual-subtype infections or
infection with recombinant FIV subtypes are common in New Zealand.
Gel electrophoresis:
Amplicons were detected by gel electrophoresis, where gel was prepared with 0,4 gram
agarose and 40 mL 0,5% TBE solution which were heated in the microwave for 1 minute.
After the gel is prepared and cooled down, 10 μL sample was mixed with 2 μL ethylbromide
dye and added in the wells of the gel. The samples run trough electrophoresis for 40 min at
100 V, together with a DNA ladder.
DNA quantification in PCR products:
After gel electrophoresis, the DNA products of the positive samples were cut out of the gel
under UV light and transferred to a gel extraction filter tube. The tubes were submerged in
liquid nitrogen for 1 minute and left at room temperature for a while. Then the samples were
centrifuged at 13000 g for 3 minutes and the filter was discard. The bottom tube contains the
DNA and the quantification of this DNA was done by a Nanodrop spectrophotometer which
measures the DNA in ng/ µL. For the Nanodrop 1,5 µL sample was load onto the optical
pedestal.
Sequencing:
For sequencing a total volume of 15 μL DNA sample was needed. Forward primer
concentration of the sample must be 3.2 pmol/15 μL. To get this concentration 1 μL forward
primer was diluted in 5.25 μL H2O, since the primer’s concentration is 20 pmol/ μL (20 μM).
After diluting the forward primer 1 μL from this dilution was added in a PCR tube. The DNA
concentration (ng/ μL) needed was 2 ng/100bp/15 μL, so 18 ng/15 μL. The DNA volume
added to the PCR tube was then calculated by dividing 18 trough the sample concentration
(ng/ μL). H2O was added to make up the volume to 15 μL. The same was done for the reverse
primer. The samples were submitted to Massey Genome Service and sequence analysis and
alignments were performed to subtype the virus.
Virus quantification:
Separation procedure:
For virus quantification PBMC’s from FIV PCR positive cats were cultured with con A to
stimulate the cell grow and virus replication. PBMC’s were derived by a separation method
using lymphoprep. First blood from FIV positive cats and from healthy donor cats were taken
and collected in citrate tubes. This blood was diluted with the same amount of sodium
11
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
chloride 0,9%. Then the diluted blood was carefully layered over Lymphoprep (6 ml diluted
blood over 3 ml lymphoprep) in a 12-15 mm centrifuge tube. It was centrifuged at 800 g for
20 minutes at room temperature. After centrifugation the PBMC’s form a distinct band at the
sample/medium interface. The distinct band was removed with a Pasteur’s pipette without
removing the upper layer (Fig 3.). The fraction was diluted with sodium chloride 0,9% and
centrifuged at 250 g for 10 minutes. The supernatant was aspirated carefully, 10 ml PBS was
added and centrifuged at 300 g for 10 minutes twice. After the second centrifugation, the
pellet was resuspended in 1 ml advanced RPMI media (2% FBS, 1% PenStrep and 1 %
glutamax) [b].
Fig. 3 separating the blood using
LymphoprepTM [24].
Cell counting:
After the cells were separated and mixed with media, 50 μL of tryphan blue and 50 μL sample
was mixed together and counted on the haemocytometer. The 10X objective of the
microscope was focused on the grid lines of the haemocytometer on one set of 16 corner
square (Fig.4). The number of living cells in 3 sets of this 16 corner squares was counted (1, 3
and 7).
Fig. 4 Hemocytometer used for counting cells: Nine large
squares: 1,3,7 and 9 are corners; 2,4 6 and 8 are sides and 5
is the center [25].
The haemocytometer is designed so that the number of cells in one corner squares is
equivalent to the number of cells x 104 / ml, so the total cell number can be calculated by the
formula: Cell number = average per box X dilution X 104.
After calculating the cells, a dilution with media was made to get a sample concentration of 1
X 106 cells/ml for the healthy cells and a concentration of 3-5 x 106 cells/ml for the infected
cells.
Cells culture and stimulation:
After calculating the cells and the preferred concentration was made, the samples were
collected and transferred to a 24 well culture plate. Both the healthy and infected PBMC’s
were cultured with 5 μg/ml ConA per ml media added and incubated at 37°C , 5% CO2 [26].
After 3 days the healthy PBMC samples were collected from the well, transferred to a 15 ml
centrifuge tube and centrifuged at 280 g for 10 minutes. 500 μL supernatant was aspirated and
the pellet was resuspended. Prewarmed media was add, to make up a total volume of 7ml. For
every 1 ml of new media added, 5 μg/ml ConA was added. 1 mL of the healthy PBMC was
add to each sample of infected PBMC in the 24 well plate and all cells were incubated at 37°C
, 5% CO2. Half medium change was performed every 3-4 days and the media aspirated was
frozen at -70°C. 5 μg/ml ConA was adjusted every second time the media was changed [27].
12
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
To compare virus production in FIV+ PBMC’s with and without donor PBMC’s, one FIV+
sample was cultured without the add of healthy donor PBMC’s. Half medium change was
performed every 3-4 days and the media aspirated was frozen at -70°C. 5 μg/ml ConA was
adjusted every second time the media was changed [27].
Viral RNA-extraction:
To quantify the amount of virus produced by the cells the viral RNA must be purificated from
the supernatant of the viral culture. RNA extraction was performed using a Viral Nucleic
Acid Extraction Kit IIR. 200 μL samples was add into a 1,5 ml microcentrifuge tube together
with 400 μL VB lysis buffer. The samples were incubated at room temperature for 10
minutes. Then 450 μL of AD buffer was added to the sample and shaken strongly. 600 μL of
the samples was transferred to the VB column and centrifuged at 14-16.000 g for 1 minute.
The flow-trough liquid was discarded and the VB column was placed back in a 2 ml
collection tube. The sample that was remained was transferred to the column and centrifuged
again at 14-16.000 g for 1 minute. After centrifugation the collection tube was discarded and
the column was transferred to a new collection tube. Now the samples were washed twice.
First by adding 400 μL buffer and centrifugation at 14-16.000 g for 30 seconds and second by
adding 600 μL washing buffer and centrifuged at 14-16.000 g for 30 seconds. At last the
sample was centrifuged at 14-16.000 g for 3 minutes to dry the column matrix. To elute the
nucleic acid the column was placed in a 1.5 ml microcentrifuge tube, 50 μL of RNase free
water was added and it was left at room temperature for 3 minutes or until the water was
absorbed by the matrix. Finally the sample was centrifuged at 14-16.000 g for 1 minute to
elute the purified nucleic acid [c]. After the RNA was extracted from the samples,
quantification of the RNA was done by a Nanodrop spectrophotometer. For measurement of
the RNA 1,5 μL sample was load onto the optical pedestal. The instrument automatically
detects the highest concentration and utilizes a specific pathlenght to calculate the amount of
absorbance. The absorbance at 260 nm represents the RNA in the sample, the absorbance at
280 the amount of contamination and the absorbance at 230 nm the amount of protein. The
machine also calculates the ratio 260/280 and 260/230. A 260/280 ratio between 1.8 and 2 is
generally accepted as pure RNA. The 260/230 ratio is a secondary measure of nucleic acid
purity and should range between 1.8 and 2.2. The sample concent μLation is also measured in
ng/ μL which is based on the absorbance at 260 nm and the selected analysis constant (H2O).
The positive control used for the real time RT-PCR was harvest from a commercial FIV
vaccine, were RNA was extracted using three different methods. The first method was as
described above. The second method was by using a TRIzolR RNA purification kit. Hereby
250 μL sample was add to 750 μL TRIzol by pippeting it up and down, which causes lysis of
the cells and dissolving cell components, but keeps the integrity of the RNA intact. The
sample was left at room temperature for 5 minutes. After this homogenisation step 200 μL
chloroform was add and shaken vigorously for 15 seconds. The samples were then left at
room temperature for 2-15 minutes and centrifuged at 12000 g for 15 minutes at 2-8 °C. After
centrifugation the sample consist of three different layers. The upper layer is an aqueous
phase, which contains the RNA, the second layer is an interphase, which contains the DNA
and proteins and the last layer is an organic phase. The aqueous phase was transferred to a
centrifuge tube, mixed with 1 μL glycogel and 500 μL isopropyl and left at room temperature
for 10 minutes. Then the sample was centrifuged at 12000 g for 10 minutes. After
centrifugation the supernatant was discharged and the RNA was found as a gel-like pellet on
one side of the bottom of the tube. The RNA was then washed with 1 ml 75% ethanol and
centrifuged at 7500 g for 5minutes. To solute the RNA, the ethanol was discharged and the
13
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
eppendorf tube was dried by air for approximately 15 minutes. The RNA was dissolved in 20
μL RNA-ase free water and incubated at 55°C for 10 minutes [d].
The third RNA extraction method was by using a Qiagen RN-easyR mini kit. By this method
250 µl of sample was added to 875 µl buffer RLT and vortexed for one minute, which causes
lysis of the cells to release the RNA. To increase selective binding of the RNA to the column,
625 μL 100% ethanol was add to the sample. 600 µl of the sample was transferred to a RNeasy column and centrifuged at 8000 g for 15 seconds. The flow trough liquid was then
discharged and the same steps were done again twice. To remove contaminants the RNA was
washed a few times with buffer. To remove the buffer the column was add in a new 2 ml tube
and centrifuged for 1 minute at full speed. To collect the RNA the column was placed in a
new tube again, 30 µl RNase-free water was add and the sample was centrifuged for 1 minute
at 8000 g. After centrifugation the collection tube contains the RNA and the column was
discharged [e]. RNA quantification was done using a NanodropR spectrophotometer.
Virusquantification:
For virusquantification different real time RT-PCR methods were used, where in all the
methods the amount of florescence was measured during each PCR-cycle. In the first method,
FRET real time RT PCR, the PCR-products were detected using two fluorescently resonance
energy transfer (FRET) sequence specific probes, which binds to the PCR product. The first
probe is a donor and the second probe an acceptor (Fig. 5A) When the two probes are into
close proximity, which happens during the annealing step of the PCR, the energy can transfer
from the donor to the acceptor with a fluorescent signal as result (Fig. 5B). Because the
quantity of signal is equal to the quantity of target sequence, the amount of fluorescent can be
used in real time RT-PCR to quantify the amount of target sequence. The fluorescent signal
stops when the probes are removed from their target sequences, which takes place during the
extension step of PCR (Fig.5C) [f].
Fig. 5. FRET probe principle
[f]
The RNA used for the real time RT-PCR in this research was extracted from the virus culture
supernatant and must first be reverse transcribed into cDNA before real time RT-PCR can be
performed. In the real time RT PCR that is used in this research both reverse transcriptase and
real time PCR takes place in the same tube, which is called a one step real time RT PCR. The
primers used are a foreword primer and a reverse primer, which can bind FIV subtype A, B, C
and E and were made to hybridize at the 5’ end of the gag gene. The probes used were a F1ABCE donor probe and a F2-ABCE acceptor probe, they were designed to hybridize the
polymorphic region of the gag PCR target and were selected for the highest differentiation of
FIV subtypes (Table. 2) [28]. The cycle used in this real time RT-PCR was 30 minutes at 95
°C in which cDNA synthesis takes place, followed by initial denaturation at 95 °C for 2
minutes. This was followed by 50 cycles of: denaturation for 3 seconds at 95 °C, primer
annealing at 64 °C for 10 seconds and primer extension at 72° 10 seconds. Then there was a
final extension at 95 °C for 1 minute. The reaction was performed with 2,0 μL of template
14
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
RNA, 2.5 μL One step Mastermix, 0,25 μL forward primer and 0,25 μL reverse primer, 1 μL
probe F1, 2 μL probe F2 , 1.5 μL H2O and 0,5 μL RT-enzyme.
Table 2. primers and probes used in this RT-PCR
Primer/probe
Upstream primer
Downstream primer
F1-ABCE
F2-ABCE
Sequence (5’  3’)
ATGGGGAAYGGACAGGGGCGAGA
TCTGGTATRTCACCAGGTTCTCGTCCTGTA
TACTCTTSSCCCCTACTCCTACAGCA-(6-FAM)
(LC-Red 640)-CATTACTACATCTYTTWATGGCCAYTTTCCA
The Y acts as a C or T, R as a A or G, S as a C or G and the W as a A or T.
In the other one step real time RT PCR methods, the mastermix used contains a fluorescent
dye which binds double stranded DNA. During the PCR cycles the amount of double stranded
DNA increases and therefore more fluorescent signal will be measured. To confirm if the
fluorescent signal is from the specific DNA a melting curve is made, since the different DNA
sequences melts at different temperatures.
Another real time RT PCR method that was used was a two step real time PCR, were the
RNA was first converted in cDNA using a High Fidelity cDNA synthesis kitR. Wherefore 9.4
μL RNA was add together with 2 μL Random Hexane primer 600 pmol/ μL. This was heated
for 10 minutes at 65°C in a block cycler, which denaturates RNA secondary structures. The
samples were cooled on ice immediately. To these samples the RT mix was add, which
consists of 4 μL transcriptor high fidelity reverse transcription reaction buffer, 0.5 μL
protector RN-ase inhibitor (40U/ μL), 2 μL deoxynucleotide mix (10 mM), 1 μL DTT and 1,1
μL transcription high fidelity reverse transcriptase. The reagents were mixed with the
template to make a total of 20 μL and then placed in a block cycler for 30 minutes at 50°C.
After 30 minutes the transcriptor high fidelity reverse transcriptase was inactivated by heating
it at 85°C for 5 minutes and placed on ice. The synthesised cDNA was used in a real time RT
PCR, were the mastermix used contains a fluorescent dye which binds double stranded cDNA
(g).
Results:
Blood samples of twenty cats, that were tested positive on FIV by the snap-ELISA test, were
taken. Samples tested negative on the first PCR, were done a second time to confirm that the
samples were negative.
Samples:
MUVTH 001, MUVTH 002, CVK 001, CVK 002, RVC006, MUVTH003, RVC001,
RVC002, RVC003, RVC004, RVC005, RVC007, RVC008, RVC009, SRV001, SRV002,
CVK003, SRV003, SRV004 and CSV001
DNA extraction and PCR was done on the samples and they were run trough the
gelelectrophoresis.
Gelelectrophoresis:
Samples MUVTH 002, CVK 001 and CVK 002 tested positive on Env gene PCR but negative
on the Gag gene (Fig.6).
15
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Fig. 6 Gelelectrophoresis Env-gene
and Gag-gene
A= DNA-ladder, 1= MUVTH 002, 2=
CVK 001, 3= CVK 002, 4= positive
control, 5= negative control, 6=
MUVTH 002, 7= CVK 001, 8= CVK
002, 9= MUVTH001, 10= positive
control, 11= negative control
Fig. 7 Gelelectrophoresis Gag-gene
A= DNA-ladder, 1= MUVTH 001,
2= MUVTH 2, 3= CVK 001, 4=
CVK 002, 5= positive control, 6=
negative control
Fig. 8 Gelelectrophoresis Gaggene
A= DNA-ladder, 1= MUVTH 001
,2= MUVTH 2, 3= CVK 001, 4=
CVK 002, 5= positive control, 6=
negative control
All samples tested negative for the Gag gene were tested a second time. The second time all
the samples were negative on the Gag gene again (Fig.7). The Gag PCR was repeated again
by adjusting the cycle: For the first round a initial incubation at 95°C for 10 min, followed by
35 cycles of 95°C for 15 s; 60°C for 20 s, and 72°C for 1 min; with a final extension at 72°C
for 7 min was done. For the second round a initial incubation at 95°C for 10 min, followed by
35 cycles of 95°C for 15 s; 64°C for 20 s, and 72°C for 1 min; with a final extension at 72°C
for 7 min was done. After adjusting the cycle all the samples were still negative on Gag PCR
(Fig.8). Because the Gag gene PCR still wasn’t working properly, further research was done
only on the Env gene.
Fig. 9 Gelelectrophoresis Env-gene
A= DNA-ladder, 1=RVC 006, 2= MUVTH 003, 3= RVC 001, 4= RVC
002, 5= RVC 003, 6= RVC 004, 7= RVC 005, 8= RVC 007, 9= RVC 008,
10= RVC 009, 11=SRV001, 12= SRV002, 13= CSV 001, 14= positive
control, 15= negative control
Fig. 10 Gelelectroforesis Env-gene
A= DNA-ladder, 1= RVC 006, 2= RVC
003, 3= SRV002, 4= CVK 003, 5= SRV
003, 6= SRV 004, 7= CSV 001, 8= positive
control, 9= negative control
Further samples were tested on the Env gene. Samples tested positive for FIV proviral DNA
Env gene were: MUVTH003, RVC001, RVC002, RVC004, RVC005, RVC007, RVC008,
RVC009 and SRV001. The samples RVC 006, RVC 003 and SRV 002 tested negative (Fig
9). To ensure the samples were negative, they were tested a second time together with 3 new
samples. The samples tested positive for FIV proviral DNA Env gene were: CVK003,
SRV003, SRV004, CSV001 and the samples tested negative the first time were negative the
16
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
second time as well (Fig.10). Of the twenty FIV Elisa positive blood samples, 17 samples
tested positive for FIV proviral DNA Env gene.
DNA quantification in PCR products:
Quantification of the DNA was done by a Nanodrop spectrophotometer which measures the
DNA in ng/ μL. Table 2 shows the RNA concentration in the sample measured by Nanodrop.
Table 2: DNA quantification in the FIV positive PCR
products done by Nanodrop spectrophotometer
Sequencing:
Subtype classification demonstrated that the samples MUVTH001, RVC007, RVC008,
SRV001 and MUVTH003 accumulated within subtype C (Fig. 11). And that most of these
sequences are more similar to each other than to representative subtype C FIV isolates such as
CABCpady02C. Sample RVC 009 is found within subtype A. The subtyping of samples
RVC001, RVC002, RVC004 and RVC 005 didn’t work, neither did the samples MUVTH001,
MUVTH002, CVK001, CVK002, CVK003, SRV003, SRV004 and CSV001.
Figure 11: Sequencing tree inferred from the
alignment of V3-V5 Env nucleotide sequences of
FIV.
The subtypes A, B, C, D and E are indicated. The
New Zealand isolates from this study are designated
C for MUVTH001, RVC007, RVC008, SRV001
MUVTH003 and A for RVC009.
The isolate name for the Env nucleotide sequences
for published sequences included in this study are:
- Subtype A: Petaluma Env gene,
USCAsam01A and MAS47922.
- Subtype B : TM2 Env gene, Aomori2
- Subtype C : CABCpady02C,
CABCpbar02C, STR211009 and TVF001
- Subtype D : Shizuoka and Fukuoka
- Subtype E : LP-3 and LP-20
17
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Unfortunately there wasn’t enough time left to submit samples RVC001, RVC002, RVC004,
RVC 005, MUVTH001, MUVTH002, CVK001, CVK002, CVK003, SRV003, SRV004 and
CSV001 for sequencing again.
Virus quantification:
Counting the cells and cell culturing:
Healthy donor PBMC’s:
PBMC cells from 4 healthy donor cats (4 ml blood each) were prepared using Lymphoprep
and added all together. After 1 ml of media was added with the PBMC’s, the cells were
counted on a hematocytometer. In three boxes 532 living cells were counted, which gives a
cell number of 3.55 x 106/ml. To get a 1 x 106 solution 2.5 ml media was added, together with
0,5 μg/μL ConA. To optimalize cell grow another 3.5 ml media and 0,5 μg/μL ConA was
added, which gives a concentration of 5 x 105 cells/ml. The sample was transferred to a
culture flask and incubated for 3 days.
FIV positive PBMC’s:
PBMC cells from 4 ml MUVTH003 blood (FIV PCR positive) were prepared, mixed with 1
ml of media and counted on a hematocytometer. In three boxes there were 410 living cells.
Cell number = (410/3)*2*104, so the cell number in 1 ml media is 2.7 x 106. To get a 1 x 106
solution 1.8ml media was added. 1.4 ml sample, together with 0.5μg/μL ConA, this was
transferred to a well and incubated. After three days a full media change was done and the
cells were counted again, and 181 cells were dead and 79 were living, so the percentage living
cells after three days was 30.4 %. On this sample every 3-4 days 500 μL new media was add
and the media aspirated was frozen. Every second media change 10μL ConA was adjusted.
PBMC cells from 9 FIV PCR positive cats (RVC 002, CVK 001, MUVTH 002, CVK 002,
RVC 007, MUVTH 003, RVC 009, RVC 008 and RVC 005) were prepared separately. Two
samples didn’t work (RVC005 and RVC 007). The PBMC’s were mixed with 1 ml of media
and one sample was counted on a hematocytometer. In three boxes there were 180 living
cells. Cell number = (180/3)*2*104, so the cell number of this sample in 1 ml media was 1.2 x
106. No extra media was added. Seven 1.0 ml samples, together with 0.5μg/μL ConA, were
transferred to a 24 well culture plate (1 ml per well) and incubated for 3 days
After three days the healthy PBMC’s were centrifuged, the supernatant was aspirated and 4
ml of new media was add together with 40 μL ConA. Then 1 ml of the donor PBMC’s were
add to each sample of infected PBMC’s, to make a total of 2 ml in each well. These samples
were incubated for another 3 days. Three days after the healthy cells were cultured together
with the infected cells, half of the supernatant was frozen and new media was add, together
with 20 μL ConA and incubated. After nine days half a media change was done, were half
the supernatant was frozen and 20 μL ConA was add. The samples were incubated for two
days and then again half a media change was done and half the supernatant was frozen. After
these days a half medium change was performed every 3-4 days and the media aspirated was
frozen at -70°C. 5 μg/ml ConA was adjusted every second time the media was changed. After
the beginning of the virusisolation protocol, virusquantification was started on the
supernatants.
Viral RNA quantification in the supernatant:
Quantification of the RNA was done by a Nanodrop spectrophotometer which measures the
RNA in ng/ μL, the RNA in the sample (260), the amount of contamination (280), the amount
of protein (230) and the ratio between RNA and contamination. Table 3, 4, 5 and 6 show the
18
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
outcomes of samples MUVTH002, MUVTH003, RVC002, RVC008, RVC009, CVK001 and
CVK002 on different time point after incubation.
The RNA concentrations measured by the NanodropR spectrophotometer are also listed in a
graphic (Fig.12). This gives a clear vision about the amount of RNA at the different time
points the supernatant was harvest of the samples. The graphic shows that the RNA
concentration in samples MUVTH002, RVC002, RVC008 CVK001, and CVK002 increase
18 days after incubation and decrease after day 18. Samples MUVTH003 and CVK001
increase after day 15 and are still increased 21 days after incubation.
Fig. 12 RNA quantification at
different days after incubation.
16
RNA concentration (ng/μl)
RNA concentration (ng/μl)
RNA quantification
RNA quantification
14
12
10
8
16
MUVTH002
MUVTH002
MUVTH003
MUVTH003
RVC002
RVC002
RVC008
RVC008
RVC009
RVC009
CVK001
CVK001
CVK002
CVK002
14
12
10
8
6
6
4
4
2
2
0
0
6
6
15
18
15
18
days after incubation
days after incubation
21
21
RNA concentration (ng/ μL)
To compare virus production in PBMC cultured cells with and without donor PBMC’s, one
FIV+ sample was incubated without adding healthy donor PBMC’s. The supernatant was
R
aspirated off at RNA
different
time points
quantification
FIV+and the RNA was quantified using Nanodrop
spectrophotometer. The outcomes are shown in Figure 13 and Table 7.
7
6
3
5
6
4
10
3
14
2
17
1
26
0
28
3
6
10
14
17
26
28
31
31
days after incubation
Fig.13 RNA quantification in FIV+ sample without
donor PBMC’s
19
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
The positive control used for the real time RT-PCR was harvest from a commercial Fel-OVaxR FIV vaccine, were RNA was extracted using three different methods. The RNA
concentration was measured using a NanodropR spectrophotometer and the outcomes are
shown in Table 8.
Table 8 viral RNA quantification in positive control in three different methods
Sample ID
ng/ µL 260
280
FIV+ A
17.50
0.437
0.212
+
FIV B
234.97 5.874
2.991
+
FIV C
11.55
0.289
0.196
A= Viral Nucleic Acid Extraction Kit IIR
260/280
2.07
1.96
1.47
260/230
0.04
1.84
0.29
B= TRIzolR RNA purification kit
C= Qiagen RN-easyR mini kit
Virusquantification real time RT-PCR:
Quantification of the virus was done by a one step FRET real time RT-PCR, which measures
the fluorescent signal. First the positive controls were measured, unfortunately these samples
didn’t work (Fig.14), so the cycle was adjusted. After adjusting the cycle the samples still
didn’t work. Therefore the DNA from PCR FIV positive samples and the three RNA FIV
positive control samples were measured using a different real time RT PCR mastermix and
cycle. DNA from 5 PCR FIV positive samples, MUVTH003, RVC002, RVC008, SRV001
and RVC009, were tested. The mix used for this RT PCR was Accumelt mix, which contains
a fluorescents dye that binds double stranded DNA and then sends out a fluorescent signal.
The cycle used for this RT PCR was 3 minutes at 95 °C, followed by 95 °C for 5 seconds,
58°C for 10 seconds and 72° 8 seconds. In total there were 35 cycles. The results of this real
time RT PCR were all positive and are shown in Fig.15 & 16. After this real time RT PCR
was done, an one step real time RT PCR was done on the FIV positive control RNA samples
and one PCR FIV positive DNA sample, using the same cycle and same mix. All these
samples were negative (results not shown). At last a two step real time PCR was performed,
were cDNA was first made out of the RNA of the 3 positive control samples. This cDNA was
used as a template and together with the Accumelt mastermix, water and the primers these
samples were measured in the real time RT PCR system (Fig.17&18). Fig. 17&18 shows that
all the samples were positive and that all the samples melt at the same temperature.
Fig. 14 Real time RT-PCR on the positive control samples.
Red = Viral Nucleic Acid Extraction Kit IIR, Green = TRIzolR RNA purification kit, Blue = Qiagen RN-easyR
mini kit, Purple = Negative control
20
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Fig. 15 ECOTM real time PCR system DNA samples:
MUVTH003, RVC002, RVC008, SRV001 and RVC009
MUVTH003 (1:10), RVC002 (1:10), RVC008 (1:10), SRV001 (1:10) and RVC009 (1:10)
Fig. 16 ECOTM real time PCR system DNA samples melt curve:
MUVTH003, RVC002, RVC008, SRV001 and RVC009
MUVTH003 (1:10), RVC002 (1:10), RVC008 (1:10), SRV001 (1:10) and RVC009 (1:10)
21
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Fig. 17 ECOTM real time PCR system cDNA samples:
Positive control RNA method A, Positive control RNA method B, Positive control RNA method C
Fig. 18 ECOTM real time PCR system cDNA samples melting curve:
Positive control RNA method A, Positive control RNA method B, Positive control RNA method C
Discussion:
Feline immunodeficiency virus (FIV) affects domesticated housecats all over the world, with
a significant diversity among the five different subtypes. FIV has been identified as an
endemic disease with a prevalence that varies among geographic locations. In New Zealand
the prevalence range from 6,8% in clinically healthy cats to 20,9-27,3% in cats with clinical
symptoms.
To test the hypothesis that variation in pathogenicity exists among FIV subtypes in New
Zealand, there is currently a long term study to determine if the commonly diagnosed isolates
of FIV subtype C in New Zealand causes disease to the same extent as New Zealand isolates
of other subtypes. In addition to the above this study was performed.
22
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
PCR method:
The first aim of this study was to determine whether cats tested positive with an FIV ELISA
are really infected with FIV and to sequence the subtypes these cats are infected with. In total
20 cats that were tested positive on FIV with the ELISA were used in this research. Blood
from these cats was taken and examined by a Gag PCR and an Env PCR reaction. From the
20 samples that were collected, 17 samples were tested positive on FIV with the Env PCR.
The original plan was to also test these 20 samples also with a Gag PCR. However the first 4
samples that were tested with a Gag PCR tested negative as well did the positive control. In
total three test runs were done with these samples and the positive control, but none of these
gave positive results. For this reason further samples that were collected from the FIV ELISA
positive cats were only tested with the Env PCR. The failure of the Gag PCR might be due to
the primers that were used. It is possible that these primers were not working at all or that
they were not able to recognise the sequence. It is also possible that the cycle used for PCR
still isn’t right and should again be adjusted. Unfortunately there was not enough time to find
out why the Gag PCR did not work. In following research the Gag gene should be examined
by using a positive control, like the FIV vaccine, to see if the Gag gene does work on this
sample.
Sequencing:
Since the gel electrophoresis doesn’t confirm the DNA amplified is the FIV DNA, the
samples were also submitted for sequencing. The 17 samples that were tested positive with
the Env PCR were therefore send to the Massey Genome Service for sequencing. Among the
17 samples analysed from PCR FIV positive cats, subtype C was present in 5 samples and
subtype A was prevalent in 1 sample. Unfortunately sequencing of the other twelve samples
didn’t work. It is possible that the quality of the DNA in the samples wasn’t right, since too
much DNA can cause a lack in resolution and too little will not produce a sequence signal at
all. Even when the DNA extracted was enough, DNA could have been lost during the
sequencing steps. The bad sequencing results could also be due to contamination and salt left
over from the initial sample preparation. It is also possible that the primers were
frozen/thawed to many times and that therefore the primers were degradated and not working
properly anymore. However this is not so likely since there were also samples treated with
these primers that did work.
Although not enough samples were subtyped to make a final conclusion, these present study
results add to the finding of other studies that the predominant FIV subtype in New Zealand
appears to be subtype C and A. This knowledge can be useful in understanding more about
feline immunodeficiency virus. Also it will help in the development of therapeutic,
preventative and diagnostic strategies. Further research about the phylogenetic analyse of the
subtypes is necessary to decide if it is possible to extrapolate research done on subtype B and
A to New Zealand subtypes.
Cell culture:
The second aim of this study was to determine what would be the optimal time from infection
to harvest of the supernatant with the most produced virus, by FIV infected cells. At the
beginning of this research the intention was to infect MYA-1 cells in vitro, using PBMC and
plasma from naturally FIV infected cats and assay the cell supernatant at defined time points
to quantify the level of virus produced by the cells. Unfortunately the IL-2 that was needed to
grow the cells wasn’t delivered by the time this research was performed. Therefore cell
culture was done using PBMC’s from healthy and FIV infected blood instead of MYA-1 cells
and no IL-2 was used during the cell culture. Although other studies cultured PBMC’s before
23
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
without IL-2, it isn’t the optimal condition to grow PBMC’s. Besides missing IL-2 the cell
culture was performed during the Christmasbreak and it wasn’t possible to change the media
every 3-4 days. Therefore it is possible that the virus culture supernatants used in this research
aren’t representative for the determination of the best suitable time from infection to harvest
of the supernatant with the most produced virus by FIV infected PBMC’s.
Virusquantification:
To determine the optimal time from infection to harvest of the supernatant, virus
quantification using real time RT-PCR was used. RNA was extracted from the supernatant
using Viral Nucleic Acid Extraction Kit IIR and then the RNA was measured by the
NanodropR. Fig.12 shows that the RNA concentration in 5 of the 7 samples increases on day
18 and decrease after that. This indicates that the best moment to harvest of the supernatant is
at 18 days after incubation. Some samples had a lower 260/280 ratio’s which may indicate
the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.
The 260/230 ratio’s were lower than 1.8 in all the samples, this may indicate the presence of
co-purified contaminants. It should also be kept in mind that the amount of RNA measured in
the samples is very dependent on how the RNA extraction method is performed, so it is
difficult to make a final conclusion. Also to compare the results a known RNA control sample
should have been adjusted. For this reason it is important to quantify the RNA by real time
RT-PCR as well. To compare virus production in PBMC cultured cells with and without
donor PBMC’s, one FIV+ sample was incubated without adding healthy donor PBMC’s. Fig
13 shows that in this sample the most RNA is found 17 days after incubation. However again
the amount of RNA measured in the samples is very dependent on how the RNA extraction
method is performed, so it is difficult to make a final conclusion. For this reason it is
important to quantify the RNA by real time RT-PCR as well.
Real time RT-PCR:
The positive control used for the real time RT-PCR was harvest from a commercial Fel-OVaxR FIV vaccine. From this vaccine the RNA was extracted using three different methods.
Table 8 shows that the most pure RNA was extracted using the TRIzolR or method, because
both methods show a 280/260 ratio near 2. However the 260/230 ration of the Viral Nucleic
Acid Extraction Kit IIR method was very low and had a lower RNA concentration. For this
reason this sample extracted by TRIzolR was used as a positive control for real time RT-PCR.
However for a good comparison between the different methods, more samples should be
examined.
Before virusquantification of the samples was done, the positive control samples were
measured by FRET real time RT-PCR, to make sure the cycle selected was suitable to use for
the virus culture supernatant samples. Fig. 14 shows that the cycle chosen wasn’t working on
the positive control samples. After the cycle was adjusted a second FRET real time RT-PCR
was performed, however this cycle didn’t work either. This could be due to the primers, the
cycle, the RNA samples or the mix that was used for the real time RT PCR. To determine if
the primers used for the FRET RT PCR were the cause of the bad FRET results the DNA
from PCR FIV positive samples and the positive control samples were measured using a
different real time RT PCR mastermix and cycle. Fig.15 shows that all the samples were
positive and the melting point were all round 83°C, so the fluorescent signal is likely from the
FIV DNA. This means that the primers used for this PCR worked. When the real time RT
PCR was performed with the same cycle and mix but with the RNA samples all the samples
were negative. It is therefore likely that the negative results are due to the RT-enzyme step.
To confirm this the one step real time RT PCR was replaced by a two step real time PCR.
24
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Fig.17 shows that in the two step real time PCR the samples were all positive and that the
melting points were round 83°C, so the fluorescent signal is likely from the FIV cDNA. These
results add to the finding that the RT-enzyme step isn’t working for the RNA samples.
Unfortunately there wasn’t enough time to figure out why the RT-enzyme step wasn’t
working properly, so virus quantification of the virus culture supernatant could not be
performed during this research project.
Acknowledgments:
The author gratefully acknowledge Massey University for the opportunity to do research at
their department and especially many thanks to the supervisors Dr. Nick Cave, Dr. Magda
Dunowska and Dr. Alison Stickney for teaching the techniques used in this research, their
enthusiasm and helpful advise about the research.
Also many thanks to Dr. Herman Egberink for the examination of this report and the helpful
feedback given.
A gratefully acknowledge to Drs. Cindy Reijne for assisting with the techniques used during
this research and for reading the report.
At last thanks to all the veterinarians and owners that made it possible to collect blood from
FIV infected cats, without them this research could not have been performed.
25
Molecular subtyping and quantification of Feline Immunodeficiency Virus from domestic cats in New Zealand
Drs. B.A Ravensbergen
Sources and manufactures:
a.
b.
c.
d.
e.
f.
g.
Roche-applied scienceR High pure PCR template preparation kit
LymphoprepTM
Viral Nucleic Acid Extraction Kit IIR
TRIzolR RNA purification kit
Qiagen RN-easyR mini kit
Qiagen R Real-time PCR brochure
High Fidelity cDNA synthesis kitR
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