The Journal of American Science, 2(2), 2006, Baba, et al, West Nile Virus in Mosquitoes and Febrile Patients
West Nile Virus in Mosquitoes and Febrile Patients in A Semi-arid
Zone, Nigeria
Marycelin M. Baba *, Marie-Francois Saron **, Ousmane Diop **, Christian Mathiot **, J. A. Adeniji ***, O. D.
Olaleye ***
* Marycelin M Baba, Department of Medical Laboratory Science, College of Medical Sciences, University of
Maiduguri, P. M. B. 1069, Maiduguri, Borno State, Nigeria, Telephone/Fax: 234 76 235688, E-mail:
marycelinb@yahoo.com
** Department of Virology, Institut Pasteur De Dakar, Senegal
*** Department of Virology, College of Basic Medical Sciences, University of Ibadan
ABSTRACT: Several arboviruses are frequently considered in the etiology of acute febrile illness in some African
countries excluding Nigeria. This study was designed to determine the role of West Nile (WN) virus in febrile cases
in Nigeria. Sera from 973 febrile patients suspected of malaria and typhoid fever from a semi-arid zone (Sahel
savanna zone), Nigeria were tested for West Nile virus (WNV) and Yellow fever virus (YFV) IgM and IgG by
capture ELISA. WN virus IgM positive sera were also tested by PRNT and RT-PCR. In addition, 52 pools of Culex
quinquefasciatus/Mansonia spp from both Sahel savanna and Rain forest zones were tested for viral RNA by RTPCR and cultured for virus isolation. Twelve (1.2%) of the 973 sera had WN virus IgM antibodies. Mixed
infections of DEN-2 and WN virus observed in two samples by MAC-ELISA, eventually had neutralizing antibody
for WNV. Overall, PRNT and ELISA results were in concordance. The high prevalence of WN virus IgG (80.16%)
obtained in this study indicates the endemicity of Flaviviruses in the environment. The prevalence of WNV IgM and
IgG antibodies and the ages as well as the gender of the patients were not significantly different. Out of 12 WN virus
IgM positive sera, 4 (33.3%) had WN viral RNA. However, negative and border line sera for WNV IgM showed
detectable WN viral RNA. No WNV was isolated from the mosquito vector tested. It is advisable to confirm WNV
IgM positive sera with PRNT and to complement both (ELISA and PRNT) with RT-PCR when the time of onset of
illness is not considered in sample collection. Also, there is a need to include arboviruses in the differential
diagnosis of febrile illnesses in Nigeria. [The Journal of American Science. 2006;2(2):28-34].
KEYWORDS: West Nile virus, Antibodies, Febrile patients, Nigeria
role of West Nile virus among febrile illnesses
suspected of malaria and typhoid fever in Nigeria with
the view to encouraging some arboviruses in the
differential diagnosis of these cases.
INTRODUCTION
Many illnesses in Africa that present with fever are
documented as fever (pyrexia) of unknown origin
(PUO), especially if they fail to respond to antimicrobial drugs [15]. In that report, several arboviruses
are frequently considered in the etiology of acute febrile
illness in some African countries. As observed in India
(Voluntary Health Association of India-unpublished
data), the knowledge and experience of general
practitioners, pediatricians and other specialists in
Nigeria, regarding the nature and management of
arbovirus diseases seem inadequate. Therefore,
ignorance of doctors could lead to under as well as over
diagnosis of the disease and inappropriate treatment.
This speculation is supported by a report that WN virus
infection is so nonspecific that it often escapes medical
attention [13]. Two main factors reported [15] to favor
the emergence of vector borne diseases are common in
this study site. Such factors include high population of
mosquito vectors and pool of susceptible individuals.
The presence of these factors made us envisage that
arboviruses could play a significant role in severe
febrile illness. This study was designed to determine the
MATERIALS AND METHODS:
STUDY SITE: Serum samples were collected from
patients who visited University of Maiduguri Teaching
Hospital (UMTH), Nigeria for medical attention. The
hospital is a tertiary Health Institution, located in Borno
State, Nigeria and serves as a reference center for six
States in Northeastern Nigeria, and neighboring West
African countries (Chad, Niger and Cameroon).
Mosquitoes were caught from, Maiduguri, a Sahel
savanna zone (semi-arid) in Borno State and Ibadan
(Rain Forest), Oyo State, Nigeria. The experiment was
carried out at the Virology Department of Institut
Pasteur de Dakar, Senegal.
Study population: A total of 973 blood samples
from Patients with febrile illness in 2001, sent to the
Immunology laboratory of UMTH, for Widal tests were
used for the study. The records of the above-mentioned
hospital showed that, the clinical manifestations on
28
The Journal of American Science, 2(2), 2006, Baba, et al, West Nile Virus in Mosquitoes and Febrile Patients
these cases by the time of sample collection included:
fever, headache, abdominal discomfort, and diarrhea.
About 5ml of blood was collected by venu- puncture
from these patients. The blood was allowed to clot at
room temperature and the serum was carefully collected
after centrifugation at 8,000rpm for 5 minutes and
stored at –20oC until tested.
days. The presence of the virus was determined by the
use of indirect Immunofluorescence assay as described
by Beckwith et. al.[1].
RT - PCR IN SERA AND MOSQUITOES
THE
EXTRACTION
OF
RNA
FROM
SERUM/MOSQUITO
SUSPENSION/TISSUE
CULTURE EXTRACT
RNA extraction was carried out according to the
specifications of the kit’s (Q1 a Amp viral RNA Mini
Kit) manufacturer. For each batch of mosquito
suspension/serum/ extracted, positive controls (cell
culture of the seed virus concerned) and uninoculated
cell as negative control were included.
SEROLOGY
Stock antigens were prepared in mouse brain from
stock viruses supplied by WHO Collaborating Centre
for Reference and Research on Arboviruses (CRORA),
IPD, Senegal. All reactants were appropriately
standardized.
Detection of IgM antibodies: An IgM capture
ELISA (MAC– ELISA) as previously described by
Sathish et. al., [16] was used for the detection of IgM
antibodies against WNV and YFV.and for testing WNV
IgM positive sera against dengue viruses 1-4. The virus
with a higher Optical density (OD) was considered the
infectious agent as reported by CDC West Nile Home
[3]
Detection of IgG antibodies: For the detection of
IgG antibodies against WNV and YFV, an IgG capture
ELISA was used as previously described by Chunge et
al., [5]. Binding of the IgG antibodies was detected
using goat anti-human IgG antibodies labeled
horseradish peroxidase. Unfortunately these samples
were not confirmed by Plaque reduction neutralization
technique (PRNT).
Interpretation of results: The standard deviation
of a battery of negative sera was calculated. A value of
three standard deviations from the mean was used as the
cut -off value to minimize false results as suggested by
[7]
Plaque reduction neutralization test (PRNT):
This test was performed as previously described (11)
using Vero cells. Serum samples were tested by using a
starting dilution of 1:20. Titers were expressed as the
reciprocal of serum dilutions reducing the number of
plagues that were (greater than or equal to) 80% for the
twelve WNV IgM positive samples.
RT- PCR for detection of West Nile virus (The
method previously described for dengue [10] was
adopted)
A single round of amplification was carried out. All
relevant aspects of the RT-PCR (Mgcl2, primers, RT,
Taq polymerase, number of cycles, and annealing
temperatures) were initially optimized by using
quantitated purified WN virus RNA to achieve a
maximum level of sensitivity before testing the field
samples. The reaction product was electrophoresed on a
1% composite agarose gel in 0.4 M Tris- 0.05 M
sodium acetate- 0.01 M EDTA buffer.The gel was
stained with ethidium bromide. The resulting DNA
band was visualized on a UV transilluminator. With the
primers used, the size of the resulting DNA band
characteristic for WNV was 327 pb. Background bands
at higher or lowers than 327 pb were ignored.
The sequence of the primers include:
WN240: 5’ GAG GTT CTT CAA ACT CCA T-3’
WN132: 5’ GAA AAC ATC AAG TAT GAG G-3’
RESULTS
IgM CAPTURE ELISA FOR WEST NILE
VIRUS
The prevalence of WNV IgM antibodies is
presented in Figure 1. Twelve (1.2%) of the 973 sera
from Sahel savanna zone had WN virus IgM antibodies.
Mixed infections of DEN-2 and WN virus were
observed in two samples .The IgM levels were lower
than IgG in most cases (Figure 1). Probably, samples
were collected when the WN virus IgM started to drop
while the IgG antibodies were increasing. Samples 1
and 6 could be cases of anamnestic response for WN
virus. Samples 13 and 14 were positive and negative
control sera respectively kindly donated by CRORA
Plaque reduction neutralization test on WNV IgM
positive sera: Incidentally, all the sera that were positive
by MAC-ELISA were found positive by PRNT. Two
sera which showed mixed infections of WNV and
dengue by MAC-ELISA were later confirmed to be
Mosquito processing
The field- caught mosquitoes were identified to the
species level when possible. Morphologic characters
essential for accurate species identification were often
damaged through the collection and shipping process.
The identified mosquitoes were placed in 12x 75mm
tubes in pools of 50. Each pool was tested by RT-PCR
assay using a set of primer and with a cell culture assay.
The cell culture assay was conducted by inoculating
100l aliquot of clarified supernatant from the mosquito
pool onto sub confluent AP-61 cell line from Aedes
pseudocutallaris in T-25 flasks and incubating for 8-10
29
The Journal of American Science, 2(2), 2006, Baba, et al, West Nile Virus in Mosquitoes and Febrile Patients
positive for WNV by PRNT. Failure to carry out PRNT
for all the samples limits this study from giving the
precise status of these patients with regard to WNV
infections in Nigeria. This is because a negative acutephase specimen is inadequate for ruling out such an
infection underscoring confirmation by demonstrating
virus-specific serum IgG antibodies in the same or later
specimen
VIRUS ISOLATION FROM MOSQUITOES
No WN virus was isolated from Culex (51 pools)
and Mansonia (1 pool) species tested.
RT-PCR ON CULEX MOSQUITOES/ IgM
POSITIVE SERA FOR WNV
Forty of 52 pools of mosquitoes (Culex
quinquefasciatus) tested showed WN viral RNA (Figure
2 with each Lane representing the different pools of
mosquito / serum as the case may be). However, one
WNV IgM negative serum was positive by RT-PCR and
one serum, which was at border line by IgM, capture
ELISA also showed detectable WN viral RNA. Samples
that showed non-specific bands were not considered.
Table 1 shows the results of RT-PCR on WNV IgM
positive. TITAN (Combination of reverse- transcription
of viral RNA and subsequent Taq polymerase
amplification in a single reaction vessel) seemed to
exhibit higher degree of sensitivity and specificity
compared with separate reverse-transcription and PCRs
. Sequencing of RT-PCR results was beyond the scope
of this study.
AGE AND GENDER DISTRIBUTION OF
PATIENTS WITH WNV IgM ANTIBODIES
The prevalence of these antibodies and the ages as
well as the gender of the patients were not significantly
different (X2=P>0.05).
THE PREVALENCE OF YELLOW FEVER
IgM AND IgG ANTIBODIES
No YFV IgM was detected in 973 sera tested.
Nevertheless, significant number of patients (86.9%),
used for the study had YFV IgG antibodies due
probably to on- going vaccination or periodic outbreaks
of natural infection in the environment.
1.6
1.4
1.2
OD Values
1
IgM
0.8
IgG
0.6
0.4
0.2
0
1
2
3
4
5
6
7
8
9
10
11
12
Sam ples
Figure 1. West Nile IgM and corresponding IgG. Antibodies
30
13
14
The Journal of American Science, 2(2), 2006, Baba, et al, West Nile Virus in Mosquitoes and Febrile Patients
Figure 2. RT-PCR on culex mosquitoes (WNV)
Table 1. The mosquito vector and the ecological zones where they were caught
Species
Sex
No. of pools
Location
Period
Culex
quinquefaciatus
Males
31
Maiduguri (Sudan/
Sahel savanna)
September to
December
Culex
quinquefaciatus
Culex species
Females
9
Males
8
September to
December
July to December
Culex species
Females
13
Mansonia
Females
1
Maiduguri (Sudan/
Sahel Savanna)
Ibadan (Rain
Forest)
Ibadan (Rain
Forest)
Ibadan (R. Forest)
TOTAL
July to December
July to December
52 pools
only out-door activities but large-scale tree planting. It
is a common practice in Sahel savanna zone to combat
high degree of desertification and drought by large-scale
tree planting. These trees usually provide shades for the
Residents of the study site to gather in groups to
socialize and discuss issues of common interest. It was
observed in this study that Culex quinquefasciatus
which yielded WN virus RNA, were caught
predominantly in areas of large –scale tree planting near
human habitations. Therefore, tree-planting which
serves a useful purpose could also pose serious threat to
public health by providing breeding sites to mosquito
vectors in agreement with a previous report [12]. No
DISCUSSION
In Nigeria most cases of PUOs are hardly
investigated for arboviruses. Febrile illnesses are
routinely investigated for malaria and /or typhoid. This
study shows that 12 (1.2%) of the 973 sera tested had
WN virus IgM antibodies by MAC-ELISA in agreement
with previous reports [15]. Also, the prevalence of WN
virus IgM and the age of the patients were not
significant. Yet age is one of the factors that influence
the degree of the virulence of the virus [6]. However,
this study did not associate the severity of infection with
age. High rate of exposure to mosquito bites and
subsequent virus infection could be attributed to not
31
The Journal of American Science, 2(2), 2006, Baba, et al, West Nile Virus in Mosquitoes and Febrile Patients
significant difference between the presence of this
antibody and the sex of the patients was observed in
agreement with Olaleye et al. [15]. Probably the hungry
mosquito vector has no respect for sex in its quest for
blood meals.
Although, MAC-ELISA is t he most efficient
diagnostic method for WNV in serum [14], the
longevity of the IgM response during WNV infection
compromises its ability to discriminate individuals
recently and previously infected [3, 10]. In conformity
with that observation CDC (4) and Washington State
Department of Health (18) reported the criteria for
Laboratory diagnosis of WNV to include the
demonstration of virus –specific IgM antibodies in
serum by antibody capture ELISA and confirmation by
demonstrating virus-specific IgG antibodies in the same
or later specimen by another serologic method [e.g.
PRNT or Hemagglutination inhibition (HI)] The former
report also noted that patients who have been recently
vaccinated against or recently infected with related
flaviviruses (e.g. YF, Japanese encephalitis, dengue)
may have positive WNV MAC-ELISA. However, in
this study, all the sera except the twelve (12) IgM
positive were not tested by PRNT and this limits this
study from giving the precise status of these patients
with regards to WNV infection in the environment.
Therefore there is need for further study using a more
specific as well as sensitive serological techniques to
determine precisely, the current status of WN virus
infections in Nigeria. Nevertheless, in comparison with
Bradley et al [2], the PRNT and ELISA data in this
study were in concordance although in the former
report, epitope-blocking ELISA was used. This
observation could be attributed to higher IgG values
compared to IgM antibodies at the time of sample
collection (Figure 1). Although with this finding, WNV
infections are evident in the country but detailed study
is still needed to confirm this claim. The high
prevalence of WNV IgG (80.16%) obtained in this
study may not be genus-specific but indicates the
endemicity of WNV and/or possibly other crossreacting Flaviviruses in the environment in agreement
with previous reports [4].
Two sera which tested positive for WNV and DEN
IgM antibodies in MAC-ELISA actually tested positive
for WNV by PRNT in agreement with previous report
[4, 18]. This observation further supports the need to
test all WNV IgG positive sera by PRNT for a more
accurate result.
YFV IgM was not detected in all the 973 sera
tested, but a significant number (86.9%) of these
patients had IgG antibodies to YFV due probably to
current active immunization or past exposure to natural
infection. . .
No WNV was isolated from the field caught
mosquitoes. Probably storage and transport conditions
were not adequate for the rescue of infectious virus in
these vectors. Therefore, where virus isolation failed in
this study, RT-PCR became useful by detecting RNA to
WN virus in agreement with Lanciotti, et al. [9].
Although sequencing the bands (Figure 2) would have
yielded a more specific results than what is presented in
this report, there was no facility for such a test where
this experiment was carried out. However, the band
produced by the reference strain was used to measure
the test sample. The bands produced by the control
virus (a reference virus which had undergone several
passages in the laboratory) could not possible be of the
same size as the virus (which probably was not an
intact virus) in the test sample. The detection of WN
viral RNA in both mosquito pools and IgM positive sera
in this study agreed favorably with previous reports
[14]. Thus RT-PCR may increase the ability to detect
WN virus activity early in the season when infection
rates are very low (with low IgM or IgG level). In
addition, cases of WN virus IgM negative and border
line by ELISA had WN viral RNA (Table 2). Therefore,
IgM negative sera (960) in this study could be false (this
probably accounts for the low prevalence rate observed
in the present study) since testing all the IgM negative
sera by RT-PCR was not feasible due to the high cost of
reagents. Generally RT-PCR complements the low
specificity but high sensitivity of MAC-ELISA [17] and
even PRNT especially when the time of onset of the
disease is not considered during sample collection.
Since the non-specific prodromal phase of West
Nile virus infection could be mistaken for malaria/
typhoid fevers, which are hyper endemic in this
environment, it is imperative to include this virus (and
possibly other endemic arboviruses) in the differential
diagnosis of febrile illnesses in Nigeria. Surveillance
with good laboratory services serves as an “early
warning system” against impending outbreak of
arbovirus infections.
32
The Journal of American Science, 2(2), 2006, Baba, et al, West Nile Virus in Mosquitoes and Febrile Patients
Table 2. RT-PCR on WNV IgM positive sera
NO
SERA
S:/ NO
SAMPLE NO
RTPCR
IgM (OD VALUES)
1
1243
positive
0.581
2
3515
negative
0.254
3
2172
negative
0.302
4
3545
negative
0.277
5
3494
negative
0.29
6
3679
negative
0.763
7
3322
positive
0.427
8
2725
negative
0.32
9
3460
positive
0.478
10
3255
positive
0.49
11
3481
negative
0.25
12
3500
negative
0.276
13
3562
positive
0,154 (negative)
14
3222
positive
0,226 (border line)
4.
ACKNOWLEDGMENT
I wish to express my profound gratitude to
TWOWS (Third World Organization for Women in
Science) and Management of Institut Pasteur De Dakar,
Senegal for sponsoring this project. Also the technical
support of University of Maiduguri Teaching Hospital
(UMTH) is highly appreciated
5.
6.
7.
Correspondence to:
Marycelin M Baba
Department of Medical Laboratory Science
College of Medical Sciences
University of Maiduguri
P. M. B. 1069
Maiduguri, Borno State, Nigeria,
Telephone/Fax: 234 76 235688
E-mail: marycelinb@yahoo.com
8.
9.
10.
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