WNV-specific IgG Research Articles

The value of West Nile virus RNA detection by real-time RT-PCR in urine samples from patients with neuroinvasive forms.

Routine laboratory screening is based on the detection of WNV specific IgM and IgG in blood and cerebrospinal fluid. Confirmation is then classically applied by real timeRT-PCR (rRT-PCR)in Cerebrospinal fluid (CSF), which often gives negative results due to too short virorachia and late sampling.rRT-PCR was applied-for the first time for routine diagnosis purpose-on urine samples. During 2018 outbreak in Tunisia, 107 patients presented WNV neurologic symptoms and were positive for WNV serology. Of them, 95 patients were sampled for urine and 35 were sampled for CSF. Qualitative rRT-PCR was performed on both type of samples. WNV RNA was detected in 50.5% of urine samples (48/95) and in 2.8% of CSF samples (1/35). WNV RNA was detectable from day1 to day41from symptom onset, however, positive urine rate was 53.1% during the first 10days from symptom onset. The proportions of urine-positive and urine-negative samples, based on day of collection, showed no statistical difference (p > 0.005). Cycle threshold (Ct)values ranged from 12 to 39, with no correlation with the day of collection. The lowest Ctvalue was detected for urine sampled on day 5 after symptom onset. A statistically significant difference was found between age groups of confirmed and non confirmed cases (p < 0.001). Our study reported the use of rRT-PCR on urine samples as a confirmatory diagnostic tool for WNV "probable cases" during an outbreak. Our findings underlined the reliability and therapidity of this confirmatory tool, even late, and showed its superiority on CSF investigation.

Seroprevalence of West Nile Virus among Healthy Blood Donors from Different National Populations Residing in Qatar

ObjectiveTo estimate the age- and nationality-specific West Nile virus (WNV) seroprevalence in select Middle East and North Africa (MENA) populations residing in Qatar. MethodsSera were collected from male blood donors attending Hamad Medical Corporation. A total of 1,948 sera were tested for anti-WNV antibodies using Serion ELISA classic IgG and IgM kits. ResultsOverall, seroprevalence estimates of WNV-specific IgG and IgM antibodies were 10.4% and 3.3%, respectively. Country-specific WNV-specific IgG seroprevalence was estimated to be 37.0% (34/92) in Sudanese, 33.0% in Egyptians (66/200), 13.0% (26/200) in Indians, 10.6% (11/104) in Iranians, 10.2% (14/137) in Yemenis, 9.2% (18/195) in Pakistanis, 7.0% (14/199) in Jordanians, 5.4% (6/111) in Filipinos, 2.5% (5/200) in Palestinians, 2.5% (5/200) in Syrians, 1.5% (3/200) in Qataris, and 0.9% (1/110) in Lebanese. Seroprevalence of WNV-specific IgM was lowest in Iranians (0/77), Lebanese (0/108), and Filipinos (0/107) at 0.0%, and was highest in Sudanese at 10.0% (8/80). While there seemed to be apparent trends in the prevalence of WNV-IgM and WNV-IgG antibodies, none of these trends were found to be statistically significant. ConclusionThe findings support the circulation of WNV in human populations in different countries of the MENA region. Seroprevalence was highest in Sudanese and Egyptians and lowest in Qataris and nationals of the Levant. The findings call for further animal, vector, and human studies, such as studying the actual prevalence of the viral RNA in blood donors to assess the risk of viral transmission through blood donation and for a better characterization of the epidemiology of this infection in this part of the world.

BAFF Produced by Neutrophils and Dendritic Cells Is Regulated Differently and Has Distinct Roles in Antibody Responses and Protective Immunity against West Nile Virus.

B cell activating factor (BAFF) is essential for B cells to develop and respond to Ags. Dysregulation of BAFF contributes to the development of some autoimmune diseases and malignancies. Little is known about when, where, and how BAFF is produced in vivo and about which BAFF-producing cells contribute to B cell responses. To better understand BAFF functions, we created BAFF reporter (BAFF-RFP) mice and Baff floxed (Bafffl/fl ) mice. Splenic and bone marrow neutrophils (Nphs) from BAFF-RFP mice expressed the highest constitutive levels of BAFF; other myeloid subsets, including conventional dendritic cells (cDCs) and monocyte (MO) subsets, expressed lower levels. Treatment of BAFF-RFP mice with polyinosinic:polycytidylic acid increased BAFF expression in splenic Ly6Chi inflammatory MOs, CD11bhi activated NK subset, and in bone marrow myeloid precursors. Postinfection with West Nile virus (WNV), BAFF increased in CD8- cDCs and Nphs, and BAFF+ CD11bhi NK cells expanded in draining lymph nodes. The cell- and tissue-specific increases in BAFF expression were dependent on type I IFN signaling. MAVS also was required or contributed to BAFF expression in dendritic cell and MO subsets, respectively. Mice with deletion of Baff in either cDCs or Nphs had reduced Ab responses after NP-Ficoll immunization; thus, BAFF produced by both cDCs and Nphs contributes to T cell-independent Ab responses. Conversely, mice with a cDC Baff deficiency had increased mortality after WNV infection and decreased WNV-specific IgG and neutralizing Ab responses. BAFF produced by Nphs and cDCs is regulated differently and has key roles in Ab responses and protective immunity.

Dendritic cell-associated MAVS is required to control West Nile virus replication and ensuing humoral immune responses.

Mitochondrial antiviral signaling protein (MAVS) is a critical innate immune signaling protein that directs the actions of the RIG-I-like receptor (RLR) signaling pathway of RNA virus recognition and initiation of anti-viral immunity against West Nile virus (WNV). In the absence of MAVS, mice die more rapidly after infection with the pathogenic WNV-Texas (TX) strain, but also produce elevated WNV-specific IgG concomitant with increased viral burden. Here we investigated whether there was a B cell intrinsic role for MAVS during the development of protective humoral immunity following WNV infection. MAVS-/- mice survived infection from the non-pathogenic WNV-Madagascar (MAD) strain, with limited signs of disease. Compared to wildtype (WT) controls, WNV-MAD-infected MAVS-/- mice had elevated serum neutralizing antibodies, splenic germinal center B cells, plasma cells and effector T cells. We found that when rechallenged with the normally lethal WNV-TX, MAVS-/- mice previously infected with WNV-MAD were protected from disease. Thus, protective humoral and cellular immune responses can be generated in absence of MAVS. Mice with a conditional deletion of MAVS only in CD11c+ dendritic cells phenocopied MAVS whole body knockout mice in their humoral responses to WNV-MAD, displaying elevated virus titers and neutralizing antibodies. Conversely, a B cell-specific deletion of MAVS had no effect on immune responses to WNV-MAD compared to WT controls. Thus, MAVS in dendritic cells is required to control WNV replication and thereby regulate downstream humoral immune responses.

Protection of mice deficient in mature B cells from West Nile virus infection by passive and active immunization.

B cell activating factor receptor (BAFFR)-/- mice have a profound reduction in mature B cells, but unlike μMT mice, they have normal numbers of newly formed, immature B cells. Using a West Nile virus (WNV) challenge model that requires antibodies (Abs) for protection, we found that unlike wild-type (WT) mice, BAFFR-/- mice were highly susceptible to WNV and succumbed to infection within 8 to 12 days after subcutaneous virus challenge. Although mature B cells were required to protect against lethal infection, infected BAFFR-/- mice had reduced WNV E-specific IgG responses and neutralizing Abs. Passive transfer of immune sera from previously infected WT mice rescued BAFFR-/- and fully B cell-deficient μMT mice, but unlike μMT mice that died around 30 days post-infection, BAFFR-/- mice survived, developed WNV-specific IgG Abs and overcame a second WNV challenge. Remarkably, protective immunity could be induced in mature B cell-deficient mice. Administration of a WNV E-anti-CD180 conjugate vaccine 30 days prior to WNV infection induced Ab responses that protected against lethal infection in BAFFR-/- mice but not in μMT mice. Thus, the immature B cells present in BAFFR-/- and not μMT mice contribute to protective antiviral immunity. A CD180-based vaccine may promote immunity in immunocompromised individuals.

A nationwide seroprevalence screening for West Nile virus and Tick-borne encephalitis virus in the population of Bulgaria.

To assess local circulation and risk for human infections with West Nile virus (WNV) and Tick-borne encephalitis virus (TBEV) in Bulgaria, a nationwide seroprevalence study was conducted. In total, 1451 residents of all 28 districts in Bulgaria were tested for WNV-specific and TBEV-specific IgG antibodies. The survey found overall seroprevalence of 1.5% and 0.6%, respectively. The highest WNV seroprevalence was found in Sofia Province and districts near the river Danube. TBEV circulation was detected among residents of six districts. The results showed that the two virus infections seem to be more wide-spread in the country as has been described.

Newcastle disease virus-vectored West Nile fever vaccine is immunogenic in mammals and poultry.

BackgroundWest Nile virus (WNV) is an emerging zoonotic pathogen which is harmful to human and animal health. Effective vaccination in susceptible hosts should protect against WNV infection and significantly reduce viral transmission between animals and from animals to humans. A versatile vaccine suitable for different species that can be delivered via flexible routes remains an essential unmet medical need. In this study, we developed a recombinant avirulent Newcastle disease virus (NDV) LaSota strain expressing WNV premembrane/envelope (PrM/E) proteins (designated rLa-WNV-PrM/E) and evaluated its immunogenicity in mice, horses, chickens, ducks and geese.ResultsMouse immunization experiments disclosed that rLa-WNV-PrM/E induces significant levels of WNV-neutralizing antibodies and E protein-specific CD4+ and CD8+ T-cell responses. Moreover, recombinant rLa-WNV-PrM/E elicited significant levels of WNV-specific IgG in horses upon delivery via intramuscular immunization, and in chickens, ducks and geese via intramuscular, oral or intranasal immunization.ConclusionsOur results collectively support the utility of rLa-WNV-PrM/E as a promising WNV veterinary vaccine candidate for mammals and poultry.

The role of MAVS signaling in humoral immune responses to West Nile virus.

Abstract MAVS is a key regulator of innate immune responses to RNA viruses, such as West Nile virus (WNV). Previous work identified that MAVS is important for the development of adaptive immunity to WNV. MAVS KO mice are highly susceptible to infection with lethal WNV-Tx. MAVS KO mice produced significantly increased levels of WNV-specific IgM and IgG compared to WT mice, however, a corresponding increase in neutralizing antibodies (NAbs) was not observed. As MAVS KO mice succumb rapidly to WNV-Tx, it was not possible in our initial study to define the role of MAVS in the development of the germinal centers (GCs) as well as long-term plasma cell (PC) and memory B cell responses. Using the less pathogenic WNV-MAD, we examined the development of humoral responses in the absence of MAVS. MAVS KO mice survived infection with WNV-MAD, and as with WNV-Tx infection, produced increased levels of IgM and IgG but comparatively low levels of NAbs. This increase in serum Ab levels was maintained up to 9 weeks post-infection (pi). Moreover, the number of WNV-specific Ab secreting cells in the spleen and bone marrow were increased in MAVS KO mice compared to WT mice. Using flow cytometry we found that MAVS KO mice have a slight delay in expansion of GL7+ GC B cells, but by day 10 pi the number of GC B cells was about 10-fold higher than in WT mice. MAVS KO mice also showed a rapid expansion of IgM−CD138hi PCs that was significantly increased compared to WT mice. We are currently testing the hypothesis that these results are due to an intrinsic dysregulation of MAVS KO B cells rather than simply due to an increase in viral load. We are also examining both extrafollicular and GC responses to identify the basis for the rapid IgG antibody responses in MAVS KO mice. (Supported by NIH grant AI83019).

Incidence of West Nile virus infection in the Dallas-Fort Worth metropolitan area during the 2012 epidemic.

The 2012 West Nile virus (WNV) epidemic was the largest since 2003 and the North Texas region was the most heavily impacted. We conducted a serosurvey of blood donors from four counties in the Dallas-Fort Worth area to characterize the epidemic. Blood donor specimens collected in November 2012 were tested for WNV-specific antibodies. Donors positive for WNV-specific IgG, IgM, and neutralizing antibodies were considered to have been infected in 2012. This number was adjusted using a multi-step process that accounted for timing of IgM seroreversion determined from previous longitudinal studies of WNV-infected donors. Of 4971 donations screened, 139 (2·8%) were confirmed WNV IgG positive, and 69 (1·4%) had IgM indicating infection in 2012. After adjusting for timing of sampling and potential seroreversion, we estimated that 1·8% [95% confidence interval (CI) 1·5-2·2] of the adult population in the Dallas-Fort Worth area were infected during 2012. The resulting overall estimate for the ratio of infections to reported WNV neuroinvasive disease (WNND) cases was 238:1 (95% CI 192-290), with significantly increased risk of WNND in older age groups. These findings were very similar to previous estimates of infections per WNND case, indicating no change in virulence as WNV evolved into an endemic infection in the United States.

Outbreak of West Nile virus infection among humans in Serbia, August to October 2012

We describe the first reported outbreak of West Nile virus (WNV) infection in humans in Serbia in August to October 2012 and examine the association of various variables with encephalitis and fatal outcome. Enzyme-linked immunosorbent assay (ELISA) was used for detection of WNV-specific IgM and IgG antibodies in sera and cerebrospinal fluid. A total of 58 patients (mean age: 61 years; standard deviation: 15) were analysed: 44 were from Belgrade and its suburbs; 52 had neuroinvasive disease, of whom 8 had meningitis, while 44 had encephalitis. Acute flaccid paralysis developed in 13 of the patients with encephalitis. Age over 60 years and immunosuppression (including diabetes) were independently associated with the development of encephalitis in a multivariate analysis: odds ratio (OR): 44.8 (95% confidence interval (CI): 4.93–408.59); p=0.001 (age over 60 years); OR: 10.76 (95% CI: 1.06–109.65); p=0.045 (immunosuppression including diabetes). Respiratory failure requiring mechanical ventilation developed in 13 patients with encephalitis. A total of 35 patients had completely recovered by the time they were discharged; nine patients died. The presence of acute flaccid paralysis, consciousness impairment, respiratory failure and immunosuppression (without diabetes) were found to be associated with death in hospital in a univariate analysis (p<0.001, p=0.007, p<0.001 and p=0.010, respectively).

A Hamster-Derived West Nile Virus Isolate Induces Persistent Renal Infection in Mice

BackgroundWest Nile virus (WNV) can persist long term in the brain and kidney tissues of humans, non-human primates, and hamsters. In this study, mice were infected with WNV strain H8912, previously cultured from the urine of a persistently infected hamster, to determine its pathogenesis in a murine host.Methodology/Principal FindingsWe found that WNV H8912 was highly attenuated for neuroinvasiveness in mice. Following a systemic infection, viral RNA could be detected quickly in blood and spleen and much later in kidneys. WNV H8912 induced constitutive IL-10 production, upregulation of IFN-β and IL-1β expression, and a specific IgM response on day 10 post-infection. WNV H8912 persisted preferentially in kidneys with mild renal inflammation, and less frequently in spleen for up to 2.5 months post infection. This was concurrent with detectable serum WNV-specific IgM and IgG production. There were also significantly fewer WNV- specific T cells and lower inflammatory responses in kidneys than in spleen. Previous studies have shown that systemic wild-type WNV NY99 infection induced virus persistence preferentially in spleen than in mouse kidneys. Here, we noted that splenocytes of WNV H8912-infected mice produced significantly less IL-10 than those of WNV NY99-infected mice. Finally, WNV H8912 was also attenuated in neurovirulence. Following intracranial inoculation, WNV persisted in the brain at a low frequency, concurrent with neither inflammatory responses nor neuronal damage in the brain.ConclusionsWNV H8912 is highly attenuated in both neuroinvasiveness and neurovirulence in mice. It induces a low and delayed anti-viral response in mice and preferentially persists in the kidneys.

Impaired Virus Clearance, Compromised Immune Response and Increased Mortality in Type 2 Diabetic Mice Infected with West Nile Virus

Clinicoepidemiological data suggest that type 2 diabetes is associated with increased risk of West Nile virus encephalitis (WNVE). However, no experimental studies have elucidated the role of diabetes in WNV neuropathogenesis. Herein, we employed the db/db mouse model to understand WNV immunopathogenesis in diabetics. Nine-week old C57BL/6 WT and db/db mice were inoculated with WNV and mortality, virus burden in the periphery and brain, and antiviral defense responses were analyzed. db/db mice were highly susceptible to WNV disease, exhibited increased tissue tropism and mortality than the wild-type mice, and were unable to clear the infection. Increased and sustained WNV replication was observed in the serum, peripheral tissues and brain of db/db mice, and heightened virus replication in the periphery was correlated with enhanced neuroinvasion and replication of WNV in the brain. WNV infection in db/db mice was associated with enhanced inflammatory response and compromised antiviral immune response characterized by delayed induction of IFN-α, and significantly reduced concentrations of WNV-specific IgM and IgG antibodies. The compromised immune response in db/db mice correlated with increased viremia. These data suggest that delayed immune response coupled with failure to clear the virus leads to increased mortality in db/db mice. In conclusion, this study provides unique mechanistic insight into the immunopathogenesis of WNVE observed in diabetics and can be used to develop therapeutics for the management of WNVE among diabetic patients.

Intrinsic adjuvanting of a novel single-cycle flavivirus vaccine in the absence of type I interferon receptor signaling

Type I interferons (IFNs) are critical for controlling pathogenic virus infections and can enhance immune responses. Hence their impact on the effectiveness of live-attenuated vaccines involves a balance between limiting viral antigen expression and enhancing the development of adaptive immune responses. We examined the influence of type I IFNs on these parameters following immunization with RepliVAX WN, a single-cycle flavivirus vaccine (SCFV) against West Nile virus (WNV) disease. RepliVAX WN-immunized mice produced IFN-α and displayed increased IFN-stimulated gene transcription in draining lymph nodes (LN). SCFV gene expression was over 100 fold-higher on days 1-3 post-infection in type I IFN receptor knockout mice (IFNAR(-/-)) compared to wild-type (wt) mice indicating a profound IFN-mediated suppression of SCFV gene expression in the wt animals. IFNAR(-/-) mice produced nearly equivalent levels of WNV-specific serum IgG and WNV-specific CD4(+) T cell responses compared to wt mice. However, significantly higher numbers of WNV-specific CD8(+) T cells were produced by IFNAR(-/-) mice and a significantly greater percentage of these T cells from IFNAR(-/-) mice produced only IFN-γ following antigen-specific re-stimulation. This altered cytokine expression was not associated with increased antigen load suggesting the loss of type I IFN receptor signaling was responsible for the altered quality of the CD8(+) effector T cell response. Together, these results indicate that although type I IFN is not essential for the intrinsic adjuvanting of RepliVAX WN, it plays a role in shaping the cytokine secretion profiles of CD8(+) effector T cells elicited by this SCFV.

Immunogenicity of RepliVAX WN, a novel single-cycle West Nile virus vaccine

We recently reported that immunization with RepliVAX WN, a single-cycle West Nile virus (WNV) vaccine, protected mice against WNV challenge. We have extended these studies by characterizing the RepliVAX WN-elicited antibody and T cell responses. WNV-specific IgG antibody responses comprised predominantly of IgG2c and IgG2b subclasses were detected 8 months after immunization. Vigorous WNV-specific CD4+ and CD8+ T cell responses directed at both structural and nonstructural WNV proteins were detected which were characterized by cytolytic activity and secretion of IFN-γ and TNF-α. Importantly, RepliVAX WN immunization resulted in vigorous CD8+ memory T cell responses detected at 8 months after immunization.

First serological evidence of West Nile virus in human rural populations of Gabon

To investigate West Nile virus (WNV) circulation in rural populations in Gabon, we undertook a large serological survey focusing on human rural populations, using two different ELISA assays. A sample was considered positive when it reacted in both tests. A total of 2320 villagers from 115 villages were interviewed and sampled. Surprisingly, the WNV-specific IgG prevalence was high overall (27.2%) and varied according to the ecosystem: 23.7% in forested regions, 21.8% in savanna, and 64.9% in the lakes region. The WNV-specific IgG prevalence rate was 30% in males and 24.6% in females, and increased with age. Although serological cross-reactions between flaviviruses are likely and may be frequent, these findings strongly suggest that WNV is widespread in Gabon. The difference in WNV prevalence among ecosystems suggests preferential circulation in the lakes region. The linear increase with age suggests continuous exposure of Gabonese populations to WNV. Further investigations are needed to determine the WNV cycle and transmission patterns in Gabon.

Oral Administration of Active Hexose Correlated Compound Enhances Host Resistance to West Nile Encephalitis in Mice

West Nile virus (WNV) poses a serious threat to public health, especially to the elderly and the immuno-compromised. Neither vaccines nor treatments are available for humans. Active hexose correlated compound (AHCC) is an extract of Lentinula edodes of the Basidiomycete family of fungi rich in α-glucans. In this study, we evaluated the effect of AHCC on host susceptibility in the murine model of WNV infection. Mice orally administered with AHCC (600 mg/kg) every other day for 1 wk before and at d 1 and 3 postinfection were assessed using viremia levels, survival rate, and protective immunity. AHCC administration in young (6- to 8-wk-old) mice attenuated viremia and mortality following lethal WNV infection. WNV-specific IgM and IgG production and γδ T cell expansion were also enhanced in these mice. Aged (21- to 22-mo-old) mice were more susceptible to WNV infection than young mice, partially due to the dysfunction of γδ T cell subsets. AHCC administration in aged mice enhanced the protective Vγ1+ T cell response as well as WNV-specific IgG but not IgM antibodies production. AHCC administration in aged mice attenuated viremia levels but led to no difference in mortality rate. Overall, our data suggests that AHCC enhances protective host immune responses against WNV infection in young and aged mice. Dietary supplementation with AHCC may be potentially immunotherapeutic for WNV-susceptible populations.

Virus and Antibody Dynamics in Acute West Nile Virus Infection

The dynamics of the early stages of West Nile virus (WNV) infection can be assessed by follow-up studies of viremic blood donors. A total of 245 donors with WNV viremia were followed up weekly for 4 weeks and then monthly for up to 6 additional months or until seroconversion. Plasma samples were tested for WNV RNA by transcription-mediated amplification (TMA) and for WNV-specific IgM and IgG antibodies. RNA persistence was investigated by 6 replicate TMA tests; samples that were viremic for >40 days were tested for WNV-neutralizing activity. Follow up of 35 additional viremic donors for up to 404 days was conducted to evaluate persistence of WNV-specific antibody. The median time from RNA detection to IgM seroconversion was 3.9 days; to IgG seroconversion, 7.7 days; to RNA negativity by single-replicate TMA, 13.2 days; and to RNA negativity by 6-replicate TMA, 6.1 additional days after results of single-replicate TMA are negative. For 4 donors in whom RNA persisted for >40 days after the index donation, all samples obtained after this threshold were also positive for WNV IgG and neutralizing activity. The mean times to IgM and IgA negativity were 156 and 220 days, respectively. IgM and IgG develop rapidly after viremia and before RNA levels become undetectable, which occurred a mean of 13.2 days after the index donation among donors in this study. WNV RNA detection by replicate TMA rarely persists for >40 days after the index donation and is accompanied by WNV-specific neutralizing antibody, consistent with an absence of WNV transmission via transfusion of seropositive blood components.

Interferon and Interferon-Induced Chemokine Expression Is Associated with Control of Acute Viremia in West Nile Virus-Infected Blood Donors

To understand early host responses controlling West Nile virus (WNV) infection, acutely viremic blood donors, identified by nucleic acid amplification testing, were enrolled and monitored for RNA-clearance and WNV-specific IgM and IgG antibodies. Viral load and chemokine and cytokine assays were performed on serial samples from donors whose index and first follow-up samples tested negative for IgM. A total of 84% of the specimens obtained from viremic donors before IgM/IgG seroconversion demonstrated a decreasing viral load. Levels of interferon (IFN)-α ere significantly increased before IgM seroconversion, relative to those in control specimens. CXCL10 and CCL2 were significantly elevated in donor specimens obtained before IgM seroconversion, compared with those obtained after IgM seroconversion. These findings suggest that IFN-mediated innate immunity plays a key role in initial control of WNV replication.

Development of a Novel, Rapid, and Sensitive Immunochromatographic Strip Assay Specific for West Nile Virus (WNV) IgM and Testing of Its Diagnostic Accuracy in Patients Suspected of WNV Infection

The West Nile virus (WNV), detected in the Western hemisphere in 1999, has since spread rapidly across North America into all 48 continental states of the US, 7 Canadian provinces, many Latin American countries, and throughout Mexico. More than 24 000 people in the US have tested positive for WNV infection; 9845 of these cases resulted in serious neuroinvasive disease and more than 959 were fatal (1). Guidelines for surveillance, prevention, and control of WNV are available(2). WNV, a member of the Flaviviridae family, belongs to the Japanese encephalitis serocomplex that includes Japanese encephalitis and St. Louis encephalitis viruses (3). WNV is transmitted to humans by mosquitoes. The transmission cycle involves mosquitoes and birds. In rare instances, the virus may be transmitted from human to human through organ donation or blood transfusion or from pregnant mother to fetus(4)(5)(6). WNV infections in a pediatric population have also been reported(7). Many patients remain asymptomatic or have only mild symptoms. Others report having fatigue, rash, fever, headache, and muscle weakness and may require hospitalization(8). The more severe form of the disease is meningoencephalitis manifested by typical signs of central nervous system infection and, in a minority of cases, development of a flaccid paralysis(9). Symptomatic patients will demonstrate an early antibody response of the IgM type during the 1st 4 days of illness, and nearly all patients will have detectable IgM antibodies by 7 to 8 days after the onset of illness (10). WNV-specific serum IgG is detectable by 3 weeks postinfection(11). The virus itself is usually no longer detectable by the time WNV-specific serum IgM appears, although both IgM and IgG may persist for more than a year(12). Laboratory-based ELISAs designed to detect WNV-specific IgM are now accepted as a …

Live-attenuated recombinant equine herpesvirus type 1 (EHV-1) induces a neutralizing antibody response against West Nile virus (WNV)

The immunogenicity in horses of a recombinant equine herpesvirus type 1 (EHV-1) vaccine expressing West Nile virus (WNV) prM and E proteins was studied. To construct the recombinant EHV-1, two-step en passant mutagenesis was employed for manipulation of a bacterial artificial chromosome (BAC) of vaccine strain RacH. Recombinant EHV-1 stably expressed the WNV prM and E proteins as demonstrated by indirect immunofluorescence and Western blotting. In addition, growth properties in vitro of the EHV-1/WNV recombinant were found to not be significantly different from those of the parental virus. To determine if vaccination of horses induces an antibody response, 10 horses were allocated in two groups. Group A consisted of six horses that were vaccinated three times with the recombinant EHV-1/WNV virus in 28- to 31-day intervals. Group B consisted of four horses that were sham-vaccinated using the same regimen. Serum was collected on days 0, 31, 45 and 66. Plaque reduction neutralization test and IgG(T)- and IgGb-specific WNV E antibody-capture ELISAs were used. After a single vaccination (day 31), at least four of the six horses from group A had detectable levels of serum neutralizing antibodies against WNV, and three horses retained SN titers until the end of the study. None of the horses in the control group B sero-converted. On days 31 and 45, five of the six horses in group A had a marked increase of WNV-specific IgG(T), and at least four exhibited modestly elevated WNV-specific IgGb titers. From the results, we concluded that the EHV-1 vectored virus is able to express the WNV structural proteins and that vaccination of horses results in the induction of WNV E-protein-specific IgG(T), IgGb, and neutralizing antibodies.