Early rise of West Nile fever in Israel, June 2024.

This report describes an unusual surge of West Nile fever in Israel in June 2024, during which 125 cases were diagnosed, compared with 4 cases on average during June in previous years (2014-23). Of the cases, 64 (62.1%) had neuroinvasive disease and 12 (9.6%) died; the 2024 case fatality rate was not significantly elevated vs the average rate in 2014-23. The early rise could be related to a temperature increase in spring and early summer of 2024.

A Hyperactive Kunjin Virus NS3 Helicase Mutant Demonstrates Increased Dissemination and Mortality in Mosquitoes.

The unwinding of double-stranded RNA intermediates is critical for the replication and packaging of flavivirus RNA genomes. This unwinding activity is achieved by the ATP-dependent nonstructural protein 3 (NS3) helicase. In previous studies, we investigated the mechanism of energy transduction between the ATP and RNA binding pockets using molecular dynamics simulations and enzymatic characterization. Our data corroborated the hypothesis that motif V is a communication hub for this energy transduction. More specifically, mutations T407A and S411A in motif V exhibit a hyperactive helicase phenotype, leading to the regulation of translocation and unwinding during replication. However, the effect of these mutations on viral infection in cell culture and in vivo is not well understood. Here, we investigated the role of motif V in viral replication using West Nile virus (Kunjin subtype) T407A and S411A mutants (T407A and S411A Kunjin, respectively) in cell culture and in vivo We were able to recover S411A Kunjin but unable to recover T407A Kunjin. Our results indicated that S411A Kunjin decreased viral infection and increased cytopathogenicity in cell culture compared to wild-type (WT) Kunjin. Similarly, decreased infection rates in surviving S411A Kunjin-infected Culex quinquefasciatus mosquitoes were observed, but S411A Kunjin infection resulted in increased mortality compared to WT Kunjin infection. Additionally, S411A Kunjin infection increased viral dissemination and saliva positivity rates in surviving mosquitoes compared to WT Kunjin infection. These data suggest that S411A Kunjin increases viral pathogenesis in mosquitoes. Overall, these data indicate that NS3 motif V may play a role in the pathogenesis, dissemination, and transmission efficiency of Kunjin virus.IMPORTANCE Kunjin and West Nile viruses belong to the arthropod-borne flaviviruses, which can result in severe symptoms, including encephalitis, meningitis, and death. Flaviviruses have expanded into new populations and emerged as novel pathogens repeatedly in recent years, demonstrating that they remain a global threat. Currently, there are no approved antiviral therapeutics against either Kunjin or West Nile viruses. Thus, there is a pressing need for understanding the pathogenesis of these viruses in humans. In this study, we investigated the role of the Kunjin virus helicase on infection in cell culture and in vivo This work provides new insight into how flaviviruses control pathogenesis and mosquito transmission through the nonstructural protein 3 helicase.

A Molecular Determinant of West Nile Virus Secretion and Morphology as a Target for Viral Attenuation.

West Nile virus (WNV), a member of the Flavivirus genus and currently one of the most common arboviruses worldwide, is associated with severe neurological disease in humans. Its high potential to reemerge and rapidly disseminate makes it a bona fide global public health problem. The surface membrane glycoprotein (M) has been associated with Flavivirus-induced pathogenesis. Here, we identified a key amino acid residue at position 36 of the M protein whose mutation impacts WNV secretion and promotes viral attenuation. We also identified a compensatory site at position M-43 whose mutation stabilizes M-36 substitution both in vitro and in vivo Moreover, we found that introduction of the two mutations together confers a full attenuation phenotype and protection against wild-type WNV lethal challenge, eliciting potent neutralizing-antibody production in mice. Our study thus establishes the M protein as a new viral target for rational design of attenuated WNV strains.IMPORTANCE West Nile virus (WNV) is a worldwide (re)emerging mosquito-transmitted Flavivirus causing fatal neurological diseases in humans. However, no human vaccine has been yet approved. One of the most effective live-attenuated vaccines was empirically obtained by serial passaging of wild-type yellow fever Flavivirus However, such an approach is not acceptable nowadays, and the development of a rationally designed vaccine is necessary. Generating molecular infectious clones and mutating specific residues known to be involved in Flavivirus virulence constitute a powerful tool to promote viral attenuation. WNV membrane glycoprotein is thought to carry such essential determinants. Here, we identified two residues of this protein whose substitutions are key to the full and stable attenuation of WNV in vivo, most likely through inhibition of secretion and possible alteration of morphology. Applied to other flaviviruses, this approach should help in designing new vaccines against these viruses, which are an increasing threat to global human health.

Sensitivity of Quails (Coturnix coturnix), Siskins (Carduelis spinus), and Frogs (Rana ridibunda) to West Nile Virus.

The level of viremia and features of the course of experimental infection caused by West Nile virus were studied in two species of migratory birds, siskins Сarduelis spinus and quails Coturnix coturnix, and in one species of amphibians, frogs Rana ridibunda. In quails, the virus caused a fatal disease; histological analysis revealed pathological changes in the heart, kidneys, liver, and brain stem. In siskins and frogs, virus antigen was detected in cloacal smears despite the absence of clinical manifestations, the level of viremia was sufficient to infect insect vectors during bloodsucking. These findings suggest that siskins and frogs can be potential reservoirs of West Nile virus and play a role in its circulation.

Immune Predictors of Mortality After Ribonucleic Acid Virus Infection.

BACKGROUND: Virus infections result in a range of clinical outcomes for the host, from asymptomatic to severe or even lethal disease. Despite global efforts to prevent and treat virus infections to limit morbidity and mortality, the continued emergence and re-emergence of new outbreaks as well as common infections such as influenza persist as a health threat. Challenges to the prevention of severe disease after virus infection include both a paucity of protective vaccines as well as the early identification of individuals with the highest risk that may require supportive treatment. METHODS: We completed a screen of mice from the Collaborative Cross (CC) that we infected with influenza, severe acute respiratory syndrome-coronavirus, and West Nile virus. RESULTS: The CC mice exhibited a range of disease manifestations upon infections, and we used this natural variation to identify strains with mortality after infection and strains exhibiting no mortality. We then used comprehensive preinfection immunophenotyping to identify global baseline immune correlates of protection from mortality to virus infection. CONCLUSIONS: These data suggest that immune phenotypes might be leveraged to identify humans at highest risk of adverse clinical outcomes upon infection, who may most benefit from intensive clinical interventions, in addition to providing insight for rational vaccine design.

Acute and Delayed Deaths after West Nile Virus Infection, Texas, USA, 2002-2012.

Infection with West Nile virus (WNV) has a well-characterized acute disease process. However, long-term consequences are less understood. We searched death records for 4,142 residents of Texas, USA, infected with WNV during 2002-2012 and identified 557 (13%) deaths. We analyzed all-cause and cause-specific deaths after WNV infection by calculating standardized mortality ratios and using statewide mortality data. Acute-phase deaths (<90 days after symptom onset) occurred in 289 (7%) of case-patients; of those deaths, 289 (92%) were cases of West Nile neuroinvasive disease (WNND). Convalescent-phase deaths (>90 days after symptom onset) occurred in 268 (7%) of the remaining 3,853 case-patients; 210 (78%) of these deaths occurred in patients with WNND. Convalescent-phase WNND case-patients showed excess deaths from infectious and renal causes; case-patients <60 years of age had increased risk for all-cause death, specifically from renal, infectious, digestive, and circulatory causes. We provide population-level evidence of increased risk for death after WNV infection resulting in WNND.

Macroecology of birds potentially susceptible to West Nile virus.

Zoonotic diseases transmitted by wildlife affect biological conservation, public and animal health, and the economy. Current research efforts are aimed at finding wildlife pathogens at a given location. However, a meta-analytical approach may reveal emerging macroecological patterns in the host-pathogen relationship at different temporal and spatial scales. West Nile virus (WNV) is a pathogen with worldwide detrimental impacts on bird populations. To understand macroecological patterns driving WNV infection, we aimed to recognize unknown competent reservoirs using three disease metrics-serological prevalence (SP), molecular prevalence (MP) and mortality (M)-and test if these metrics are correlated with the evolutionary history, geographical origin of bird species, viral strain, time-space and methodology. We performed a quantitative review of field studies on birds sampled for WNV. We obtained 4945 observations of 949 species from 39 countries. Our analysis supported the idea that MP and M are good predictors of reservoir competence, and allowed us to identify potential competent reservoirs. Furthermore, results indicated that the variability of these metrics was attributable to phylogeny, time-space and sample size. A macroecological approach is needed to recognize susceptible species and competent reservoirs, and to identify other factors driving zoonotic diseases originating from wildlife.

Interleukin-17A Promotes CD8+ T Cell Cytotoxicity To Facilitate West Nile Virus Clearance.

CD8+ T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic-mediator gene expression and promoting CD8+ T cell cytotoxicity against WNV infection in mice. We find that IL-17A-deficient (Il17a-/-) mice are more susceptible to WNV infection and develop a higher viral burden than wild-type (WT) mice. Interestingly, the CD8+ T cells isolated from Il17a-/- mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo Importantly, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 postinfection, significantly reduces the viral burden and increases survival, suggesting a therapeutic potential for IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8+ T cell cytotoxicity, which may have broad implications in other microbial infections and cancers. IMPORTANCE: Interleukin-17A (IL-17A) and CD8+ T cells regulate diverse immune functions in microbial infections, malignancies, and autoimmune diseases. IL-17A is a proinflammatory cytokine produced by diverse cell types, while CD8+ T cells (known as cytotoxic T cells) are major cells that provide immunity against intracellular pathogens. Previous studies have demonstrated a crucial role of CD8+ T cells in recovery from West Nile virus (WNV) infection. However, the role of IL-17A during WNV infection remains unclear. Here, we demonstrate that IL-17A protects mice from lethal WNV infection by promoting CD8+ T cell-mediated clearance of WNV. In addition, treatment of WNV-infected mice with recombinant IL-17A reduces the viral burden and increases survival of mice, suggesting a potential therapeutic. This novel IL-17A-CD8+ T cell axis may also have broad implications for immunity to other microbial infections and cancers, where CD8+ T cell functions are crucial.

West Nile Virus Infection in American Singer Canaries: An Experimental Model in a Highly Susceptible Avian Species.

This study investigated the susceptibility of American singer canaries ( Serinus canaria) to West Nile virus (WNV) infection. Adult canaries were inoculated with 105, 102, and 101 plaque forming units (PFU) of WNV. All birds became infected and mortality occurred by 5 days postinoculation. The load of viral RNA as determined by RT-qPCR was dose dependent, and was higher at all doses than the level of viral RNA detected in American crows ( Corvus brachyrhynchos) challenged with 105 PFU of WNV. In a subset of birds, viremia was detected by virus isolation; canaries inoculated with 101 PFU of WNV developed viremia exceeding 1010 PFU/mL serum, a log higher than American crows inoculated with 105 PFU of virus. In canaries euthanized at 3 days postinoculation, WNV was isolated at >107 PFU of virus/100 mg of lung, liver, heart, spleen, and kidney tissues. Pallor of the liver and splenomegaly were the most common macroscopic observations and histologic lesions were most severe in liver, spleen, and kidney, particularly in canaries challenged with 102 and 101 PFU. Immunoreactivity to WNV was pronounced in the liver and spleen. IgG antibodies to WNV were detected in serum by enzyme immunoassay in 11 of 21 (52%) challenged canaries and, in 4 of 5 (20%) of these sera, neutralization antibodies were detected at a titer ≥ 1:20. American singer canaries provide a useful model as this bird species is highly susceptible to WNV infection.

Critical West Nile Neuroinvasive Disease.

BACKGROUND: Data to guide neurointensivists seeing patients with West Nile Neuroinvasive disease (WNND) are lacking. We present a comparatively large series of patients with WNND admitted to the intensive care unit (ICU) and provide data on their early diagnosis, triage to the ICU and predictors of short-term outcomes. METHODS: We retrospectively identified patients aged ≥ 18 years old with WNND from January 1999 to November 2016. Demographic and clinical data, the modified Rankin Scale at discharge and disposition were collected. Univariate analysis was performed to find predictors of ICU admission and to assess the impact of ICU admission on the short-term outcomes. P values < 0.05 were considered significant. RESULTS: Among 26 patients, 16 were admitted to the ICU. Age < 60 years and the presentation with encephalitis and acute flaccid paralysis predicted ICU admission (P = 0.044 and 0.0007). Among patients requiring ICU admission, four died and no one was discharged home. ICU admission predicted longer hospital stay (P = 0.021), inhospital death (P = 0.034), survival with inability to walk independently (P = 0.0094), and discharge disposition other than home (P = 0.007). In the ICU group, older age was associated with longer hospital stay (P = 0.0001) and inhospital death (P = 0.035). CONCLUSION: WNND requiring ICU care has a high morbidity and mortality, especially among older patients. Survivors are highly disabled at discharge, but many improve over time. Therefore, more data on the long-term prognosis of survivors are needed to guide the goals of care in the acute setting.

West Nile Virus Lineage 2 in Horses and Other Animals with Neurologic Disease, South Africa, 2008-2015.

During 2008-2015 in South Africa, we conducted West Nile virus surveillance in 1,407 animals with neurologic disease and identified mostly lineage 2 cases in horses (7.4%, 79/1,069), livestock (1.5%, 2/132), and wildlife (0.5%, 1/206); 35% were fatal. Geographic correlation of horse cases with seropositive veterinarians suggests disease in horses can predict risk in humans.

A theoretical model of the West Nile Virus survival data.

BACKGROUND: In this work, we develop a theoretical model that explains the survival data in West Nile Virus infection. RESULTS: We build a model based on three cell populations in an infected host; the collateral damage cells, the infected dividing cell, and the infected non-dividing cells. T cell-mediated lysis of each of these populations is dependent on the level of MHC-1 upregulation, which is different in the two infected cell populations, interferon-gamma and free virus levels. CONCLUSIONS: The model allows us to plot a measure of host health versus time for a range of initial viral doses and from that infer the dependence of minimal health versus viral dose. This inferred functional relationship between the minimal host health and viral dose is very similar to the data that has been collected for WNV survival curves under experimental conditions.

Epidemiologic and clinical parameters of West Nile virus infections in humans: a scoping review.

BACKGROUND: Clinical syndromes associated with West Nile virus (WNV) infection range from fever to neuroinvasive disease. Understanding WNV epidemiology and disease history is important for guiding patient care and healthcare decision-making. The objective of this review was to characterize the existing body of peer-reviewed and surveillance literature on WNV syndromes and summarize epidemiologic and clinical parameters. METHODS: We followed scoping review methodology described by the Joanna Briggs Institute. Terms related to WNV epidemiology, hospitalization, and surveillance were searched in four bibliographic databases (MEDLINE, EMBASE, Scopus, and CINAHL) for literature published from January 1999 to December 2015. RESULTS: In total, 2334 non-duplicated titles and abstracts were screened; 92 primary studies were included in the review. Publications included one randomized controlled trial and 91 observational studies. Sample sizes ranged from under 25 patients (n = 19) to over 400 patients (n = 28). Eight studies were from Canada, seven from Israel, and the remaining (n = 77) from the United States. N = 17 studies were classified as outbreak case investigations following epidemics; n = 37 with results of regional/national surveillance and monitoring programs. Mean patient ages were > 40 years old; three studies (3%) focused on the pediatric population. Patients with encephalitis fared worse than patients with meningitis and fever, considering hospitalization, length of stay, discharge, recovery, and case-fatality. Several studies examined risk factors; however, age was the only risk factor for neuroinvasive disease/death consistently identified. Overall, patients with acute flaccid paralysis or encephalitis fared worse than patients with meningitis and West Nile fever in terms of hospitalization and mortality. Among the included studies, proportion hospitalized, length of stay, proportion discharged home and case-fatality ranged considerably. CONCLUSION: Our review highlights the heterogeneity among reporting clinical WNV syndromes and epidemiologic parameters of WNV-related illness. Presently, there is potential for further synthesis of the risk factors of WNV-illness and mortality; undertaking further analysis through a systematic review and meta-analysis may benefit our understanding of risk factors for emerging mosquito-borne diseases. Future research on the burden of WNV can build on existing evidence summarized in this review, not only to support our understanding of endemic WNV, but also to strengthen research on emerging arboviruses with similar clinical manifestations.

West Nile Review: 15 Years of Human Disease in South Dakota, 2002-2016.

During the past 15 years, 2002-2016, West Nile virus (WNV) has emerged in South Dakota resulting in 509 neuroinvasive disease (NID) cases, 745 hospitalizations and 38 deaths. Culex tarsalis is the state's primary mosquito vector. South Dakota's average annual incidence of WNV-NID and death rate are the highest of any state in the U.S. WNV cases have been reported from all counties in the state. All age groups have been infected with cases peaking in the 40-44 year age group, but deaths peaking in cases 70 years and older. Although South Dakota's WNV season lasts six months, May-October, the first week of August has been the peak week of WNV disease onsets. West Nile is now enzootic in South Dakota. Every citizen, local mosquito control programs, medical and public health infrastructures must continue to prevent and respond to annual WNV outbreaks, and prepare for the next arboviral disease to emerge.

Susceptibility of Carrion Crows to Experimental Infection with Lineage 1 and 2 West Nile Viruses.

West Nile virus (WNV) outbreaks in North America have been characterized by substantial die-offs of American crows (Corvus brachyrhynchos). In contrast, a low incidence of bird deaths has been observed during WNV epidemic activity in Europe. To examine the susceptibility of the western European counterpart of American crows, we inoculated carrion crows (Corvus corone) with WNV strains isolated in Greece (Gr-10), Italy (FIN and Ita09), and Hungary (578/10) and with the highly virulent North American genotype strain (NY99). We also inoculated American crows with a selection of these strains to examine the strains' virulence in a highly susceptible bird species. Infection with all strains, except WNV FIN, resulted in high rates of death and high-level viremia in both bird species and virus dissemination to several organs. These results suggest that carrion crows are highly susceptible to WNV and may potentially be useful as part of dead bird surveillance for early warning of WNV activity in Europe.

Seasonality and survival associated with three outbreak seasons of West Nile virus disease in Oklahoma--2003, 2007, and 2012.

West Nile virus (WNV) activity has fluctuated in the south-central United States since its introduction. Seasonal outbreaks are common, with three in Oklahoma during 2003, 2007, and 2012. Morbidity and mortality rates vary during each outbreak. Long-term neurologic sequelae in association with West Nile virus disease (WNVD) are well-described, but limited information is available about delayed mortality among acute WNV infection survivors. A retrospective cohort analysis of all confirmed and probable WNVD cases reported to the Oklahoma State Department of Health (OSDH) during 2003, 2007, and 2012 was performed. OSDH surveillance data and mortality data from Oklahoma's vital statistics database were used to construct a descriptive epidemiologic analysis of the geography, temporality, severity, and associated mortality for each outbreak season. A Kaplan-Meier survival curve and standardized mortality ratios (SMRs) were calculated to measure survival of the 2003 and 2007 WNVD cohorts. Seventy-nine cases during 2003, 107 cases during 2007, and 180 cases during 2012 met inclusion criteria. Median ages of the 2003, 2007, and 2012 cohorts were 48, 58, and 59 years, respectively; race, sex, and symptom information were not substantially different. Each outbreak season had a different severity, temporality, and geography. Age- and sex-adjusted SMRs for the combined 2003 and 2007 cohorts censored at 5 years was 0.9 (95% confidence interval 0.51-1.75); no substantial difference was observed between the survival curves. Although similar patterns of long-term mortality were evident on the survival curves, SMRs did not demonstrate increased 5-year cumulative risk for death for patients surviving acute WNV infection.

Prospective investigation of the impact of West Nile Virus infections in renal diseases.

An increased incidence of chronic kidney disease (CKD) after West Nile Virus (WNV) infections has been suggested but the association of WNV infections with renal damage remain inconclusive. This study was undertaken to characterize WNV infections in individuals with acute kidney injury (AKI) and CKD, and to evaluate hemodialysis as a probable transmission route. A total of 463 plasma and urine samples were collected from 45 AKI and 77 CKD patients. Nested and real-time polymerase chain reaction (PCR) assays were employed for viral RNA detection. Specific immunoglobulins were investigated via immunofluorescence and plaque reduction neutralization assays. Consecutive pre and post-dialysis samples were evaluated in CKD cases. WNV RNA and specific immunoglobulins were detected in 7 (5.7%) and 5 (4.1%) individuals, respectively. The AKI patients with WNV RNA in blood and urine had underlying diseases requiring immunosuppressive therapy and demonstrated moderate to high viral loads. No clinical symptom related to WNV infection were observed in CKD cases with detectable viral nucleic acids. All WNV sequences were characterized as lineage 1 clade 1a and several amino acid substitutions with unknown impact were noted. Detailed epidemiologic investigation of WNV RNA positive CKD cases revealed probable vector-borne virus exposure, without the evidence for transmission via hemodialysis.

The efficacy of inactivated West Nile vaccine (WN-VAX) in mice and monkeys.

BACKGROUND: West Nile virus (WNV) belonging to the genus Flavivirus of the family Flaviviridae causes nervous system disorder in humans, horses and birds. Licensed WNV vaccines are available for use in horses but not for humans. We previously developed an inactivated West Nile virus vaccine (WN-VAX) using a seed virus from West Nile virus (WNV NY99) that was originally isolated in New York City in 1999. In this study, we report the immunogenicity of WN-VAX in both mice and non-human primates. FINDINGS: The WN-VAX immunized mice showed protection against lethal infection with WNV NY99. The challenge test performed on mice passively immunized with serum from other mice that were previously immunized with WN-VAX confirmed that the neutralizing antibody titers of more than 1log10 protected the passively immunized mice from WNV lethal infection. Furthermore, monkeys (Macaca fascicularis) immunized three times with 2.5 µg, 5 µg or 10 µg/dose of WN-VAX exhibited neutralizing antibodies in their sera with titers of more than 2log10 after the second immunization. CONCLUSIONS: The WN-VAX was protective in mice both by active and passive immunizations and was immunogenic in monkeys. These results suggest that the vaccine developed in this study may be a potential WNV vaccine candidate for human use.

Do citation trends reflect epidemiologic patterns? Assessing MRSA, emerging and re-emerging pathogens, 1963-2014.

BACKGROUND: A rapid rise in PubMed citations on methicillin-resistant Staphylococcus aureus (MRSA) occurred after 2000, but the relationship of trends in citation to epidemiologic trends for infectious disease is not known. METHODS: We queried PubMed(R), for citations to the following: MRSA, HIV/AIDS, Staphylococcus aureus, severe acute respiratory syndrome, Lyme disease, avian influenza, West Nile virus, Chikungunya, Ebola virus and Middle Eastern respiratory syndrome. Incidence or mortality data were tabulated. RESULTS: We identified 560,225 citations in 1963-2014. There were two distinct qualitative citation patterns. Type I pathogens showed a decade of initial exponential growth. Type II pathogens showed a sudden spike in citations in a year or two, followed by a relative decline. MRSA most closely resembled a Type I pathogen. CONCLUSIONS: The Type I pattern pathogens had varied trends in disease incidence in the years following the exponential growth and subsequent decline in the number of citations. Their differing epidemiologic patterns did not correlate with their pattern of citations. We conclude that citation trends on MRSA cannot be used to determine past epidemiologic trends and also that the citation trend for MRSA in 1995-2011 most closely resembled that for HIV in 1981-1998.

Diagnostic exercise: High mortality in a flock of chukar partridge chicks (Alectoris chukar) in California.

Mortality of 20% of a flock of 1000 chukar partridge chicks occurred over a 6-week period in Northern California from August to September 2012. Affected birds were 2 to 42 days old and died without premonitory clinical signs or after showing ruffled feathers and anorexia for 24 to 72 hours. Three carcasses were submitted for necropsy, 2 birds had hemorrhagic tracheitis grossly, and all 3 had lymphoplasmacytic and histiocytic myocarditis with myocardial necrosis microscopically. The differential diagnoses and the diagnostic workup to achieve a final diagnosis are discussed. The detection of 2 zoonotic agents in these birds makes this an interesting case from a public health perspective.