West Nile Virus Strains Research Articles

Continuous and Dynamic Circulation of West Nile Virus in Mosquito Populations in Bucharest Area, Romania, 2017-2023.

West Nile virus (WNV) is a mosquito-borne pathogen with a worldwide distribution. Climate change and human activities have driven the expansion of WNV into new territories in Europe during the last two decades. Romania is endemic for WNV circulation since at least 1996 when the presence of lineage 1 was documented during an unprecedented outbreak. Lineage 2 was first identified in this country during a second significant human outbreak in 2010. Its continuous circulation is marked by clade replacement, and even co-circulation of different strains of the same clade was observed until 2016. The present study aims to fill the information gap regarding the WNV strains that were circulating in Romania between 2017 and 2023, providing chiefly viral sequences obtained from mosquito samples collected in the Bucharest metropolitan area, complemented by human and bird viral sequences. WNV was detected mainly in Culex pipiens mosquitoes, the vectors of this virus in the region, but also in the invasive Aedes albopictus mosquito species. Lineage 2 WNV was identified in mosquito samples collected between 2017 and 2023, as well as in human sera from patients in southern and central Romania during the outbreaks of 2017 and 2018. Both 2a and 2b sub-lineages were identified, with evidence of multiple clusters and sub-clusters within sub-lineage 2a, highlighting the complex and dynamic circulation of WNV in Romania, as a consequence of distinct introduction events from neighboring countries followed by in situ evolution.

Eilat virus (EILV) causes superinfection exclusion against West Nile virus (WNV) in a strain-specific manner in Culex tarsalis mosquitoes.

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the USA. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vector Culex tarsalis is also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells and C. tarsalis mosquitoes. The titres of both WNV strains - WN02-1956 and NY99 - were suppressed by EILV in C6/36 cells as early as 48-72 h post-superinfection at both m.o.i. values tested in our study. The titres of WN02-1956 at both m.o.i. values remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titres. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In C. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titres at 3 days post-superinfection, but this effect disappeared at 7 days post-superinfection. In contrast, WN02-1956 infection titres were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, in C. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains.

Sensitivity Characteristics of Genotype 2 West Nile Virus Strains to Anti-Viral Drugs

Sensitivity Characteristics of Genotype 2 West Nile Virus Strains to Anti-Viral Drugs

Next-generation sequencing survey of acute febrile illness in Senegal (2020-2022).

Acute febrile illnesses (AFI) in developing tropical and sub-tropical nations are challenging to diagnose due to the numerous causes and non-specific symptoms. The proliferation of rapid diagnostic testing and successful control campaigns against malaria have revealed that non-Plasmodium pathogens still contribute significantly to AFI burden. Thus, a more complete understanding of local trends and potential causes is important for selecting the correct treatment course, which in turn will reduce morbidity and mortality. Next-generation sequencing (NGS) in a laboratory setting can be used to identify known and novel pathogens in individuals with AFI. In this study, plasma was collected from 228 febrile patients tested negative for malaria at clinics across Senegal from 2020-2022. Total nucleic acids were extracted and converted to metagenomic NGS libraries. To identify viral pathogens, especially those present at low concentration, an aliquot of each library was processed with a viral enrichment panel and sequenced. Corresponding metagenomic libraries were also sequenced to identify non-viral pathogens. Sequencing reads for pathogens with a possible link to febrile illness were identified in 51/228 specimens, including (but not limited to): Borrelia crocidurae (N = 7), West Nile virus (N = 3), Rickettsia felis (N = 2), Bartonella quintana (N = 1), human herpesvirus 8 (N = 1), and Saffold virus (N = 1). Reads corresponding to Plasmodium falciparum were detected in 19 specimens, though their presence in the cohort was likely due to user error of rapid diagnostic testing or incorrect specimen segregation at the clinics. Mosquito-borne pathogens were typically detected just after the conclusion of the rainy season, while tick-borne pathogens were mostly detected before the rainy season. The three West Nile virus strains were phylogenetically characterized and shown to be related to both European and North American clades. Surveys such as this will increase the understanding of the potential causes of non-malarial AFI, which may help inform diagnostic and treatment options for clinicians who provide care to patients in Senegal.

Different viral genes modulate virulence in model mammal hosts and Culex pipiens vector competence in Mediterranean basin lineage 1 West Nile virus strains.

West Nile virus (WNV) is a single-stranded positive-sense RNA virus (+ssRNA) belonging to the genus Orthoflavivirus. Its enzootic cycle involves mosquito vectors, mainly Culex, and wild birds as reservoir hosts, while mammals, such as humans and equids, are incidental dead-end hosts. It was first discovered in 1934 in Uganda, and since 1999 has been responsible for frequent outbreaks in humans, horses and wild birds, mostly in America and in Europe. Virus spread, as well as outbreak severity, can be influenced by many ecological factors, such as reservoir host availability, biodiversity, movements and competence, mosquito abundance, distribution and vector competence, by environmental factors such as temperature, land use and precipitation, as well as by virus genetic factors influencing virulence or transmission. Former studies have investigated WNV factors of virulence, but few have compared viral genetic determinants of pathogenicity in different host species, and even fewer have considered the genetic drivers of virus invasiveness and excretion in Culex vector. In this study, we characterized WNV genetic factors implicated in the difference in virulence observed in two lineage 1 WNV strains from the Mediterranean Basin, the first isolated during a significant outbreak reported in Israel in 1998, and the second from a milder outbreak in Italy in 2008. We used an innovative and powerful reverse genetic tool, e.g., ISA (infectious subgenomic amplicons) to generate chimeras between Israel 1998 and Italy 2008 strains, focusing on non-structural (NS) proteins and the 3'UTR non-coding region. We analyzed the replication of these chimeras and their progenitors in mammals, in BALB/cByJ mice, and vector competence in Culex (Cx.) pipiens mosquitoes. Results obtained in BALB/cByJ mice suggest a role of the NS2B/NS3/NS4B/NS5 genomic region in viral attenuation in mammals, while NS4B/NS5/3'UTR regions are important in Cx. pipiens infection and possibly in vector competence.

Abrupt Increase in Detection of Locally Acquired West-Nile-Virus-Lineage-2-Mediated Neuroinvasive Disease in a Previously Non-Endemic Area of Southern Italy (2023).

West Nile virus (WNV) is a public health concern in Europe. Rising temperatures and the migration of potential vectors promote the spread of viruses to previously unaffected areas. In 2023, the Apulia region of Southern Italy experienced an unexpected increase in West Nile neuroinvasive disease (WNND); no such cases had been reported in the previous 10 years. Overall, eight autochthonous cases of WNV infection were identified between July and October 2023, six of which were WNND. All cases were male (median age, 73 years). Two of the cases were blood donors. All WNND cases were hospitalized and all recovered within a few weeks. Surveillance data showed that, in the Apulia region, WNV Lineage 2 was detected in humans, mosquitoes, and horses. Based on the number of WNND cases reported, we can assume that a high number of infections occurred during the summer period. Changes in the climate in the region over recent years could be considered among the main drivers of the rapid increase in WNV infections. Therefore, integrated surveillance should be strengthened to avoid the potential massive spread of WNV in Southern Italy. Moreover, the implementation of whole-genome sequencing of WNV strains, as well as seroepidemiological studies in the area, will facilitate a better understanding of circulation dynamics.

Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics

The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, in-vitro infection experiments in mammalian (Vero B4), goose (GN-R) and mosquito cell lines (C6/36, CT) were performed to investigate potential effects of co-infections in vectors and vertebrate hosts. The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate.

West Nile virus infections in Hungary: Epidemiological update and phylogenetic analysis of the Hungarian virus strains between 2015 and 2022.

Following the introduction of the West Nile virus (WNV) into Hungary in 2004, it has shortly become one of the most important human arbovirus infections, with a gradually increasing number of cases. The study aimed to summarize the current epidemiological situation in Hungary and sequence the WNV PCR-positive clinical specimens and virus isolates by next-generation whole genome sequencing (NGS) to obtain a detailed phylogenetic analysis of the circulating virus strains. Whole blood and urine samples from confirmed WNV-infected patients and WNV isolates were investigated by reverse transcription PCR assays. Genome sequencing was carried out by Sanger-method, followed by NGS on the Illumina MiSeq platform. Altogether 499 human infections were diagnosed between 2004 and 2022. A particularly remarkable increase in human WNV infections was observed in 2018, while the number of reported cases significantly decreased during the COVID-19 pandemic. Between 2015 and 2022, 15WNV isolates, and 10 PCR-positive clinical specimens were investigated by NGS. Phylogenetic analysis revealed that the major European WNV lineage 2 clades, namely the Eastern European (or Russian) and the Central European (or Hungarian) clades, are presented in Hungary. Strains of the Balkan and other European clusters within the Central European clade are co-circulating in the country, following a characteristic geographical distribution. In Hungary, the presence and co-circulation of multiple lineage 2 WNV strains could be identified in the last few years. Therefore, in light of the 2018WNV outbreak, sequence-based typing of the currently circulating strains could highly support outbreak investigations.

Experimental infections in red-legged partridges reveal differences in host competence between West Nile and Usutu virus strains from Southern Spain.

West Nile virus (WNV) and Usutu virus (USUV) are emerging zoonotic arboviruses sharing the same life cycle with mosquitoes as vectors and wild birds as reservoir hosts. The main objective of this study was to characterize the pathogenicity and course of infection of two viral strains (WNV/08 and USUV/09) co-circulating in Southern Spain in a natural host, the red-legged partridge (Alectoris rufa), and to compare the results with those obtained with the reference strain WNV/NY99. WNV inoculated birds were monitored for clinical and analytical parameters (viral load, viremia, and antibodies) for 15 days post-inoculation. Partridges inoculated with WNV/NY99 and WNV/08 strains showed clinical signs such as weight loss, ruffled feathers, and lethargy, which were not observed in USUV/09-inoculated individuals. Although statistically significant differences in mortality were not observed, partridges inoculated with WNV strains developed significantly higher viremia and viral loads in blood than those inoculated with USUV. In addition, the viral genome was detected in organs and feathers of WNV-inoculated partridges, while it was almost undetectable in USUV-inoculated ones. These experimental results indicate that red-legged partridges are susceptible to the assayed Spanish WNV with pathogenicity similar to that observed for the prototype WNV/NY99 strain. By contrast, the USUV/09 strain was not pathogenic for this bird species and elicited extremely low viremia levels, demonstrating that red-legged partridges are not a competent host for the transmission of this USUV strain.

Gone (and spread) with the birds: Can chorotype analysis highlight the spread of West Nile virus within the Afro-Palaearctic flyway?

West Nile virus (WNV) is a globally significant vector-borne disease that is primarily transmitted between birds and mosquitoes. Recently, there has been an increase in WNV in southern Europe, with new cases reported in more northern regions. Bird migration plays a crucial role in the introduction of WNV in distant areas. To better understand and address this complex issue, we adopted a One Health approach, integrating clinical, zoological, and ecological data. We analyzed the role of migratory birds in the Palaearctic-African region in the spread of WNV across Africa and Europe. We categorized bird species into breeding and wintering chorotypes based on their distribution during the breeding season in the Western Palaearctic and the wintering season in the Afrotropical region, respectively. By linking these chorotypes to the occurrence of WNV outbreaks in both continents throughout the annual bird migration cycle, we investigated the relationship between migratory patterns and virus spread. We demonstrate that WNV-risk areas are interconnected through the migration of birds. We identified a total of 61 species that potentially contribute to the intercontinental spread of the virus or its variants, as well as pinpointed high-risk areas for future outbreaks. This interdisciplinary approach, which considers the interconnectedness of animals, humans, and ecosystems, represents a pioneering effort to establish connections between zoonotic diseases across continents. The findings of our study can aid in anticipating the arrival of new WNV strains and predicting the occurrence of other re-emerging diseases. By incorporating various disciplines, we can enhance our understanding of these complex dynamics and provide valuable insights for proactive and comprehensive disease management strategies.

Higher replication potential of West Nile virus governs apoptosis induction in human neuroblastoma cells.

Theextent of neuronal cell damage caused by West Nile virus (WNV) infection governs the disease severity ranging from mild, febrile illness to fatal encephalitis. Availability of naturally occurring genetic variants is helpful to study viral factors governing differential pathogenesis. During WNV infection, apoptosis serves as a virulence determinant positively contributing to viral pathogenesis. We investigated the levels of apoptosis induced by a low neurovirulent WNV lineage 5 strain 804994 and a high neurovirulent lineage 1 strain 68856 in human neuroblastoma cells, IMR-32. Our investigations clearly show the correlation between higher multiplication capacities of 68856 with higher levels of cytopathology induced by apoptosis. We observed activation of both the extrinsic and intrinsic apoptotic pathways during WNV infection. Infection with higher neurovirulent strain resulted in higher upregulation of pro-apoptotic proteins including death receptors (DR), adaptor protein, BH3-only regulatory proteins and higher cleavage of initiator caspases of both pathways. These results suggest that the virulence of a WNV strain may correlate with its higher replication fitness and ability to cause more cellular damage.

Simultaneous Coinfections with West Nile Virus and Usutu Virus in Culex pipiens and Aedes vexans Mosquitoes

The mosquito-borne zoonotic flaviviruses West Nile virus (WNV) and Usutu virus (USUV) are endemic in many European countries and emerged in Germany in recent years. Due to the increasing overlap of their distribution areas and their similar epidemiology, coinfections of WNV and USUV are possible. Indeed, coinfections in vertebrate hosts as a rare event have already been reported from some countries including Germany. However, it is largely unknown whether and to what extent coinfections could affect the vector competence of mosquitoes for WNV and USUV. For this purpose, the mosquito species Culex pipiens biotype pipiens, Culex pipiens biotype molestus, and Aedes vexans were orally infected in mono- and simultaneous coinfections with German strains of WNV and USUV. Mosquitoes were incubated for 14 days at 26°C, 85% relative humidity, and a 16 : 8 light-dark photocycle, before they were dissected and forced to salivate. The results showed a decrease in USUV susceptibility in Culex pipiens biotype pipiens, an increase in USUV susceptibility in Aedes vexans, and no obvious interaction between both viruses in Culex pipiens biotype molestus. Vector competence for WNV appeared to be unaffected by a simultaneous occurrence of USUV in all tested mosquito species. Coinfections with both viruses were only found in Culex mosquitoes, and cotransmission of WNV and USUV was observed in Culex pipiens biotype molestus. Overall, our results show that viral interactions between WNV and USUV vary between mosquito species, and that the interaction mainly occurs during infection and replication in the mosquito midgut. The results of this study confirm that to fully understand the interaction between WNV and USUV, studies with various mosquito species are necessary. In addition, we found that even mosquito species with a low susceptibility to both viruses, such as Ae. vexans, can play a role in their transmission in areas with cocirculation.

Genomic Analysis of West Nile Virus Lineage 1 Detected in Mosquitoes during the 2020-2021 Outbreaks in Andalusia, Spain.

Emerging infectious diseases are one of the most important global health challenges because of their impact on human and animal health. The vector-borne West Nile virus (WNV) is transmitted between birds by mosquitos, but it can also infect humans and horses causing disease. The local circulation of WNV in Spain has been known for decades, and since 2010, there have been regular outbreaks in horses, although only six cases were reported in humans until 2019. In 2020, Spain experienced a major outbreak with 77 human cases, which was followed by 6 additional cases in 2021, most of them in the Andalusian region (southern Spain). This study aimed to characterize the genomes of the WNV circulating in wild-trapped mosquitoes during 2020 and 2021 in Andalusia. We sequenced the WNV consensus genome from two mosquito pools and carried out the phylogenetic analyses. We also compared the obtained genomes with those sequenced from human samples obtained during the outbreak and the genomes obtained previously in Spain from birds (2007 and 2017), mosquitoes (2008) and horses (2010) to better understand the eco-epidemiology of WNV in Spain. As expected, the WNV genomes recovered from mosquito pools in 2020 were closely related to those recovered from humans of the same outbreak. In addition, the strain of WNV circulating in 2021 was highly related to the WNV strain that caused the 2020 outbreak, suggesting that WNV is overwintering in the area. Consequently, future outbreaks of the same strain may occur in in the future.

Serological evidence of circulation of West Nile virus in equids in Algerian eastern drylands and its epidemiological risk factors

In order to determine the prevalence of equine infectious anemia virus (EIAV), Usutu virus (USUV), and West Nile virus (WNV) in eastern Algerian drylands, 340 sera from distinct equids have been collected from 2015 to 2017. Serological analysis for the presence of antibodies against EIAV and flaviviruses was performed using commercially available ELISAs. Sera detected positive, doubtful, or negative close to the doubtful threshold in flavivirus ELISA were tested by the virus neutralization test (VNT), using WNV and USUV strains. The prevalence of WNV antibodies with ELISA was 11.47% (39/340) against 13.53% (46/340) by WNV VNT. EIAV antibodies were not detected in any samples. WNV seroprevalence varies with species, breed and location of horses. Only, one equid was positive for both WNV and USUV neutralizing antibodies. This is the first screening on equids sera of EIAV and USUV in Algeria. This study indicate that WNV and possibly USUV have circulated/are circulating in the Algerian equine population, unlike EIAV does not seem to be present.

Re-Introduction of West Nile Virus Lineage 1 in Senegal from Europe and Subsequent Circulation in Human and Mosquito Populations between 2012 and 2021.

West Nile virus (WNV) is a virus of the Japanese encephalitis antigenic complex and belongs to the family Flaviviridae of the genus flavivirus. The virus can cause infection in humans which in most cases is asymptomatic, however symptomatic cases exist and the disease can be severe causing encephalitis and meningoencephalitis. The virus is maintained in an enzootic cycle involving mosquitoes and birds, humans and other mammals such as horses can be accidental hosts. A mosquito-based arbovirus surveillance system and the sentinel syndromic surveillance network (4S) have been in place since 1988 and 2015 respectively, to better understand the transmission dynamics of arboviruses including WNV in Senegal. Arthropod and human samples have been collected from the field and analysed at Institut Pasteur de Dakar using different methods including RT-PCR, ELISA, plaque reduction neutralization test and viral isolation. RT-PCR positive samples have been analysed by Next Generation Sequencing. From 2012 to 2021, 7912 samples have been analysed and WNV positive cases have been detected, 20 human cases (19 IgM and 1 RT-PCR positive cases) and 41 mosquito pools. Phylogenetic analyzes of the sequences of complete genomes obtained showed the circulation of lineage 1a, with all these recent strains from Senegal identical to each other and very close to strains isolated from horse in France in 2015, Italy and Spain. Our data showed lineage 1a endemicity in Senegal as previously described, with circulation of WNV in humans and mosquitoes. Phylogenetic analyzes carried out with the genome sequences obtained also revealed exchanges of WNV strains between Europe and Senegal which could be possible via migratory birds. The surveillance systems that have enabled the detection of WNV in humans and arthropods should be extended to animals in a one-health approach to better prepare for global health threats.

West Nile Virus Lineage 2 Overwintering in Italy.

In January 2022, West Nile virus (WNV) lineage 2 (L2) was detected in an adult female goshawk rescued near Perugia in the region of Umbria (Italy). The animal showed neurological symptoms and died 15 days after its recovery in a wildlife rescue center. This was the second case of WNV infection recorded in birds in the Umbria region during the cold season, when mosquitoes, the main WNV vectors, are usually not active. According to the National Surveillance Plan, the Umbria region is included amongst the WNV low-risk areas. The necropsy evidenced generalized pallor of the mucous membranes, mild splenomegaly, and cerebral edema. WNV L2 was detected in the brain, heart, kidney, and spleen homogenate using specific RT-PCR. Subsequently, the extracted viral RNA was sequenced. A Bayesian phylogenetic analysis performed through a maximum-likelihood tree showed that the genome sequence clustered with the Italian strains within the European WNV strains among the central-southern European WNV L2 clade. These results, on the one hand, confirmed that the WNV L2 strains circulating in Italy are genetically stable and, on the other hand, evidenced a continuous WNV circulation in Italy throughout the year. In this report case, a bird-to-bird WNV transmission was suggested to support the virus overwintering. The potential transmission through the oral route in a predatory bird may explain the relatively rapid spread of WNV, as well as other flaviviruses characterized by similar transmission patterns. However, rodent-to-bird transmission or mosquito-to-bird transmission cannot be excluded, and further research is needed to better understand WNV transmission routes during the winter season in Italy.

Epidemiology of Mosquito-Borne Viruses in Egypt: A Systematic Review.

There are at least five common mosquito-borne viruses (MBVs) recorded in Egypt, including dengue virus (DENV), Rift Valley fever virus (RVFV), West Nile virus (WNV), Chikungunya virus, and Sindbis virus. Unexpected outbreaks caused by MBVs reflect the deficiencies of the MBV surveillance system in Egypt. This systematic review characterized the epidemiology of MBV prevalence in Egypt. Human, animal, and vector prevalence studies on MBVs in Egypt were retrieved from Web of Science, PubMed, and Bing Scholar, and 33 eligible studies were included for further analyses. The monophyletic characterization of the RVFV and WNV strains found in Egypt, which spans about half a century, suggests that both RVFV and WNV are widely transmitted in this nation. Moreover, the seropositive rates of DENV and WNV in hosts were on the rise in recent years, and spillover events of DENV and WNV to other countries from Egypt have been recorded. The common drawback for surveillance of MBVs in Egypt is the lack of seroprevalence studies on MBVs, especially in this century. It is necessary to evaluate endemic transmission risk, establish an early warning system for MBVs, and develop a sound joint system for medical care and public health for managing MBVs in Egypt.

Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia

The Kunjin strain of West Nile virus (WNVKUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNVKUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNVKUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNVKUN and MVEV transmission by FTATM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNVKUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTATM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTATM card system as a sensitive and accurate method to monitor the transmission of WNVKUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a “One Health” issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTATM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities.

Detection of West Nile Virus in Overwintering Mosquitoes in the Volgograd Region

Introduction: West Nile fever is a zoonotic, vector-borne viral infection caused by West Nile virus. The possibility of persistence of West Nile virus in overwintering mosquitoes in regions with a temperate climate is of great importance for understanding the mechanisms of pathogen circulation. Objective: To detect West Nile virus in mosquitoes during the inter-epizootic period in the Volgograd Region. Materials and methods: In 2013–2021, we collected overwintering mosquitoes in different locations of the Volgograd Region using a battery-powered aspirator with a Krishtal’s trap to detect West Nile virus RNA in them using a real-time reverse transcription polymerase chain reaction. An isolate (WNV Volgograd_o16/19) was obtained from sample o16/19 (Cx. pipiens, collected on April 2, 2019) with detected West Nile virus RNA using a VERO cell culture. After that, total RNA was isolated from the filtered cell supernatant of that isolate. Metagenomic sequencing of the sample was performed using a high-throughput Illumina MiSeq sequencer, Illumina Inc., USA. Results: In total, we collected 4,070 mosquitoes in wintering shelters and tested 157 pools of the insects for West Nile virus RNA. The latter was detected in two pools of Culex pipiens and in one pool of Anopheles maculipennis complex. The phylogenetic analysis showed that the WNV Volgograd_о16/19 strain isolated from the pool of wintering mosquitoes belonged to lineage 2 of West Nile virus. We also established its belonging to the monophyletic clade of West Nile virus strains isolated in the Volgograd, Astrakhan, and Rostov regions in the years 2007 and 2018–2020. Conclusions: We were first to detect West Nile virus in overwintering mosquitoes in the Volgograd Region. Our findings confirm the hypothesis that lineage 2 strains of encephalitic West Nile virus persist in mosquitoes during the inter-epizootic period and can be transmitted from mosquito to bird in springtime as one of the mechanisms of forming autochthonous foci in WNV endemic areas of the Russian Federation in the absence of the annual import of this infection.

Emergence of West Nile Virus Lineage-2 in Resident Corvids in Istanbul, Turkey.

West Nile fever is a vector-borne viral disease affecting animals and humans causing significant health and economic problems globally. This study was aimed at investigating circulating West Nile virus (WNV) strains in free-ranging corvids in Istanbul, Turkey. Brain, liver, and kidney were collected from corvids (n = 34) between June 2019 and April 2020 and analyzed for the presence of WNV-specific RNA by quantitative RT-PCR. In addition, histopathologic and immunohistochemical examinations were also performed. Samples found to be positive by qRT-PCR were partially sequenced. WNV-specific RNA was detected in 8 of 34 corvids analyzed, which included 7 hooded crows (Corvus cornix) and 1 Eurasian magpie (Pica pica). Phylogenetic analysis based on partial WNV sequences from the 8 WNV-positive corvids identified in this study revealed that all sequences clustered within the WNV lineage-2; they were at least 97% homologues to WNV lineage-2 sequences from Slovakia, Italy, Czechia, Hungary, Senegal, Austria, Serbia, Greece, Bulgaria, and Germany. WNV sequences showed a divergence (87.94-94.46%) from sequences reported from Romania, Central African Republic, South Africa, Madagascar, Israel, and Cyprus, which clustered into a different clade of WNV lineage-2. Common histopathologic findings of WNV-positive corvids included lymphoplasmacytic hepatitis, myocarditis, and splenitis. The liver and heart were found to be the tissues most consistently positive for WNV-specific antigen by immunohistochemistry, followed by the kidney and brain. This study demonstrates for the first time the existence of WNV virus belonging to the genetic lineage-2 in resident corvids in Istanbul, Turkey. We hypothesize that the WNV strains circulating in Istanbul are possibly the result of a spillover event from Europe. Since WNV is a zoonotic pathogen transmitted by mosquito vectors, the emergence of WNV in Istanbul also poses a risk to humans and other susceptible animals in this densely populated city and needs to be addressed by animal and public health authorities.