Widespread Circulation of Flaviviruses in Horses and Birds in Northeastern Spain (Catalonia) between 2010 and 2019

The surveillance for West Nile virus (WNV) in Catalonia (northeastern Spain) has consistently detected flaviviruses not identified as WNV. With the aim of characterizing the flaviviruses circulating in Catalonia, serum samples from birds and horses collected between 2010 and 2019 and positive by panflavivirus competition ELISA (cELISA) were analyzed by microneutralization test (MNT) against different flaviviruses. A third of the samples tested were inconclusive by MNT, highlighting the limitations of current diagnostic techniques. Our results evidenced the widespread circulation of flaviviruses, in particular WNV, but also Usutu virus (USUV), and suggest that chicken and horses could serve as sentinels for both viruses. In several regions, WNV and USUV overlapped, but no significant geographical aggregation was observed. Bagaza virus (BAGV) was not detected in birds, while positivity to tick-borne encephalitis virus (TBEV) was sporadically detected in horses although no endemic foci were observed. So far, no human infections by WNV, USUV, or TBEV have been reported in Catalonia. However, these zoonotic flaviviruses need to be kept under surveillance, ideally within a One Health framework.     

A Sentinel Serological Study in Selected Zoo Animals to Assess Early Detection of West Nile and Usutu Virus Circulation in Slovenia

Monitoring infectious diseases is a crucial part of preventive veterinary medicine in zoological collections. This zoo environment contains a great variety of animal species that are in contact with wildlife species as a potential source of infectious diseases. Wild birds may be a source of West Nile virus (WNV) and Usutu (USUV) virus, which are both emerging pathogens of rising concern. The aim of this study was to use zoo animals as sentinels for the early detection of WNV and USUV in Slovenia. In total, 501 sera from 261 animals of 84 animal species (including birds, rodents, lagomorphs, carnivores, ungulates, reptiles, equids, and primates) collected for 17 years (2002–2018) were tested for antibodies to WNV and USUV. Antibodies to WNV were detected by indirect immunofluorescence tests in 16 (6.1%) of 261 animals representing 10 species, which were sampled prior to the first active cases of WNV described in 2018 in Slovenia in humans, a horse, and a hooded crow (Corvus cornix). Antibodies to USUV were detected in 14 out of 261 animals tested (5.4%) that were positive prior to the first positive cases of USUV infection in common blackbirds (Turdus merula) in Slovenia. The study illustrates the value of zoological collections as a predictor of future emerging diseases.     

West Nile Virus Vaccination Protects against Usutu Virus Disease in Mice

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that can cause neuroinvasive disease in humans. WNV and USUV circulate in both Africa and Europe and are closely related. Due to antigenic similarity, WNV-specific antibodies and USUV-specific antibodies have the potential to bind heterologous viruses; however, it is unclear whether this interaction may offer protection against infection. To investigate how prior WNV exposure would influence USUV infection, we used an attenuated WNV vaccine that contains the surface proteins of WNV in the backbone of a dengue virus 2 vaccine strain and protects against WNV disease. We hypothesized that vaccination with this attenuated WNV vaccine would protect against USUV infection. Neutralizing responses against WNV and USUV were measured in vitro using sera following vaccination. Sera from vaccinated CD-1 and Ifnar1^−/− mice cross-neutralized with WNV and USUV. All mice were then subsequently challenged with an African or European USUV strain. In CD-1 mice, there was no difference in USUV titers between vaccinated and mock-vaccinated mice. However, in the Ifnar1^−/− model, vaccinated mice had significantly higher survival rates and significantly lower USUV viremia compared to mock-vaccinated mice. Our results indicate that exposure to an attenuated form of WNV protects against severe USUV disease in mice and elicits a neutralizing response to both WNV and USUV. Future studies will investigate the immune mechanisms responsible for the protection against USUV infection induced by WNV vaccination, providing critical insight that will be essential for USUV and WNV vaccine development.     

Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission

The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention.     

Changes in seroprevalence of West Nile virus across Illinois in free-ranging birds from 2001 through 2004

Of the 5,236 birds sampled for antibodies to West Nile virus (WNV) in Illinois from 2001 through 2004, 348 (6.6%) birds were seropositive. Our multiple year surveillance identified several avian species that had particularly high percentages of seropositive individuals. The importance of these species in the enzootic and/or epizootic transmission of WNV is discussed relative to their regional abundance and literature on host competency. The species with the highest exposure rates to WNV differed both temporally and regionally. In general, birds that bred or were born in Illinois were more likely to have antibodies than transient birds. There was also a significant difference in the seroprevalence between adults (12.1%) and juveniles (5.5%), indicating that the acquired antibody response from previous years is a critical concern when interpreting seroprevalence rates in wild-caught birds. The most common hosts for St. Louis encephalitis virus were also the most common hosts for WNV, which strongly supports the role of similar vectors for both flaviviruses. Avian species with high WNV seroprevalence rates tended to be those that bred throughout the year, have open cup nests, and live in close proximity to humans.     

First evidence of simultaneous occurrence of West Nile virus and Usutu virus neuroinvasive disease in humans in Croatia during the 2013 outbreak

Purpose

We report on first evidence of simultaneous occurrence of West Nile virus (WNV) and Usutu virus (USUV) neuroinvasive infection in humans in Croatia during the transmission season 2013.

Methods

From June to December 2013, a total of 95 patients with clinically suspected WNV infection (WNV fever and neuroinvasive disease) were tested for WNV IgM/IgG antibodies using enzyme-linked immunosorbent assay. Twenty-six reactive samples were further tested by virus neutralization test for confirmation.

Results

WNV neuroinvasive infection was confirmed in 20 patients, while in three patients USUV neutralizing antibodies were detected. Cases occurred during the 11-week interval (from 24 July to 07 October 2013). Both WNV and USUV cases were distributed in three north-western Croatian counties. In addition to human cases, recent asymptomatic WNV infection (detection of IgM antibodies) was recorded in 9/3,460 (0.3 %) tested sentinel horses. Infected animals were recorded in two eastern and one north-western county.

Conclusions

Our results indicate co-circulation of WNV and USUV in Croatia. WNV infection could be misdiagnosed with other emerging infectious diseases presenting with neurological symptoms such as USUV infection.     

Avian hosts of West Nile virus in Puerto Rico

West Nile virus (WNV) ecology in neotropical ecosystems is poorly understood, and vertebrate hosts responsible for infecting mosquitoes remain unidentified throughout the Caribbean Basin. After a period of intense WNV transmission among sentinel chickens near Ceiba, Puerto Rico, we measured abundance of resident birds and species-specific prevalence of WNV infection. Taking the product of these measures indicates the relative number of WNV infections by species. Greater Antillean grackle (Quiscalus niger) accounted for the most WNV infections among birds in our 100-km(2) study site. In urban habitats, the house sparrow (Passer domesticus) was frequently infected. Immature birds less than one year of age were more likely to have detectable WNV-reactive antibodies than older birds of the same species.     

First serological evidence of West Nile virus activity in horses in Serbia

West Nile virus (WNV), the most widely distributed flavivirus worldwide, has lately reemerged in Europe, causing worrisome outbreaks in humans and horses. Serological analysis by enzyme-linked immunoassay and plaque reduction neutralization test showed for the first time in Serbia that 12% of 349 horses presented specific neutralizing WNV antibodies, which in one case also cross-neutralized Usutu virus (USUV). This is the first time that anti-USUV high neutralizing antibody titers are reported in horses. All these data indicate that WNV and USUV are circulating in the region and advise on the convenience of implementing surveillance programs.     

Longitudinal studies of West Nile virus infection in avians, Yucatán State, México

Following the introduction of West Nile virus (WNV) into North America in 1999, surveillance for evidence of infection with this virus in migratory and resident birds was established in Yucatán State, México in March 2000. Overall, 8611 birds representing 182 species and 14 orders were captured and assayed for antibodies to WNV. Of these, 5066 (59%) birds were residents and 3545 (41%) birds were migrants. Twenty-one (0.24%) birds exhibited evidence of flavivirus infection. Of these, 8 birds had antibodies to WNV by epitope-blocking enzyme-linked immunosorbent assay. Five (0.06%) birds (gray catbird, brown-crested flycatcher, rose-breasted grosbeak, blue bunting and indigo bunting) were confirmed to have WNV infections by plaque reduction neutralization test. The WNV-infected birds were sampled in December 2002 and January 2003. The brown-crested flycatcher and blue bunting presumably were resident birds; the other WNV seropositive birds were migrants. These data provide evidence of WNV transmission among birds in the Yucatán Peninsula.     

Targeting Host Factors to Treat West Nile and Dengue Viral Infections

West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans.     

Migratory birds and the dispersal of arboviruses in California

Each spring large numbers of neotropical migrants traversing the Pacific flyway pass through the Coachella Valley enroute to northern destinations, providing an opportunity to test the hypothesis that mosquito-borne encephalitis viruses are introduced annually into California by migratory birds. A total of 5,632 sera were collected from 43 species of migrants during spring (April-June), of which 34 (0.61%) comprised of 14 species tested positive by enzyme immunoassay; only 10 were confirmed by plaque reduction neutralization tests (PRNT). In addition, of 1,109 migrants comprised of 76 species that were reported dead by the public and necropsied, 126 (11%) were positive for West Nile virus (WNV) RNA; however, only three (0.7%) of 428 birds tested during the spring were positive. Limited experimental infection studies with WNV showed that Orange-crowned Warblers were highly susceptible and frequently died, whereas most Yellow Warblers survived. Our results indicated that birds entering California rarely exhibited a history of infection and that most birds probably became infected after entering California.     

Role of California (Callipepla californica) and Gambel's (Callipepla gambelii) quail in the ecology of mosquito-borne encephalitis viruses in California, USA

Gambel's and California quail were infected repeatedly whenever western equine encephalomyelitis virus (WEEV), St. Louis encephalitis virus (SLEV), and (WNV) West Nile virus were active during summer in California. The timing of virus appearance and quail infection coincided well with the appearance of chicks in nature, leading us to hypothesize that large coveys containing these non-immune birds could be important in focal virus amplification in rural settings. However, experimental infection studies with chicks, juveniles, and adults of both quail species using sympatric strains of WEEV, SLEV, and WNV indicated that only immature birds were competent hosts for WEEV, producing viremias sufficiently elevated to efficiently infect Culex tarsalis mosquitoes. Quail were less competent hosts for WNV and were incompetent for SLEV. Large populations of quail that frequently are infected with SLEV or WNV, but produce low to moderate viremias, may serve as dead end hosts for these viruses. Due to their abundance and repeated infection, these birds may attenuate virus amplification in rural areas of California and possibly could be one reason why WNV epidemics seem to occur more frequently in urban and periurban than in rural landscapes.     

Usutu Virus: An Emerging Flavivirus in Europe

Usutu virus (USUV) is an African mosquito-borne flavivirus belonging to the Japanese encephalitis virus serocomplex. USUV is closely related to Murray Valley encephalitis virus, Japanese encephalitis virus, and West Nile virus. USUV was discovered in South Africa in 1959. In Europe, the first true demonstration of circulation of USUV was reported in Austria in 2001 with a significant die-off of Eurasian blackbirds. In the subsequent years, USUV expanded to neighboring countries, including Italy, Germany, Spain, Hungary, Switzerland, Poland, England, Czech Republic, Greece, and Belgium, where it caused unusual mortality in birds. In 2009, the first two human cases of USUV infection in Europe have been reported in Italy, causing meningoencephalitis in immunocompromised patients. This review describes USUV in terms of its life cycle, USUV surveillance from Africa to Europe, human cases, its cellular tropism and pathogenesis, its genetic relationship with other flaviviruses, genetic diversity among USUV strains, its diagnosis, and a discussion of the potential future threat to Asian countries.     

Genetic Characteristics of Avian Influenza Virus Isolated from Wild Birds in South Korea, 2019–2020

Wild aquatic birds, a natural reservoir of avian influenza viruses (AIVs), transmit AIVs to poultry farms, causing huge economic losses. Therefore, the prevalence and genetic characteristics of AIVs isolated from wild birds in South Korea from October 2019 to March 2020 were investigated and analyzed. Fresh avian fecal samples (3256) were collected by active monitoring of 11 wild bird habitats. Twenty-eight AIVs were isolated. Seven HA and eight NA subtypes were identified. All AIV hosts were Anseriformes species. The HA cleavage site of 20 representative AIVs was encoded by non-multi-basic amino acid sequences. Phylogenetic analysis of the eight segment genes of the AIVs showed that most genes clustered within the Eurasian lineage. However, the HA gene of H10 viruses and NS gene of four viruses clustered within the American lineage, indicating intercontinental reassortment of AIVs. Representative viruses likely to infect mammals were selected and evaluated for pathogenicity in mice. JB21-58 (H5N3), JB42-93 (H9N2), and JB32-81 (H11N2) were isolated from the lungs, but JB31-69 (H11N9) was not isolated from the lungs until the end of the experiment at 14 dpi. None of infected mice showed clinical sign and histopathological change in the lung. In addition, viral antigens were not detected in lungs of all mice at 14 dpi. These data suggest that LPAIVs derived from wild birds are unlikely to be transmitted to mammals. However, because LPAIVs can reportedly infect mammals, including humans, continuous surveillance and monitoring of AIVs are necessary, despite their low pathogenicity.     

High prevalence of West Nile virus: a continuing risk in acquiring infection from a mosquito bite

The prevalence of West Nile Virus (WNV) was evaluated by diplex real-time RT-PCR assay for the years 2001-2005 in Culex species of mosquitoes, several species of dead birds, and clinically suspected mammals collected in Kansas. The analysis was performed using a TaqMan-based diplex real-time RT-PCR assay targeted against two regions of the WNV genome, envelope glycoprotein gene and 3' untranslated region. The assay aided in the accurate detection of WNV in mosquitoes at high prevalence for the years 2002-2005. Similarly, high incidence of birds that tested positive for WNV was detected in 2002-2004. WNV positives in mammals by the diplex real time RT-PCR assay included horses, squirrels, mules, sheep and a mountain goat. Majority of the equine WNV positives were detected only in the year 2002. Sequence analysis of a segment of the envelope glycoprotein gene from 31 randomly selected WNV positive samples revealed variations in six samples at one or two nucleotide positions. The identity of high levels of WNV positives in Kansas parallels the recent reports on the widespread distribution of the virus in the United States. The continued detection of WNV in the mosquitoes is of significant public health concern and calls for continued surveillance and public health activities.     

Serologic evidence of West Nile Virus infection in birds, Tamaulipas State, México

Following the introduction of West Nile virus (WNV) into North America in 1999, surveillance for WNV in migratory and resident birds was established in Tamaulipas State, northern México in December 2001. Overall, 796 birds representing 70 species and 10 orders were captured and assayed for antibodies to WNV. Nine birds had flavivirus-specific antibodies by epitope-blocking enzyme-linked immunosorbent assay; four were confirmed to have antibody to WNV by plaque reduction neutralization test. The WNV-infected birds were a house wren, mourning dove, verdin and Bewick's wren. The house wren is a migratory species; the other WNV-infected birds are presumably residents. The WNV-infected birds were all captured in March 2003. These data provide the first indirect evidence of WNV transmission among birds in northern México.