Large Human Outbreak of West Nile Virus Infection in North-Eastern Italy in 2012

Human cases of West Nile virus (WNV) disease have been reported in Italy since 2008. So far, most cases have been identified in north-eastern Italy, where, in 2012, the largest outbreak of WNV infection ever recorded in Italy occurred. Most cases of the 2012 outbreak were identified in the Veneto region, where a special surveillance plan for West Nile fever was in place. In this outbreak, 25 cases of West Nile neuroinvasive disease and 17 cases of fever were confirmed. In addition, 14 WNV RNA-positive blood donors were identified by screening of blood and organ donations and two cases of asymptomatic infection were diagnosed by active surveillance of subjects at risk of WNV exposure. Two cases of death due to WNND were reported. Molecular testing demonstrated the presence of WNV lineage 1 in all WNV RNA-positive patients and, in 15 cases, infection by the novel Livenza strain was ascertained. Surveillance in other Italian regions notified one case of neuroinvasive disease in the south of Italy and two cases in Sardinia. Integrated surveillance for WNV infection remains a public health priority in Italy and vector control activities have been strengthened in areas of WNV circulation.     

Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes

Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.     

Rhabdomyolysis in patients with west nile encephalitis and meningitis

Since 1999, more than 6,500 cases of West Nile virus neuroinvasive disease (WNND) have been reported in the United States. Patients with WNND can present with muscle weakness that is often assumed to be of neurological origin. During 2002, nearly 3,000 persons with WNV meningitis or encephalitis (or both) were reported in the United States; in suburban Cook County, Illinois, with 244 persons were hospitalized for WNV illnesses. The objective of this investigation was to describe the clinical and epidemiological features of identified cases of WNV neuroinvasive disease and rhabdomyolysis. Public health officials investigated patients hospitalized in Cook County, and identified a subset of WNV neuroinvasive disease patients with elevated creatine kinase levels. Cases were defined as hospitalized persons with a WNV infection, encephalitis or meningitis, and rhabdomyolysis. Retrospective medical record reviews were conducted and data was abstracted with a standardized data collection instrument. Eight patients with West Nile encephalitis and one with West Nile meningitis were identified with rhabdomyolysis. Median age of the nine patients was 70 years (range, 45-85 years), and eight were men. For all nine patients, the peak CK level was documented a median of 2 days after hospitalization (range, 1-24 days). Median CK level during hospitalization for all case-patients was 3,037 IU (range, 1,153-42,113 IU). Six patients had history of recent falls prior to admission. Although the temporal relationship of rhabdomyolysis and neurological WNV illness suggested a common etiology, these patients presented with complex clinical conditions which may have led to development of rhabdomyolysis from other causes. The spectrum of WNV disease requires further investigation to describe this and other clinical conditions associated with WNV infection.     

Unprecedented increase of West Nile virus neuroinvasive disease,Spain, summer 2020

Cases of West Nile neuroinvasive disease (WNND) in Spain increased in summer 2020. Here we report on this increase and the local, regional and national public health measures taken in response. We analysed data from regional surveillance networks and the National Epidemiological Surveillance Network, both for human and animal West Nile virus (WNV) infection. During the 2020 season, a total of 77 human cases of WNV infection (median age 65 years; 60% males) were detected in the south-west of Spain; 72 (94%) of these cases developed WNND, presenting as meningoencephalitis, seven of which were fatal. In the previous two decades, only six human cases of WNND were detected in Spain. Reduced activities for vector control this season, together with other factors, might have contributed to the massive increase. Public health measures including vector control, campaigns to raise awareness among physicians and the general population, and interventions to ensure the safety of donations of blood products, organs, cells and tissues were effective to reduce transmission. Going forward, maintenance of vector control activities and an update of the vector-borne diseases response plan in Spain is needed.     

Identification of a possible ancestor of the subtype A1 HIV Type 1 variant circulating in the former Soviet Union

The HIV-1 subsubtype A1 epidemic of the former Soviet Union (FSU) was caused by an A1 variant apparently derived from a single introduction event. We identified an A1 variant highly related to the A1 lineage circulating in the FSU in a patient from Conakry, Republic of Guinea, who was diagnosed with HIV-1 when he moved to Italy in 2002. The most probable route of infection was two blood transfusions received in his country of origin in 1998. Full-length (9781 bp) molecular characterization revealed that this strain was evolutionarily parental to, but distinct from, the A1 lineage circulating in the FSU. Similar results were obtained analyzing partial genome sequences. A full-length sequence similarity plot and bootscanning analysis supported this evidence and excluded any potential recombination events with other HIV-1 variants. This viral strain represents the first evidence of an African patient infected by an A1 subtype related to the A1 variants spreading in FSU countries. The identification of this distinct A1 variant may support the origin of the Russian A epidemic from West Africa.     

West Nile Virus Neuroinvasive Disease

West Nile virus (WNV), first recognized in North America in 1999, was responsible for the largest arboviral epidemic of human encephalitis in history and continues to be the most frequent cause of epidemic meningoencephalitis in North America. WNV neuroinvasive disease (WNND) occurs in fewer than 1% of infected individuals, with presentations including aseptic meningitis, encephalitis, and poliomyelitis. Between 1999 and 2009, over 12,000 cases of WNND were reported in the United States, with the peak annual incidence occurring in epidemics of 2002 and 2003. In this review, we first summarize the epidemiology of WNV over the past decade and the salient clinical features of WNND, including a discussion of laboratory and radiographic findings, risk factors, morbidity, and mortality. In addition, we review recent progress in our understanding of virus and host determinants of the pathogenesis of WNND, as well as the prospects for the development of specific therapeutic targets.     

Early start of seasonal transmission and co-circulation of West Nile virus lineage 2 and a newly introduced lineage 1 strain,northern Italy,June 2022

In spring 2022, Europe faced an unprecedented heatwave, increasing the risk of West Nile virus (WNV) outbreaks. As early as 7 June 2022, WNV was detected in Culex mosquitoes in northern Italy, and – in the following days – in two blood donors, a patient with encephalitis, wild birds and additional mosquito pools. Genome sequencing demonstrated co-circulation of WNV lineage 2 and a newly introduced WNV lineage 1, which was discovered in the region in 2021.     

Pathogenesis of West Nile Virus Lineage 2 in Domestic Geese after Experimental Infection

West Nile virus (WNV) is an emerging infectious pathogen circulating between mosquitoes and birds but also infecting mammals. WNV has become autochthonous in Germany, causing striking mortality rates in avifauna and occasional diseases in humans and horses. We therefore wanted to assess the possible role of free-ranging poultry in the WNV transmission cycle and infected 15 goslings with WNV lineage 2 (German isolate). The geese were monitored daily and sampled regularly to determine viremia, viral shedding, and antibody development by molecular and serological methods. Geese were euthanized at various time points post-infection (pi). All infected geese developed variable degrees of viremia from day 1 to day 10 (maximum) and actively shed virus from days 2 to 7 post-infection. Depending on the time of death, the WN viral genome was detected in all examined tissue samples in at least one individual by RT-qPCR and viable virus was even re-isolated, except for in the liver. Pathomorphological lesions as well as immunohistochemically detectable viral antigens were found mainly in the brain. Furthermore, all of the geese seroconverted 6 days pi at the latest. In conclusion, geese are presumably not functioning as important amplifying hosts but are suitable sentinel animals for WNV surveillance.     

西尼罗病毒RT-PCR检测方法的建立及其初步应用

目的　建立西尼罗病毒 (WestNilevirus ,WNV)RT PCR检测方法 ,为WNV感染的诊断和流行病学调查奠定基础。方法　选择Vero E6细胞进行WNV培养 ,通过乳鼠脑内接种病毒培养液获得WNV感染脑组织。在病毒基因组E区和C区设计 3对引物 ,以WNV培养液 ,建立并优化病毒核酸RT PCR分析方法。然后 ,以此对感染蚊虫模拟标本和乳鼠脑组织进行检测。PCR产物测序后 ,用Blast进行同源性分析。结果　用RT PCR在WNV培养液中扩增出与预期大小一致的核苷酸片段 ,并通过改变循环数 ,模板稀释倍数等参数对该方法进行了优化 ;将建立的方法用于检测感染蚊虫模拟标本和感染乳鼠脑组织 ,均检测出WNV目的基因片段 ,且扩增效果与病毒培养液无明显差异 ;套式PCR能显著提高检测的敏感性 ( 10 8-10 9倍 )。结论　本研究建立的WNVRT PCR检测方法具有较高的敏感性 ,可用于WNV感染的流行病学调查研究     

Variation in virulence of West Nile virus strains for house sparrows (Passer domesticus)

The observation of avian mortality associated with West Nile virus (WNV) infection has become a hallmark epidemiologic feature in the recent emergence of this pathogen in Israel and North America. To determine if phenotypic differences exist among different WNV isolates, we exposed house sparrows (Passer domesticus) to low passage, lineage 1 WNV strains from North America (NY99), Kenya (KEN), and Australia (KUN; also known as Kunjin virus). House sparrows inoculated with the NY99 and KEN strains experienced similar mortality rates and viremia profiles. The KUN strain elicited significantly lower-titered viremia when compared with the other strains and induced no mortality. This study suggests that natural mortality in house sparrows due to Old World strains of WNV may be occurring where the KEN strain occurs.     

Host-range restriction of chimeric yellow fever-West Nile vaccine in fish crows (Corvus ossifragus)

We evaluated a recombinant virus chimera, ChimeriVax-WN, in which the West Nile virus (WNV) surface protein genes (pre-membrane [prM] and envelope [E]) are substituted into the genome of the 17D vaccine strain yellow fever virus (YF-17D), as a vaccine candidate for protection of birds from WNV disease. Using fish crows (Corvus ossifragus) as a model, we found that none of eight crows that received two high doses of vaccine (approximately 100,000 plaque-forming units [PFU]) developed viremia and only one developed WNV-neutralizing antibodies. When challenged with subcutaneous injection of 2,000 PFU of WNV (NY99 strain), all eight developed viremia levels similar to unvaccinated control birds (n = 4). Two of the vaccinated birds died of the infection, compared with no mortality in the four controls. To further investigate the failure of the vaccine, we inoculated chickens with both the vaccine and YF-17D and found no evidence of replication with either of these viruses. These data indicate that this vaccine candidate failed to protect birds from the morbidity and mortality attributed to WNV infections. However, if used in mammals, this recombinant viral vaccine is unlikely to inadvertently enter a natural transmission cycle with birds as amplifying hosts.     

Isolation of a complementary DNA fragment of hepatitis C virus in Taiwan revealed significant sequence variations compared with other isolates.

To clone and characterize hepatitis C virus strains present in Taiwan, RNA was extracted from liver tissue collected from a patient during the acute phase of posttransfusion non-A, non-B hepatitis. RNA was then subjected to complementary DNA synthesis and the polymerase chain reaction, using primers derived from the original nucleotide sequence of the United States hepatitis C virus strain. A complementary DNA clone, HCV-T3, containing 552 base pairs of hepatitis C virus complementary DNA sequences was isolated and characterized. The homologies in nucleotide sequence between the Taiwan isolate and either the United States or Japan isolate were 80.1% and 91.5%, respectively. However, most of the nucleotide changes occurred in the third base positions, resulting in much higher homologies in amino acid sequence of 91.8% and 97.3%, respectively. Amplification of the less conserved region of hepatitis C virus genome with the polymerase chain reaction was improved by use of primers with nucleotides matched to the local strain. Finally, in addition to the liver and serum, the viral genome was also demonstrated in the spleen tissue by similar methods, suggesting another possible target for hepatitis C viral infection. These findings indicate that there is considerable heterogeneity in hepatitis C virus genomes isolated from different areas of the world.     

Infection with Non-Lethal West Nile Virus Eg101 Strain Induces Immunity that Protects Mice against the Lethal West Nile Virus NY99 Strain

Herein we demonstrate that infection of mice with West Nile virus (WNV) Eg101 provides protective immunity against lethal challenge with WNV NY99. Our data demonstrated that WNV Eg101 is largely non-virulent in adult mice when compared to WNV NY99. By day 6 after infection, WNV-specific IgM and IgG antibodies, and neutralizing antibodies were detected in the serum of all WNV Eg101 infected mice. Plaque reduction neutralization test data demonstrated that serum from WNV Eg101 infected mice neutralized WNV Eg101 and WNV NY99 strains with similar efficiency. Three weeks after infection, WNV Eg101 immunized mice were challenged subcutaneously or intracranially with lethal dose of WNV NY99 and observed for additional three weeks. All the challenged mice were protected against disease and no morbidity and mortality was observed in any mice. In conclusion, our data for the first time demonstrate that infection of mice with WNV Eg101 induced high titers of WNV specific IgM and IgG antibodies, and cross-reactive neutralizing antibodies, and the resulting immunity protected all immunized animals from both subcutaneous and intracranial challenge with WNV NY99. These observations suggest that WNV Eg101 may be a suitable strain for the development of a vaccine in humans against virulent strains of WNV.     

Two distinct introductions of the West Nile virus in Portugal disclosed by phylogenetic analysis of genomic sequences

In this report, we describe genomic sequencing and analysis of different West Nile virus strains isolated from mosquitoes in the south of Portugal (Alentejo and Algarve) at two different time points (1971 and 2004, respectively). Phylogenetic analysis indicated different origins for the two recorded introductions of WNV in our country, with strains segregating in different sub-clades within lineage 1a. PTRoxo (isolated in 1971) was found to be very similar to an Egyptian WNV strain isolated in 1951, while the viruses isolated in 2004 formed a statistically well-supported group with WNV strains isolated over the last decade in countries lining the Mediterranean (Morocco, Italy, and France). Analyses of the putative amino acid sequences showed all the main expected features described for viral mature proteins except for the absence of the N-glycosylation site in the envelope glycoprotein of PTRoxo, which may be reflected as attenuated neurovirulence and neuroinvasive phenotypes.     

Prevalence of West Nile Virus infection in animals from two state zoos Tabasco

OBJECTIVE: To determine the prevalence of West Nile Virus (WNV) infection in animals, mosquitoes and employees from two zoos of Tabasco state, Mexico. MATERIAL AND METHODS: WNV antibodies were detected by blocking ELISA in serum samples from animals. Viral RNA was detected by RT-PCR from mosquitoes and serum samples from employees at "Yum-Ká" zoo. RESULTS: Seroprevalence in birds was 25.65% (19/74) and 85% (6/7) in reptiles from "La Venta" zoo. Thirty-one percent of birds (50/160) and 34.48% mammals (16/29) at the "Yum-Ká" zoo, were seropositive. All human serum samples from Yum-ká zoo were negative by RT-PCR. A pool of mosquitoes (Culex quinquefasciatus) was positive for WNV. CONCLUSIONS: The presence of WNV antibodies in animals from both zoos and the detection of viral genome in mosquitoes demonstrate the presence of WNV in this region and indicates a potential risk of infection in animals and humans.     

West Nile virus neuroinvasive disease incidence in the United States, 2002-2006

As the geographic range of reported human West Nile virus (WNV) disease has expanded across the United States, seasonal transmission and outbreaks have persisted over several years in many areas of the country. West Nile virus neuroinvasive disease (WNND) case reports from 2002 to 2006 were reviewed to determine which areas of the country have the highest reported cumulative incidence and whether those areas have had consistently high annual incidence. During the 5-year period examined, 9632 cases of WNND were reported nationwide. The cumulative incidence of WNND ranged from 0.2 to 32.2 per 100,000 population by state and from 0.1 to 241.2 per 100,000 population by county. States and counties with the highest cumulative incidence were primarily located in the northern Great Plains. States with consistently high annual incidence included South Dakota, North Dakota, Wyoming, New Mexico, Mississippi, Nebraska, Louisiana, and Colorado. All of these states, with the exception of New Mexico, were also among the states with the highest cumulative incidence. Counties with repeatedly high annual incidence were also primarily in the Great Plains and mid-South. The risk of WNND appears to be highest in areas where the primary WNV vectors are Culex tarsalis and Cx. quinquefasciatus mosquitoes.     

Patterns of mutation within an emerging endemic lineage of HEV‐3a

The hepatitis E virus can cause chronic infections in immuno‐suppressed patients, and cases have been on the rise globally. Viral mutations during such infections are difficult to characterize. We deep‐sequenced viral populations from 15 immunocompromised patients with chronic HEV to identify the viral lineage and describe viral mutational hotspots within and across patients. A total of 21 viral RNA samples were collected between 2012 and 2017 from a single hospital in Singapore. Sequences covering a total of 3894 bp of the HEV genome were obtained. Phylogenetic analyses identified all sequences as belonging to the HEV‐3a sub‐clade and clearly indicate a unique local lineage. Deep sequencing reveals variable viral population complexity during infections. Comparisons of viral samples from the same patients spaced 2‐19 months apart revealed rapid nucleotide replacements in the dominant viral sequence in both ribavirin treated and treatment‐naive patients. Mutational hotspots were identified within ORF3 and the PCP/HVR domain of ORF1.     

West Nile Virus Emergence and Persistence in Los Angeles,California, 2003–2008

West Nile virus (WNV) invaded Los Angeles in September 2003, and during the subsequent five-year period followed a pattern of amplification, subsidence, and resurgence. Enzootic transmission was tracked by abundance and infection incidence in Culex pipiens quinquefasciatus and Cx. tarsalis and by seroprevalence in peridomestic passerine birds, infection in dead birds, and seroconversions in sentinel chickens. Culex p. quinquefasciatus served as the primary vector of WNV, with gravid traps serving as the best sampling method and the most consistent indicator of viral activity. Spatial scan statistics applied to mosquito infection and positive dead bird data delimited three major clusters of WNV transmission, with introduction occurring in the Los Angeles Basin, and amplification and dispersal events carrying transmission to the San Fernando and Santa Clarita valleys. Los Angeles experienced major epidemics in 2004 and 2008, providing a unique opportunity to investigate specific patterns of enzootic amplification preceding epidemics.     

Interlaboratory study to evaluate the performance of laboratories involved in West Nile virus RNA screening of blood and blood components by nucleic acid amplification testing in Italy

Background

An Italian interlaboratory study was run in 2010 to assess the performance of Blood Transfusion Services in detecting the genome of West Nile virus (WNV) in plasma.

Materials and methods

Each laboratory received a panel of samples containing four samples negative for WNV and six positive samples with a nominal viral concentration close to or below the 95% detection limit of two commercially available nucleic acid amplification tests (NAT) for WNV, the PROCLEIX® WNV kit and the Cobas® TaqScreen West Nile Virus kit.

Results

Ten laboratories took part in the study. All correctly identified the positive samples with a viral concentration above the 95% detection limit. No pre- or post-analytical errors were observed.

Conclusions

The interlaboratory study run in 2010 allowed participants to assess the performance of the NAT methods applied in their seasonal routine screening of blood donations.