Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons

West Nile virus (WNV) is a zoonotic flavivirus that is transmitted by blood-suckling mosquitoes with birds serving as the primary vertebrate reservoir hosts (enzootic cycle). Some bird species like ravens, raptors and jays are highly susceptible and develop deadly encephalitis while others are infected subclinically only. Birds of prey are highly susceptible and show substantial mortality rates following infection. To investigate the WNV pathogenesis in falcons we inoculated twelve large falcons, 6 birds per group, subcutaneously with viruses belonging to two different lineages (lineage 1 strain NY 99 and lineage 2 strain Austria). Three different infection doses were utilized: low (approx. 500 TCID50), intermediate (approx. 4 log10 TCID50) and high (approx. 6 log10 TCID50). Clinical signs were monitored during the course of the experiments lasting 14 and 21 days. All falcons developed viremia for two weeks and shed virus for almost the same period of time. Using quantitative real-time RT-PCR WNV was detected in blood, in cloacal and oropharyngeal swabs and following euthanasia and necropsy of the animals in a variety of neuronal and extraneuronal organs. Antibodies to WNV were first time detected by ELISA and neutralization assay after 6 days post infection (dpi). Pathological findings consistently included splenomegaly, non-suppurative myocarditis, meningoencephalitis and vasculitis. By immunohistochemistry WNV-antigens were demonstrated intralesionally. These results impressively illustrate the devastating and possibly deadly effects of WNV infection in falcons, independent of the genetic lineage and dose of the challenge virus used. Due to the relatively high virus load and long duration of viremia falcons may also be considered competent WNV amplifying hosts, and thus may play a role in the transmission cycle of this zoonotic virus.     

West Nile virus infection of horses

West Nile virus (WNV) is a flavivirus closely related to Japanese encephalitis and St. Louis encephalitis viruses that is primarily maintained in nature by transmission cycles between mosquitoes and birds. Occasionally, WNV infects and causes disease in other vertebrates, including humans and horses. West Nile virus has re-emerged as an important pathogen as several recent outbreaks of encephalomyelitis have been reported from different parts of Europe in addition to the large epidemic that has swept across North America. This review summarises the main features of WNV infection in the horse, with reference to complementary information from other species, highlighting the most recent scientific findings and identifying areas that require further research.     

Recent progress in West Nile virus diagnosis and vaccination

ABSTRACT: West Nile virus (WNV) is a positive-stranded RNA virus belonging to the Flaviviridae family, a large family with 3 main genera (flavivirus, hepacivirus and pestivirus). Among these viruses, there are several globally relevant human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV). Since the mid-1990s, outbreaks of WN fever and encephalitis have occurred throughout the world and WNV is now endemic in Africa, Asia, Australia, the Middle East, Europe and the Unites States. This review describes the molecular virology, epidemiology, pathogenesis, and highlights recent progress regarding diagnosis and vaccination against WNV infections.     

Equine encephalomyelitis outbreak caused by a genetic lineage 2 West Nile virus in Hungary

Background: The spread of lineage 2 West Nile virus (WNV) from sub‐Saharan regions to Europe and the unpredictable change in pathogenicity indicate a potential public and veterinary health threat and requires scientific awareness. Objectives: To describe the results of clinical and virological investigations of the 1st outbreak of a genetic lineage 2 WNV encephalomyelitis in horses. Animals: Seventeen horses with neurologic signs. Methods: Information regarding signalment, clinical signs, and outcome was obtained for each animal. Serology was performed in 15 cases, clinicopathological examination in 7 cases, and cerebrospinal fluid was collected from 2 horses. Histopathology was carried out in 4 horses, 2 of which were assessed for the presence of WNV in their nervous system. Results: WNV neutralizing antibody titers were between 10 and 270 (median, 90) and the results of other serological assays were in agreement with those of the plaque reduction neutralization test. Common signs included ataxia, weakness, asymmetric gait, muscle tremors, hypersensitivity, cranial nerve deficits, and recumbency. Twelve animals survived. Amplicons derived from the infection‐positive specimens allowed molecular characterization of the viral strain. Conclusions and Clinical Importance: From our results, we conclude that this outbreak was caused by a lineage 2 WNV strain, even though such strains often are considered nonpathogenic. Neurological signs and survival rates were similar to those reported for lineage 1 virus infections. The disease occurrence was not geographically limited as had been the typical case during European outbreaks; this report describes a substantial northwestern spread of the pathogen.     

West Nile virus: North American experience

West Nile virus, a mosquito‐vectored flavivirus of the Japanese encephalitis serogroup, was first detected in North America following an epizootic in the New York City area in 1999. In the intervening 11 years since the arrival of the virus in North America, it has crossed the contiguous USA, entered the Canadian provinces bordering the USA, and has been reported in the Caribbean islands, Mexico, Central America and, more recently, South America. West Nile virus has been reported in over 300 species of birds in the USA and has caused the deaths of thousands of birds, local population declines of some avian species, the clinical illness and deaths of thousands of domestic horses, and the clinical disease in over 30 000 Americans and the deaths of over 1000. Prior to the emergence of West Nile virus in North America, St. Louis encephalitis virus and Dengue virus were the only other known mosquito‐transmitted flaviviruses in North America capable of causing human disease. This review will discuss the North American experience with mosquito‐borne flavivirus prior to the arrival of West Nile virus, the entry and spread of West Nile virus in North America, effects on wild bird populations, genetic changes in the virus, and the current state of West Nile virus transmission.     

Retrospective genetic analysis of SAT-1 type foot-and-mouth disease outbreaks in West Africa (1975-1981)

The complete 1D genome region encoding the immunogenic and phylogenetically informative VP1 gene was genetically characterized for 23 South African Territories (SAT)-1 viruses causing foot-and-mouth (FMD) disease outbreaks in the West African region between 1975 and 1981. The results indicate that two independent outbreaks occurred, the first involved two West African countries, namely Niger and Nigeria, whilst the second affected Nigeria alone. In the former epizootic, virus circulation spanned a period of 2 years, whilst in the latter virus was recovered from the field over a 3 year period. Comparison of the West African viruses with SAT-1 viruses from other regions on the continent revealed that the two West African lineages identified in this study are regionally distinct. Furthermore, variation in VP1 gene length was identified in SAT-1 viruses for the first time, further emphasizing the uniqueness of these pathogens in West Africa. This first retrospective analysis in which the molecular epidemiology of SAT-1 viruses in West Africa is reported, provides a useful measure of the regional variation of these viruses and is an essential first step in the establishment of a West African sequence database that will be a useful reference for future outbreak eventualities.     

Evidence of West Nile virus lineage 2 circulation in Northern Italy

A West Nile virus (WNV) strain belonging to lineage 2 was for the first time detected in two pools of Culex pipiens collected in the province of Udine and in tissues of a wild collared dove (Streptopelia decaocto) found dead in the province of Treviso, in North East of Italy. It was molecularly identified by group and WNV lineage specific RT-PCRs and characterized by partial sequencing of the NS3 and NS5 genes. When compared with the sequences of same fragments of NS3 and NS5 of the WNV lineage 2 strain isolated from birds of prey in Hungary (2004), the phylogenetic analysis of these sequences revealed 100% and 99% similarity, respectively. As the Hungarian strain, the NS3 selected sequence differed from the 2010 Greek isolate by one amino-acid located at 249 site which is the site involved in genetic modulation of WNV pathogenicity. The Italian and Hungarian strains have histidine rather than proline at this site. The presence of a lineage 2 strain in regions where the lineage 1 strain is still circulating, creates a new scenario with unpredictable consequences. In this situation comprehensive investigations on the occurrence, ecology, and epidemiology of these different WNV strains circulating in Italy become the highest priority.     

West Nile virus infection of Thoroughbred horses in South Africa (2000-2001)

REASONS FOR PERFORMING STUDY: West Nile virus (WNV) infection is endemic in southern Africa. With the recent emergence of WNV infection of horses in Europe and the USA the present study was performed to estimate the risk of seroconversion to WNV in a cohort of 488 young Thoroughbred (TB) horses. OBJECTIVES: To estimate the risk of seroconversion to WNV among a cohort of South African TB yearlings sold at the 2001 National Yearling Sales (NYS) and to determine whether the risk varied geographically. Two horses were also infected with a recent South African isolate of WNV to evaluate its virulence in horses. METHODS: Serum samples were collected from the cohort of 488 TB yearlings at the 2001 NYS. Serum samples that were collected from the same horses at the time that they were identified were sourced from our serum bank. Sera from 243 of the dams that were collected at the time that the foals were identified were also sourced from our serum bank. These sera were subjected to serum neutralisation (SN) tests for antibody to WNV. RESULTS: Approximately 11% of yearlings seroconverted to WNV on paired serum samples collected from each animal approximately 12 months apart. Studfarms with WNV-seropositive yearlings were widely distributed throughout South Africa and SN tests on sera from their dams indicated that exposure to WNV was even more prevalent (75%) in this population. Neurological disease was not described in any of the horses included in this study and 2 horses inoculated with a recent lineage 2 South African isolate of WNV showed no clinical signs of disease after infection and virus was not detected in their blood. CONCLUSIONS: Infection of horses with WNV is common in South Africa, but infection is not associated with neurological disease. POTENTIAL RELEVANCE: In contrast to recent reports from Europe, North Africa, Asia and North America, the results of our field and experimental studies indicated that exposure of horses to the endemic southern African strains of WNV was not associated with neurological disease.     

West Nile: worldwide current situation in animals and humans

West Nile (WN) virus is a mosquito-borne flavivirus that is native to Africa, Europe, and Western Asia. It mainly circulates among birds, but can infect many species of mammals, as well as amphibians and reptiles. Epidemics can occur in rural as well as urban areas. Transmission of WN virus, sometimes involving significant mortality in humans and horses, has been documented at erratic intervals in many countries, but never in the New World until it appeared in New York City in 1999. During the next four summers it spread with incredible speed to large portions of 46 US states, and to Canada, Mexico, Central America and the Caribbean. In many respects, WN virus is an outstanding example of a zoonotic pathogen that has leaped geographical barriers and can cause severe disease in human and equine. In Europe, in the past two decades there have been a number of significant outbreaks in several countries. However, very little is known of the ecology and natural history of WN virus transmission in Europe and most WN outbreaks in humans and animals remain unpredictable and difficult to control.     

Experimental infection of house sparrows (Passer domesticus) with West Nile virus isolates of Euro-Mediterranean and North American origins

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle involving wild birds as reservoir hosts. The virus has recently emerged in North America and re-emerged in Europe. North American WNV outbreaks are often accompanied by high mortality in wild birds, a feature that is uncommon in Europe. The reason for this difference is unknown, but the intrinsic virulence of the viruses circulating in each continent and/or the susceptibility to the disease of Palearctic as opposed to Nearctic wild bird species could play a role. To assess this question, experimental inoculations with four lineage 1 WNV strains, three from southern Europe (Italy/2008, Italy/2009 and Spain/2007) and one from North America (NY99) were performed on house sparrows (Passer domesticus), a wild passerine common in both continents. Non-significant differences which ranged from 0% to 25% were observed in mortality for the different WNV strains. Viremias lasted from 1 to 5–6 days post-inoculation (dpi) in all cases; individuals inoculated with NY99 had significantly higher titres than those inoculated with any of the Euro-Mediterranean strains. Remarkably, host competence was found to be higher for NY99 than for the other strains. Consequently, albeit being pathogenic for house sparrows, some Euro-Mediterranean strains had reduced capacity for replication in -and transmission from- this host, as compared to the NY99 strain. If applicable also to other wild bird host species, this relatively reduced transmission capacity of the Euro-Mediterranean strains could explain the lower incidence of this disease in wild birds in the Euro-Mediterranean area.     

西尼罗病毒套式RT-PCR检测方法的建立

从GenBank上调取西尼罗病毒的基因序列,经过分析,设计并合成2套套式RT-PCR引物,分别对西尼罗病毒灭活苗进行扩增,结果扩增出与目的片段大小一致的条带,将其克隆入pMD18-T-Vector中,进行序列测定,证实为WNV的基因。建立了套式RT-PCR检测方法,经各反应条件的优化后,发现2套nest-PCR引物能分别扩增出85个拷贝和62个拷贝的双链DNA,对乙型脑炎病毒、黄热病毒等11种病毒核酸进行扩增,发现具有良好的特异性,显示建立套式RT-PCR具有高效、快速、特异、灵敏的特点,可用于口岸WNV的检测和监测。     

Re-emerging of porcine respiratory and reproductive syndrome virus (lineage 3) and increased pathogenicity after genomic recombination with vaccine variant

Porcine reproductive and respiratory syndrome virus (PRRSV) was first reported in China since late 1995 and several variants were further reported in subsequence years, causing huge economic losses to the Chinese swine industry. To date, three major lineages (lineage 3, 5.1 and 8.7) of Type 2 PRRSV were reported in China based on our global genotyping. The present study provides the epidemiology of the PRRSV in South China based on the isolates collected during 2009–2012, indicating three lineages (lineage 3, 5.1 and 8.7) of Type 2 PRRSV were still circulating in this area. Our phylogenetic reconstruction indicated that lineage 3 re-emerged in 2010 formed a huge cluster with closely related to the 2004 isolates from Hong Kong. Furthermore, the inter-lineage genomic recombination between MLV vaccine strain (lineage 5) and a recently re-emerged lineage 3 virus (QYYZ) has also been found in a farm practicing MLV vaccination. Our in vivo experiment comparing the pathogenicity and clinical presentations among currently isolated viruses indicated that pigs infected with recombinant lineage 3 virus (GM2) showed persistent higher fever compared to pigs infected by its wild counterpart (QYYZ). This study enhanced our understanding on potential importance of the recombination of PRRSV along with their evolution.     

Serologic Responses of West Nile Virus Seronegative Mature Horses to West Nile Virus Vaccines

A 42-day study was conducted to assess the impact of three West Nile virus vaccines given either as separate injections or incorporated with their counterpart equine encephalitis and tetanus vaccines on serological responses under field use conditions. Two hundred forty mature, West Nile virus seronegative (<4) horses were followed serologically pre- and postprimary and secondary vaccination with six different vaccination programs, all including West Nile virus antigens. Forty horses were unvaccinated sentinel horses. All vaccines stimulated both a primary and secondary (booster) response to vaccination that was significantly higher than that of seronegative controls. However, inclusion of West Nile virus with equine encephalitis viruses and tetanus toxoid in vaccines had a significant detrimental impact on West Nile virus serum neutralization antibody production to both the primary and secondary vaccinations.