Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)

West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.     

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.     

Pathogenesis of bovine respiratory syncytial virus in experimentally infected lambs

Twenty-four 6-8-week-old conventionally reared lambs were inoculated intranasally and intratracheally with bovine respiratory syncytial virus. Infected lambs showed mild clinical signs characterized by slight serous nasal discharge, coughing, lachrymation and bronchovascular sounds on the middle part of the lung 5-9 days post-inoculation (PI). Virus was isolated in nasal swabs from 9 of 24 lambs between 3 and 7 days PI. However, virus was recovered from tracheal and lung tissue of all lambs killed between 3 and 11 days PI. Virus-specific antibodies appeared as early 6 days PI but high titres were attained 14-21 days PI. Lungs of lambs killed on different days PI had multifocal areas of consolidation. There was an increase of lymphocytes with a T-suppressor cell marker and a decrease in those with a T-helper marker in lung lavages obtained 5 days PI.     

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.     

Pathogenesis of a bovine enterovirus-1 isolate in experimentally infected calves

The pathogenesis and virulence of Bovine enterovirus-1 (BEV-1) in cattle is largely unknown. Reports concerning its virulence suggest that there might be an association between BEV-1 infections and a range of diseases in cattle that vary from respiratory to enteric to reproductive disease and infertility. In the current study, the pathogenesis associated with acute infection of BEV-1 in calves experimentally inoculated with the Oklahoma isolate of BEV-1 was described. Although interpretation of the study was limited by lack of an effective control group, results suggest that an association between inoculation of BEV-1, virus localization, and the potential development of lesions in the brain and heart probably exists. In the experiment, BEV-1 virus localized to the terminal ileum, ileocecal and cecocolonic junctions, spiral colon, and ileocecal lymph nodes; BEV-1 virus was detected in the cytoplasm of enterocytes, lamina propria macrophages, endothelium, neurons of the submucosal and myenteric plexi, and lymphocytes of the submucosal lymphoid tissue. Although no clinical signs were noted following acute infection, BEV-1 was localized in the cerebellar white matter of a calf with encephalitis and in the heart of another calf with coronary arteritis. The current study suggests that the BEV-1 isolate is infectious to young calves and that BEV-1 potentially can have a similar pathogenesis to that observed in natural or experimental enterovirus infections in other species.     

DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly,via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.)

As West Nile virus (WNV) can cause lethal diseases in raptors, a vaccination prophylaxis of free-living and captive populations is desirable. In the absence of vaccines approved for birds, equine vaccines have been used in falcons, but full protection against WNV infection was not achieved. Therefore, two DNA vaccines encoding the ectodomain of the envelope protein of WNV lineages 1 and 2, respectively, were evaluated in 28 large falcons. Four different vaccination protocols were used, including electroporation and booster-injections of recombinant WNV domain III protein, before challenge with the live WNV lineage 1 strain NY99. Drug safety, plasmid shedding and antibody production were monitored during the vaccination period. Serological, virological, histological, immunohistochemical and molecular biological investigations were performed during the challenge trials. Antibody response following vaccination was low overall and lasted for a maximum of three weeks. Plasmid shedding was not detected at any time. Viremia, mortality and levels, but not duration, of oral virus shedding were reduced in all of the groups during the challenge trial compared to the non-vaccinated control group. Likewise, clinical scoring, levels of cloacal virus shedding and viral load in organs were significantly reduced in three vaccination groups. Histopathological findings associated with WNV infections (meningo-encephalitis, myocarditis, and arteritis) were present in all groups, but immunohistochemical detection of the viral antigen was reduced. In conclusion, the vaccines can be used safely in falcons to reduce mortality and clinical signs and to lower the risk of virus transmission due to decreased levels of virus shedding and viremia, but full protection was not achieved in all groups.     

西尼罗病毒NS1基因的原核表达与抗原性鉴定

为进行西尼罗病毒(WNV)NY99株NS1蛋白的免疫学研究,通过PCR方法扩增WNV NY99株NS1基因,并将其克隆至原核表达载体pMAL-c2X上,构建出重组表达质粒pc2X-NS1,经IPTG诱导得到可溶性的麦芽糖结合蛋白(MBP).NS1融合蛋白(MBP-NS1),其分子质量约为90 ku,约占可溶性菌体蛋白质量的50%.利用直链淀粉树脂柱对MBP-NS1纯化后的重组蛋白的纯度达到60%,westem blot和间接ELISA检测证明纯化的MBP-NS1具有良好的抗原性和特异性,为进一步开展关于WNV NS1蛋白的研究奠定了基础.     

Pathogenesis and clinical signs of equine herpesvirus-1 in experimentally infected ponies in vivo.

Equine herpesvirus-1 (EHV-1) causes respiratory disease, neonatal death, abortion and neurologic disease. The main purpose of this study was to identify viral antigen in respiratory tract samples by immunoperoxidase staining. Six pony foals were selected on the basis of demonstrating seronegativity to EHV-1 by virus neutralization and housed in isolation. They were infected experimentally by administering EHV-1 nebulized ultrasonically through a face mask. Successful infection was clinically apparent as each of the foals had febrile responses, nasal discharge, and enlarged submandibular lymph nodes. Sporadic coughing was also heard. EHV-1 was isolated from nasopharyngeal swabs of 4/6 ponies and seroconversion was demonstrated in all foals. Bronchoscopic examination of the large airways revealed hyperemia. The incidence of recovery of Actinobacillus suis from nasopharyngeal swabs increased initially, with recovery of Streptococcus zooepidemicus isolates predominating at 3 wk post-infection. Cytology brushes were used to sequentially sample the respiratory tract of the infected ponies at the nasopharynx, mid-trachea and the mainstem bronchus. Bronchoalveolar lavage provided lung cells. Immunocytochemistry techniques were applied to both types of samples to locate EHV-1 antigen. Indirect immunoperoxidase staining of samples utilizing monoclonal antibodies specific for EHV-1 demonstrated viral antigen associated with cellular debris, primarily in the nasopharyngeal samples on days 3-9 post-infection.     

Pathogenicity of West Nile virus in chickens

In the fall of 1999, West Nile virus (WNV) was isolated for the first time in the Western Hemisphere during an outbreak of neurologic disease in humans, horses, and wild and zoo birds in the northeastern United States. Chickens are a potential reservoir for WNV, and little is known about the pathogenicity of WNV in domestic chickens. Seven-week-old chickens derived from a specific-pathogen-free flock were inoculated subcutaneously with 1.8 x 10(3) 50% tissue culture infectious dose of a crow isolate of WNV in order to observe clinical signs and evaluate the viremic phase, gross and microscopic lesions, contact transmission, and immunologic response. There were no observable clinical signs in the WNV-inoculated chickens during the 21-day observation period. However, histopathologic examination of tissues revealed myocardial necrosis, nephritis, and pneumonitis at 5 and 10 days postinoculation (DPI); moderate to severe nonsuppurative encephalitis also was observed in brain tissue from one of four inoculated birds examined at 21 DPI. WNV was recovered from blood plasma for up to 8 DPI. Virus titers as high as 10(5)/ml in plasma were observed at 4 DPI. Fecal shedding of virus was detected in cloacal swabs on 4 and 5 DPI only. The WNV also was isolated from myocardium, spleen, kidney, lung, and intestine collected from chickens euthanatized at 3, 5, and 10 DPI. No virus was isolated from inoculated chickens after 10 DPI. Antibodies specific to WNV were detected in inoculated chickens as early as 5 DPI by the plaque reduction neutralization test and 7 DPI by the indirect fluorescent antibody test. Chickens placed in contact with inoculated chickens at 1 DPI lacked WNV-specific antibodies, and no WNV was isolated from their blood plasma or cloacal swabs throughout the 21 days of the experiment.