Susceptibility of Chicken Embryos, Sheep, Cattle, Pigs, and Chickens to Zika Virus Infection.

The susceptibility of sheep, cattle, pigs, chickens and chicken embryos to Zika virus infection was evaluated by experimental inoculation with Zika virus Thailand strain isolated from a Canadian traveler in 2013. The inoculated animals did not develop any clinical signs of disease nor evidence of Zika virus replication in peripheral blood, cerebrospinal fluid and tissues including brain and spinal cord assessed by real-time RT-PCR. Sera were also negative for Zika virus antibodies by Zika virus neutralization assays as well as Zika virus immunoperoxidase staining of Zika infected Vero cells. Chicken embryos were inoculated by different routes including yolk sac (4 day old embryos), chorioallantoic membrane (8 day old embryos), amniotic fluid (8 day old embryos) and intravenous routes (12 day old embryos). Virus replication in chicken embryos was observed in the brain and body tissues following intravenous (IV), yolk sac (YS), chorioallantoic membrane (CAM), and amniotic fluid (AF) inoculation routes. The highest mortality was observed in embryos inoculated via yolk sac. The dead embryos showed diffuse muscular hemorrhages. The yolk sac inoculated chicken embryos showed delayed hatching and displayed neurological signs immediately after hatching. These studies demonstrate that 8 week old sheep, 6 month old cattle, 4 week old pigs, and 4 week old chickens are not susceptible to Zika virus infection when inoculated experimentally and therefore unlikely to pose a risk as Zika virus reservoirs. However, chicken embryos are highly susceptible to Zika virus resulting in clinical disease of chicks after hatching. This study demonstrates that Zika virus has a tropism for embryonic tissue and that chicken embryos can be used as a model to study Zika virus replication and pathogenesis.

Pathobiological Characterization of a Novel Reassortant Highly Pathogenic H5N1 Virus Isolated in British Columbia, Canada, 2015.

In the current study, we describe the pathobiologic characteristics of a novel reassortant virus - A/chicken/BC/FAV-002/2015 (H5N1) belonging to clade 2.3.4.4 that was isolated from backyard chickens in British Columbia, Canada. Sequence analyses demonstrate PB1, PA, NA and NS gene segments were of North American lineage while PB2, HA, NP and M were derived from a Eurasian lineage H5N8 virus. This novel virus had a 19 amino acid deletion in the neuraminidase stalk. We evaluated the pathogenic potential of this isolate in various animal models. The virus was highly pathogenic to mice with a LD50 of 10 plaque forming units (PFU), but had limited tissue tropism. It caused only subclinical infection in pigs which did result in seroconversion. This virus was highly pathogenic to chickens, turkeys, juvenile Muscovy ducks (Cairnia moschata foma domestica) and adult Chinese geese (Anser cynoides domesticus) causing a systemic infection in all species. The virus was also efficiently transmitted and resulted in mortality in naïve contact ducks, geese and chickens. Our findings indicate that this novel H5N1 virus has a wide host range and enhanced surveillance of migratory waterfowl may be necessary in order to determine its potential to establish itself in the wild bird reservoir.

Hemagglutinin-Neuraminidase Balance Influences the Virulence Phenotype of a Recombinant H5N3 Influenza A Virus Possessing a Polybasic HA0 Cleavage Site.

UNLABELLED: Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype. IMPORTANCE: To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance.

Molecular characterization of pandemic H1N1 influenza viruses isolated from turkeys and pathogenicity of a human pH1N1 isolate in turkeys.

Suspected human-to-animal transmission of the 2009 pandemic H1N1 (pH1N1) virus has been reported in several animal species, including pigs, dogs, cats, ferrets, and turkeys. In this study we describe the genetic characterization of pH1N1 viruses isolated from breeder turkeys that was associated with a progressive drop in egg production. Sequence analysis of all eight gene segments from three viruses isolated from this outbreak demonstrated homology with other human and swine pH1N1 isolates. The susceptibility of turkeys to a human pH1N1 isolate was further evaluated experimentally. The 50% turkey infectious dose (TID50) for the human isolate A/Mexico/LnDRE/4487/2009 was determined by inoculating groups of 8-10-week-old turkeys with serial 10-fold dilutions of virus by oronasal and cloacal routes. We estimated the TID50 to be between 1 x 10(5) and 1 x 10(6) TCID50. The pathogenesis of pH1N1 in oronasally or cloacally inoculated juvenile turkeys was also examined. None of the turkeys exhibited clinical signs, and no significant difference in virus shedding or seroconversion was observed between the two inoculation groups. More than 50% of the turkeys in both oronasal and cloacal groups shed virus beginning at 2 days postinoculation (dpi). All birds that actively shed virus seroconverted by 14 dpi. Virus antigen was demonstrated by immunohistochemistry in the cecal tonsils and bursa of Fabricius in two of the birds that were infected by the cloacal route. Virus transmission to naive contact turkeys was at best doubtful. This report provides additional evidence that pH1N1 can cross the species barrier and cause disease outbreaks in domestic turkeys. However, it appears that the reproductive status of the host as well as environmental factors such as concurrent infections, stress, the presence or absence of litter, and stocking density may also contribute to efficient infection and transmission of this agent.

The roles of national and provincial diagnostic laboratories in the eradication of highly pathogenic H7N3 avian influenza virus from the Fraser Valley of British Columbia, Canada.

In February 2004 a highly pathogenic avian influenza outbreak erupted in the Fraser Valley of British Columbia, Canada. The index farm was a chicken broiler breeder operation comprising two flocks, 24 and 52 wk of age. Birds in the older flock presented with a mild drop in egg production and a small increase in mortality. Pathological specimens taken from the older flock were submitted to the provincial veterinary diagnostic laboratory from which an influenza A virus was isolated. While still under investigation by the provincial veterinary authorities, a spike in mortality was observed in birds belonging to the younger flock. Diagnostic material from both flocks was forwarded to the Canadian Food Inspection Agency's National Centre for Foreign Animal Disease. A low-pathogenicity H7N3 virus was detected in the older flock and a novel highly pathogenic H7N3 virus was found in specimens collected from the younger flock. Despite destruction and disposal of birds on the index farm, the virus spread to adjacent farms. Given the high density of poultry operations in the Fraser Valley and the high level of integration amongst industry support services, a total of approximately 17 million chickens, turkeys, ducks, geese, and speciality birds were put at immediate risk. Despite movement controls the virus spread and established itself in three distinct clusters. To prevent further spread, healthy, marketable birds outside of the surveillance areas were pre-emptively slaughtered. Although highly pathogenic avian influenza is a federal responsibility, the successful control and eradication of this outbreak would not have been possible without the cooperative involvement of federal and provincial diagnostic laboratories. The success of this collaboration was partly responsible for the formation of a national avian influenza laboratory network.

Relationship between H5N2 avian influenza viruses isolated from wild and domestic ducks in British Columbia, Canada.

In the summer of 2005 a Canadian national surveillance program for influenza A viruses in wild aquatic birds was initiated. The program involved collaboration between federal and provincial levels of government and was coordinated by the Canadian Cooperative Wildlife Health Centre. The surveillance plan targeted young-of-the-year Mallards along with other duck species at six sampling locations along the major migratory flyways across Canada. Beginning in early August, cloacal swabs were taken from 704 ducks on two lakes adjacent to one another near Kamloops, British Columbia. The swabs were screened for the presence of influenza A RNA using a real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay that targets the M1 gene. Swab samples that gave positive results underwent further testing using H5- and H7-specific RRT-PCR assays. One hundred and seventy-four cloacal swab specimens gave positive or suspicious results for the presence of an H5 virus. A portion of these (28/35) were confirmed using an H5-specific conventional reverse transcription-polymerase chain reaction assay and an H5 virus was eventually isolated from 24/127 swab specimens. Neuraminidase typing revealed the presence of H5N2 and H5N9 viruses. In mid-November of 2005 an H5N2 virus was detected in a commercial duck operation in the lower mainland of British Columbia, approximately 120 km from where the H5N2-positive wild ducks were sampled. Molecular genetic analysis of the H5N2 viruses isolated from wild and domestic ducks was carried out to determine their kinship.

Intersegmental recombination between the haemagglutinin and matrix genes was responsible for the emergence of a highly pathogenic H7N3 avian influenza virus in British Columbia.

In February 2004 a highly pathogenic avian influenza (HPAI) outbreak erupted in British Columbia. Investigations indicated that the responsible HPAI H7N3 virus emerged suddenly from a low pathogenic precursor. Analysis of the haemagglutinin (HA) genes of the low and high pathogenic viruses isolated from the index farm revealed the only difference to be a 21 nt insert at the HA cleavage site of the highly pathogenic avian influenza virus. It was deduced that this insert most probably arose as a result of non-homologous recombination between the HA and matrix genes of the same virus. Over the course of the outbreak, a total of 37 isolates with, and 3 isolates without inserts were characterized. The events described here appear very similar to those which occurred in Chile in 2002 where the virulence shift of another H7N3 virus was attributed to non-homologous recombination between the HA and nucleoprotein genes.

Comparison of assays for the detection of West Nile virus antibodies in chicken serum.

Six tests for the detection of West Nile virus (WNV) antibodies in the serum of experimentally infected chickens were compared. The tests included the hemagglutination-inhibition test (HIT), immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (ELISA) with WNV-infected mouse brain antigen, immunoglobulin G (IgG) indirect ELISA with tickborne encephalitis viral antigen, the microtitre virus neutralization test, the standard plaque reduction neutralization test (PRNT), and the microtitre PRNT (micro-PRNT). Thirty adult chickens, intravenously and intramuscularly inoculated with 10(7) plaque-forming units (PFU) of WNV strain Egypt 101, were bled and given a booster of 10(7) PFU at 7,15, and 21 d postinoculation; the final blood collection was on day 28. Although the micro-PRNT is capable of detecting the highest antibody titres during both early and late infection, because of the technical complexity and time requirements of this test a combination of IgM and IgG ELISAs is recommended for serologic screening. Serum samples that give positive results in the ELISAs can then be tested by the micro-PRNT to determine the specificity of antibodies to WNV.