Yohannes Berhane

Recombinant Nipah Virus Vaccines Protect Pigs against Challenge

ABSTRACT Nipah virus (NiV), of the family Paramyxoviridae, was isolated in 1999 in Malaysia from a human fatality in an outbreak of severe human encephalitis, when human infections were linked to transmission of the virus from pigs. Consequently, a swine vaccine able to abolish virus shedding is of veterinary and human health interest. Canarypox virus-based vaccine vectors carrying the gene for NiV glycoprotein (ALVAC-G) or the fusion protein (ALVAC-F) were used to intramuscularly immunize four pigs per group, either with 108 PFU each or in combination. Pigs were boosted 14 days postvaccination and challenged with 2.5 × 105 PFU of NiV two weeks later. The combined ALVAC-F/G vaccine induced the highest levels of neutralization antibodies (2,560); despite the low neutralizing antibody levels in the F vaccinees (160), all vaccinated animals appeared to be protected against challenge. Virus was not isolated from the tissues of any of the vaccinated pigs postchallenge, and a real-time reverse transcription (RT)-PCR assay detected only small amounts of viral RNA in several samples. In challenge control pigs, virus was isolated from a number of tissues (104.4 PFU/g) or detected by real-time RT-PCR. Vaccination of the ALVAC-F/G vaccinees appeared to stimulate both type 1 and type 2 cytokine responses. Histopathological findings indicated that there was no enhancement of lesions in the vaccinees. No virus shedding was detected in vaccinated animals, in contrast to challenge control pigs, from which virus was isolated from the throat and nose (102.9 PFU/ml). Based on the data presented, the combined ALVAC-F/G vaccine appears to be a very promising vaccine candidate for swine.

Genetic and Pathobiologic Characterization of Pandemic H1N1 2009 Influenza Viruses from a Naturally Infected Swine Herd

ABSTRACT Since its initial identification in Mexico and the United States, concerns have been raised that the novel H1N1 influenza virus might cause a pandemic of severity comparable to that of the 1918 pandemic. In late April 2009, viruses phylogenetically related to pandemic H1N1 influenza virus were isolated from an outbreak on a Canadian pig farm. This outbreak also had epidemiological links to a suspected human case. Experimental infections carried out in pigs using one of the swine isolates from this outbreak and the human isolate A/Mexico/InDRE4487/2009 showed differences in virus recovery from the lower respiratory tract. Virus was consistently isolated from the lungs of pigs infected with A/Mexico/InDRE4487/2009, while only one pig infected with A/swine/Alberta/OTH-33-8/2008 yielded live virus from the lung, despite comparable amounts of viral RNA and antigen in both groups of pigs. Clinical disease resembled other influenza virus infections in swine, albeit with somewhat prolonged virus antigen detection and delayed viral-RNA clearance from the lungs. There was also a noteworthy amount of genotypic variability among the viruses isolated from the pigs on the farm. This, along with the somewhat irregular pathobiological characteristics observed in experimentally infected animals, suggests that although the virus may be of swine origin, significant viral evolution may still be ongoing.

Invasion of the Central Nervous System in a Porcine Host by Nipah Virus

ABSTRACT Nipah virus, a newly emerged zoonotic paramyxovirus, infects a number of species. Human infections were linked to direct contact with pigs, specifically with their body fluids. Clinical signs in human cases indicated primarily involvement of the central nervous system, while in pigs the respiratory system was considered the primary virus target, with only rare involvement of the central nervous system. Eleven 5-week-old piglets were infected intranasally, orally, and ocularly with 2.5 × 105 PFU of Nipah virus per animal and euthanized between 3 and 8 days postinoculation. Nipah virus caused neurological signs in two out of eleven inoculated pigs. The rest of the pigs remained clinically healthy. Virus was detected in the respiratory system (turbinates, nasopharynx, trachea, bronchus, and lung in titers up to 105.3 PFU/g) and in the lymphoreticular system (endothelial cells of blood and lymphatic vessels, submandibular and bronchiolar lymph nodes, tonsil, and spleen with titers up to 106 PFU/g). Virus presence was confirmed in the nervous system of both sick and apparently healthy animals (cranial nerves, trigeminal ganglion, brain, and cerebrospinal fluid, with titers up to 107.7 PFU/g of tissue). Nipah virus distribution was confirmed by immunohistochemistry. The study presents novel findings indicating that Nipah virus invaded the central nervous system of the porcine host via cranial nerves as well as by crossing the blood-brain barrier after initial virus replication in the upper respiratory tract.

Reassortant Highly Pathogenic Influenza A H5N2 Virus Containing Gene Segments Related to Eurasian H5N8 in British Columbia, Canada, 2014

In late November 2014 higher than normal death losses in a meat turkey and chicken broiler breeder farm in the Fraser Valley of British Columbia initiated a diagnostic investigation that led to the discovery of a novel reassortant highly pathogenic avian influenza (HPAI) H5N2 virus. This virus, composed of 5 gene segments (PB2, PA, HA, M and NS) related to Eurasian HPAI H5N8 and the remaining gene segments (PB1, NP and NA) related to North American lineage waterfowl viruses, represents the first HPAI outbreak in North American poultry due to a virus with Eurasian lineage genes. Since its first appearance in Korea in January 2014, HPAI H5N8 spread to Western Europe in November 2014. These European outbreaks happened to temporally coincide with migratory waterfowl movements. The fact that the British Columbia outbreaks also occurred at a time associated with increased migratory waterfowl activity along with reports by the USA of a wholly Eurasian H5N8 virus detected in wild birds in Washington State, strongly suggest that migratory waterfowl were responsible for bringing Eurasian H5N8 to North America where it subsequently reassorted with indigenous viruses.

Investigation into the role of potentially contaminated feed as a source of the first-detected outbreaks of porcine epidemic diarrhea in Canada.

Summary In January 2014, approximately 9 months following the initial detection of porcine epidemic diarrhea (PED) in the USA, the first case of PED was confirmed in a swine herd in south‐western Ontario. A follow‐up epidemiological investigation carried out on the initial and 10 subsequent Ontario PED cases pointed to feed as a common risk factor. As a result, several lots of feed and spray‐dried porcine plasma (SDPP) used as a feed supplement were tested for the presence of PEDV genome by real‐time RT‐PCR assay. Several of these tested positive, supporting the notion that contaminated feed may have been responsible for the introduction of PEDV into Canada. These findings led us to conduct a bioassay experiment in which three PEDV‐positive SDPP samples (from a single lot) and two PEDV‐positive feed samples supplemented with this SDPP were used to orally inoculate 3‐week‐old piglets. Although the feed‐inoculated piglets did not show any significant excretion of PEDV, the SDPP‐inoculated piglets shed PEDV at a relatively high level for ≥9 days. Despite the fact that the tested PEDV genome positive feed did not result in obvious piglet infection in our bioassay experiment, contaminated feed cannot be ruled out as a likely source of this introduction in the field where many other variables may play a contributing role.

Susceptibility of Canada geese (Branta canadensis) to highly pathogenic avian influenza virus (H5N1).

Prior exposure of Canada geese to a North American low pathogenic virus (H5N2) decreases their susceptibility to Eurasian highly pathogenic avian influenza virus (H5N1).

Animal models of henipavirus infection: A review

Hendra virus (HeV) and Nipah virus (NiV) form a separate genus Henipavirus within the family Paramyxoviridae, and are classified as biosafety level four pathogens due to their high case fatality rate following human infection and because of the lack of effective vaccines or therapy. Both viruses emerged from their natural reservoir during the last decade of the 20th century, causing severe disease in humans, horses and swine, and infecting a number of other mammalian species. The current review summarises current published data relating to experimental infection of small and large animals, including the natural reservoir species, the Pteropus bat, with HeV or NiV. Susceptibility to infection and virus distribution in the individual species is discussed, along with the pathogenesis, pathological changes, and potential routes of transmission.

Highly pathogenic avian influenza virus A (H7N3) in domestic poultry, Saskatchewan, Canada, 2007.

Epidemiologic, serologic, and molecular phylogenetic methods were used to investigate an outbreak of highly pathogenic avian influenza on a broiler breeding farm in Saskatchewan, Canada. Results, coupled with data from influenza A virus surveillance of migratory waterfowl in Canada, implicated wild birds as the most probable source of the low pathogenicity precursor virus.

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

Abstract 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/InDRE/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 × 105 and 1 × 106 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.

Characterization of H1N1 Swine Influenza Viruses Circulating in Canadian Pigs in 2009

ABSTRACT The 2009 pandemic H1N1 (pH1N1), of apparent swine origin, may have evolved in pigs unnoticed because of insufficient surveillance. Consequently, the need for surveillance of influenza viruses circulating in pigs has received added attention. In this study we characterized H1N1 viruses isolated from Canadian pigs in 2009. Isolates from May 2009 were comprised of hemagglutinin and neuraminidase (NA) genes of classical SIV origin in combination with the North American triple-reassortant internal gene (TRIG) cassette, here termed contemporary SIV (conSIV) H1N1. These conSIV H1N1 viruses were contiguous with the North American αH1 cluster, which was distinct from the pH1N1 isolates that were antigenically more related to the γH1 cluster. After the initial isolation of pH1N1 from an Alberta pig farm in early May 2009, pH1N1 was found several times in Canadian pigs. These pH1N1 isolates were genetically and antigenically homogeneous. In addition, H1N1 viruses bearing seasonal human H1 and N1 genes together with the TRIG cassette and an NA encoding an oseltamivir-resistance marker were isolated from pigs. The NS gene of one of these seasonal human-like SIV (shSIV) H1N1 isolates was homologous to pH1N1 NS, implicating reassortment between the two strains. Antigenic cross-reactivity was observed between pH1N1 and conSIV but not with shSIV H1N1. In summary, although there was cocirculation of pH1N1 with conSIV and shSIV H1N1 in Canadian pigs after May 2009, there was no evidence supporting the presence of pH1N1 in pigs prior to May 2009. The possibility for further reassortants being generated exists and should be closely monitored.