College of Agriculture & Natural Resources
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Item Partial direct contact transmission in ferrets of a mallard H7N3 influenza virus with typical avian-like receptor specificity(Springer Nature, 2009-08-14) Song, Haichen; Wan, Hongquan; Araya, Yonas; Perez, Daniel RAvian influenza viruses of the H7 subtype have caused multiple outbreaks in domestic poultry and represent a significant threat to public health due to their propensity to occasionally transmit directly from birds to humans. In order to better understand the cross species transmission potential of H7 viruses in nature, we performed biological and molecular characterizations of an H7N3 virus isolated from mallards in Canada in 2001. Sequence analysis that the HA gene of the mallard H7N3 virus shares 97% identity with the highly pathogenic avian influenza (HPAI) H7N3 virus isolated from a human case in British Columbia, Canada in 2004. The mallard H7N3 virus was able to replicate in quail and chickens, and transmitted efficiently in quail but not in chickens. Interestingly, although this virus showed preferential binding to analogs of avian-like receptors with sialic acid (SA) linked to galactose in an α2–3 linkage (SAα2–3Gal), it replicated to high titers in cultures of primary human airway epithelial (HAE) cells, comparable to an avian H9N2 influenza virus with human-like α2–6 linkage receptors (SAα2–6Gal). In addition, the virus replicated in mice and ferrets without prior adaptation and was able to transmit partially among ferrets. Our findings highlight the importance and need for systematic in vitro and in vivo analysis of avian influenza viruses isolated from the natural reservoir in order to define their zoonotic potential.Item Improved hatchability and efficient protection after in ovo vaccination with live-attenuated H7N2 and H9N2 avian influenza viruses(2011-01-21) Cai, Yibin; Song, Haichen; Ye, Jianqiang; Shao, Hongxia; Padmanabhan, Rangarajan; Sutton, Troy C; Perez, Daniel RMass in ovo vaccination with live attenuated viruses is widely used in the poultry industry to protect against various infectious diseases. The worldwide outbreaks of low pathogenic and highly pathogenic avian influenza highlight the pressing need for the development of similar mass vaccination strategies against avian influenza viruses. We have previously shown that a genetically modified live attenuated avian influenza virus (LAIV) was amenable for in ovo vaccination and provided optimal protection against H5 HPAI viruses. However, in ovo vaccination against other subtypes resulted in poor hatchability and, therefore, seemed impractical. In this study, we modified the H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype. We found that with this modification, a single dose in ovo vaccination of 18- day old eggs provided complete protection against homologous challenge with low pathogenic virus in ≥70% of chickens at 2 or 6 weeks post-hatching. Further, inoculation of 19-day old egg embryos with 10 6 EID50 of LAIVs improved hatchability to ≥90% (equivalent to unvaccinated controls) with similar levels of protection. Our findings indicate that the strategy of modifying the HA cleavage site combined with the LAIV backbone could be used for in ovo vaccination against avian influenza. Importantly, with protection conferred as early as 2 weeks post-hatching, with this strategy birds would be protected prior to or at the time of delivery to a farm or commercial operation.Item FUNCTIONAL STUDIES OF INFECTIOUS PANCREATIC NECROSIS VIRUS PROTEINS AND MECHANISM OF VIRUS-INDUCED APOPTOSIS(2003-12-04) Song, Haichen; Vakharia, Vikram N; Animal SciencesInfectious pancreatic necrosis virus (IPNV) encodes a 12 or 15-kDa nonstructural protein, known as VP5. To study the function of VP5, we generated three recombinant viruses rNVI15, rNVI15-15K, and rNVI15-ΔVP5, which could encode either 12-kDa VP5, 15-kDa VP5 or be deficient in VP5, respectively. VP5 was detected in rNVI15 and rNVI-15K infected cells but not in the cells infected with rNVI15-ΔVP5. However, the opal stop codon at nucleotide position 427 in rNVI15 virus was read-through, giving rise to a 15-kDa VP5 that is expressed poorly than rNVI15-15K virus-infected cells. All three recombinant viruses show similar replication kinetics in both Chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells. Moreover, in Sp strains, IPNV segment A could encode a novel, putative 25-kDa protein from another ORF. This 25-kDa protein could not be detected in virus-infected cells, however, we could recover a mutant virus lacking this ORF, indicating that it is not essential for virus replication. To assess the molecular basis of virus adaptation in the cell culture, virulent rNVI15 was serially passaged in CHSE cells nine times to obtain a tissue-culture adapted virus, rNVI15TC. Comparison of the deduced amino acid sequences showed only one amino acid substitution at position 221 (Ala → Thr) in VP2. However, this adaptation mutation is only acquired in CHSE cells but not in RTG-2 cells. Two chimeric viruses, rNVI15ΔVP2 and rNVI15-15KΔVP2 were also generated, in which the residues at positions 217 and 247 in VP2 of the rNVI15 and rNVI15-15K viruses were replaced by the corresponding residues of an attenuated strain, Sp103. These two viruses have similar replication kinetics as Sp103, which replicates faster than rNVI15 in vitro, indicating that residues at positions 217 and 247 of VP2 may be the important markers for virus adaptation and attenuation in vitro. By generating a reassortant virus between rNVI15-15K and Sp103, we also demonstrate that VP1 is not involved in virus cell adaptation. The signal pathways and nature of IPNV-induced apoptosis were investigated in RTG-2 cells. IPNV-induced apoptosis occurs at the late stage of viral life cycle. Caspase-3 is activated during virus infection, and inhibition of caspase-3 could partially inhibit virus-induced apoptosis. Moreover, NF-κB activation is essential for IPNV-induced apoptosis, and it is involved in interferon-induced antiviral state. Both the NF-κB inhibitor and the antioxidant could inhibit NF-κB activity and apoptosis induced by IPNV infection, but they do not affect viral replication.