Genetic And Pathobiologic Characterization Of H3N2 Canine Influenza Virus Isolated In Jiangsu

Canine influenza virus (CIV) is a newly identified, highly contagious respiratory pathogen causing cough, pneumonia and other respiratory symptoms in dogs. The genome of influenza A virus is segmented negative single-stranded RNA, which enables the dynamic evolution of virus by both antigenic drift and genetic shift. The serological surveillance in South Korea and southern China showed that the seroconversion rates for H3N2 IAVs were very high in the sampled dog population and this result suggests that avian-origin H3N2 CIVs are circulating in the two country’s dogs. Since dogs are in regular contact with their owners and other people, it is a concern that the illness could potentially spread from dogs to humans in the future. So, it is important for us to understand the virus evolution and pathobiology in order to control the virus infection. This study focused on the genetic and pathobiologic characterization of H3N2 CIV, as well as effect of a-two amino acid (2-aa) insertion in NA stalk on virus replication.1. Identification and genetic analysis of H3N2 canine influenza viruses isolated in JiangsuDuring 2009-2010, six strains of H3N2 influenza virus were isolated from dogs in Jiangsu, China. There was a high degree of genetic similarity (> 99%) in eight genes among the six Jiangsu canine isolates. All eight genes exhibited the highest homology (>99%) to a recent canine-derived subtype H3N2 influenza virus isolated in cats from South Korea, which originated from avian strain. By comparing the deduced amino acid sequences of the hemagglutinin 1(HA1) and neuraminidase (NA) genes of the six isolates against the most similar avian strains, we found that all isolates had several important mutations. In HA1, there were mutations D81N, A160T and G209 at antigenic sites; W222L at receptor-binding sites, K326R at cleavage site, and T10A and D81N at potential glycosylation sites. In NA, S372L and R432G were found at antigenic sites. Significantly, a unique 2-aa insertion in NA stalk was found in Jiangsu isolates whereas isolates of South Korea and Guangdong did not possessed the insertion. From the eight constructed phylogenetic trees, we can see that the six Jiangsu viruses were grouped together with the newly isolated canine H3N2 viruses in dogs and cats from Korea and China. Additionally, all eight genes of the six viruses were most closely related to the feline isolate from Korea. Comparing the six isolates,06 strain was somewhat unique. Firstly, it was different in the M, NP and NA genes from the other five strains. Secondly, in the phylogenetic analysis of all eight genes, the 06 strain was more distantly related to the other five strains. Thirdly,06 strain had a lower virus titer of EID50. The pathogenic characteristics of 06 strain should be further investigated.2. Pathobiologic characterization of H3N2 canine influenza virus isolated in JiangsuMouse was used as animal model system to investigate the pathobiology of CIV. We inoculated mice with 01 and 06 strains, respectively. In both infected group, viral RNA could be detected in major rodent organs, such as brain, heart, liver, spleen, kidney, and intestine, as well as the lung by nested PCR and a high proportion of animals (3/3) with detectable viral RNA in all tested organs by 6 d.p,i.. But mice inoculated with 06 strain had higher viral RNA loads in tissues at 6 and 8 d.p.i. and lost more body weight than mice inoculated with 01 strain. So we presume that 06 strain has a higher virulence. Immunohistochemical and histopathological observation showed that virus caused lesions and detectable viral antigen in all tested tissues. H3N2 canine influenza virus was surprisingly found to have ability to infect many cell cultures, such as MDCK, A549, H292, canine bronchiolar epithelia cells (CBE), canine bronchiolar smooth muscle (BSM) cells, chicken embryo fibroblasts (CEF) and caused severe cytopathy on these cells. Viral antigen could be detected in all infected cells by indirect immunofluorescence. These result revealed that H3N2 canine influenza virus had broad tissue and cell tropism.3. Establishment of reverse genetics system of H3N2 canine influenza virus and rescue of its NA mutantTo establish reverse genetics system of H3N2 CIV, full-length cDNAs of A/canine/Jiangsu/06/2011(H3N2) were amplified and cloned into the bidirectional pBD vector. The eight plasmids were co-transfected into 293T cells and then the cell lysate was inoculated to MDCK cells. After propagated 3 times in MDCK cell, the virus was successfully generated with designation of r-06. Using this reverse genetics system we rescued the mutant with deletion of 2-aa in NA stalk, which was designated as r-06/NA2. No mutation occurred in the genome of the two rescued viruses. The successful rescue of the r-06 and r-06/NA2 viruses established a foundation for the further functional studies.4. Effect of a-two amino acid insertion in NA stalk on in vitro replication of H3N2 canine influenza virusTo investigate effects of a 2-aa insertion at the end of the NA stalk on virus replication in vitro, we compared replication kinetic of r-06 (long stalk virus with 2 aa insertion) and r-06/NA2 (short stalk virus without 2 aa insertion) viruses on MDCK, CEF and BSM cells. Our data showed that r-06 exhibited higher peak viral titer and more virus yields at all the time points in all three cell cultures, as well as made much larger plaques in MDCK cell monolayers. Flow cytometry analysis confirmed the proportions of r-06-infected MDCK and BSM cells were significantly higher than that in r-06/NA2-infected cells, whereas the ratios of infected CEF cells by the two viral strains were similar. However, r-06 virus expressed significantly more NS1 protein than r-06/NA2 virus did in three cell cultures. Erythrocytes elution assay showed that,2-aa insertion increased NA ability to release virus from canine erythrocytes, but not avian erythrocytes. These observations suggest that avian-origin H3N2 CIVs that acquire 2-aa insertion in NA stalk might more likely be a result of adaptive evolution in canine hosts, and the insertion enhances virus replication ability.5. Effect of a-two amino acid insertion in NA stalk on in vivo replication of H3N2 canine influenza virusTo investigate the effect of the 2-aa insertion on virus replication in vivo, we inoculated the r-06 (long stalk virus with 2 aa insertion) and r-06/NA2 (short stalk virus without 2 aa insertion) viruses to mice and chickens. Mice of both infected groups showed an apparent decrease in weight at 4 d.p.i., but mice infected with the r-06 strain lost more body weight. By 6 d.p.i., both infected groups had a high proportion of animals (3/3) with detectable viral RNA in all tested organs. By 8 d.p.i., r-06 infected mice still exhibited a surprisingly high level of detectable viral RNA in organs, whereas r-06/NA2 infected mice showed a reduction. And viral RNA titer of the brain of r-06 infected mice was statistically higher. Interstitial pneumonia was obsereved in lungs of both groups, but r-06-infected mice showed larger area of thickened alveolar septum. In addition, r-06 strain induced local IFN-y production with faster kinetics and at higher level in mice at 2.d.p.i.. However, in chicken infection experiment, no significant difference was found between the two viral strains with almost same proportion of detectable viral loads in tested tissues. We presume that the elongation of the NA stalk by 2-aa in nature may facilitate more efficient replication and transmission of avian-origin H3N2 CIVs in mammalian but not favorable in avian.