Genetic and pathobiologic characterization of H3N2 canine influenza viruses isolated in the Jiangsu Province of China in 2009–2010

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Abstract

The newly emerging canine influenza virus (CIV) causes considerable concerns for both veterinary and public health. During 2009–2010, six strains of H3N2 influenza virus were isolated from dogs in Jiangsu Province, China. Sequence and phylogenetic analysis of eight gene segments revealed that the six viruses were most similar 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 Jiangsu isolates against the most similar avian strains, we found that all isolates had several common mutations at the receptor-binding sites, potential glycosylation sites and cleavage site in HA1, and antigenic sites in both the HA1 and NA segments. Significantly, a unique two amino acid insertion in the NA stalk was found. Experimental infection of BALB/c mice revealed that viral RNA could be detected in the major rodent organs, such as brain, heart, spleen, kidney, liver and intestine, as well as the lung. All the sampled organs from infected mice showed significant lesions and viral antigen staining. This study highlights the potential of domesticated animals to become a reservoir for influenza virus and the need for surveillance programs to detect cross-species transmission.

Introduction

Influenza A is a highly contagious disease that has led to severe local and pandemic disease outbreaks, seriously threatening human health and causing tremendous economic losses in the poultry industry. Influenza A viruses can be isolated from a wide variety of species, including humans, pigs, horses, sea mammals and birds. In 2004, dogs were discovered as new hosts, coming as a surprise because they were previously thought to be refractory to infection with influenza viruses. The first canine influenza virus (CIV) subtype H3N8 emerged in racing greyhounds in Florida in January 2004 (Crawford et al., 2005), and then seven months later, the virus was also isolated in shelter and pet dogs (Payungporn et al., 2008). Thousands of greyhound dogs, at tracks in nine states, were subsequently affected during multiple respiratory disease outbreaks from 2004 to 2006 (Yoon et al., 2005). In 2007, another canine influenza outbreak was confirmed in Korea (Song et al., 2008), but this time the causal agent was an H3N2 avian influenza virus. The H3N2 virus was isolated during outbreaks of severe respiratory disease in dogs at multiple facilities, and had been demonstrated to be capable of transmitting directly from dog to dog (Song et al., 2009). In 2010, the interspecies transmission of H3N2 CIV to cats was firstly reported in Korea (Song et al., 2011). Additionally, a fatal infection in dogs and cats with the highly pathogenic avian influenza virus (HPAIV) H5N1 was also reported in several countries in Asia (Desvaux et al., 2009, Songserm et al., 2006). However, transmission of the infection to in-contact dogs or cats was not observed (Butler, 2006). In China, the first case of H3N2 CIV was reported in Guangdong Province in 2010 (Li et al., 2010b). As all of these cases of canine influenza have caused considerable problems and concerns for both veterinary and public health, the surveillance of this virus is of great importance.

Influenza A virus (family Orthomyxoviridae) is an enveloped virus with an eight segmented negative stranded RNA genome encoding 12 viral proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), M1, M2, NS1, NS2 (NEP: nuclear export protein), PA, PB2, PB1, PB1-F2 and PB1-N40, a newly identified protein that is expressed from the PB1 segment (Wise et al., 2009). Of these proteins, HA and NA are the two major surface glycoproteins on the virion used for determination of the subtype of the virus. HA and NA are targeted by the protective immune response and can vary as a result of antigenic drift and antigenic shift (Shu et al., 1994).

In this study, we provided the molecular and biological properties of six strains of H3N2 CIV, which were isolated from the dogs with severe respiratory syndrome in the Jiangsu Province of China. Genetic sequence data from six viruses were valuable for understanding the mutation frequencies associated with replication of the virus in the canine host. Experimental infections of BALB/c mice provided insights into the pathobiological behavior of these viruses in dogs.

Section snippets

Samples

A total of 13 nasopharyngeal swabs of dogs with severe respiratory syndrome were collected from Animal Clinics of Nanjing Agricultural University in Jiangsu Province of China from November 2009 to July 2010. These dogs were kept as domestic pets and the diseases were sporadic. The samples were taken from individual dogs at different time but showing similar respiratory symptoms such as coughing, sneezing and copious nasal discharge. The pet dogs did not have contact with birds, but it is

Virus isolation

Six strains of influenza virus were isolated out of the 13 samples and designated as A/Canine/Jiangsu/01/2009 (01), A/Canine/Jiangsu/02/2010 (02), A/Canine/Jiangsu/03/2010 (03), A/Canine/Jiangsu/04/2010 (04), A/Canine/Jiangsu/05/2010 (05) and A/Canine/Jiangsu/06/2010 (06), with virus titers of 106.71 EID50/200 μl, 106.16 EID50/200 μl, 106 EID50/200 μl, 105.71 EID50/200 μl, 106.3 EID50/200 μl, and 107.2 EID50/200 μl, respectively.

Homology analysis of nucleotide sequences

All eight genes of the six virus isolates were amplified, sequenced and

Discussion

Influenza viruses are widespread in nature and have the capacity to cross interspecies barriers and adapt to new hosts by altering antigenic characteristics (Peiris et al., 2007, Webster et al., 1992). The viral HA protein is a major contributor to host range, as it is responsible for receptor-binding (Ito, 2000). Avian influenza A viruses are generally thought to preferentially bind to sialic-acid α-2,3-galactose (SAα-2,3 gal) receptor, whereas human or swine influenza A viruses preferentially

Acknowledgements

This work was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PATD) and the Program for New Century Excellent Talents in University (NCET-07-0440).

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