Elsevier

Veterinary Microbiology

Volume 175, Issues 2–4, 25 February 2015, Pages 224-231
Veterinary Microbiology

Two different genotypes of H1N2 swine influenza virus isolated in northern China and their pathogenicity in animals

https://doi.org/10.1016/j.vetmic.2014.11.031Get rights and content

Highlights

  • Two swine triple-reassortant influenza A (H1N2) viruses were isolated in China.

  • Phylogenetic analyses showed different emergence patterns for the two viruses.

  • Mice infected with the viruses showed weight loss.

  • The viruses could replicate in the nasal cavity and lung tissue of pigs.

Abstract

During 2006 and 2007, two swine-origin triple-reassortant influenza A (H1N2) viruses were isolated from pigs in northern China, and the antigenic characteristics of the hemagglutinin protein of the viruses were examined. Genotyping and phylogenetic analyses demonstrated different emergence patterns for the two H1N2 viruses, Sw/Hebei/10/06 and Sw/Tianjin/1/07. Sequences for the other genes encoding the internal proteins were compared with the existing data to determine their origins and establish the likely mechanisms of genetic reassortment. Sw/Hebei/10/06 is an Sw/Indiana/9K035/99-like virus, whereas Sw/Tianjin/1/07 represents a new H1N2 genotype with surface genes of classic swine and human origin and internal genes originating from the Eurasian avian-like swine H1N1 virus. Six-week-old female BALB/c mice infected with the Sw/HeB/10/06 and Sw/TJ/1/07 viruses showed an average weight loss of 12.8% and 8.1%, respectively. Healthy six-week-old pigs were inoculated intranasally with either the Sw/HeB/10/06 or Sw/TJ/1/07 virus. No considerable changes in the clinical presentation were observed post-inoculation in any of the virus-inoculated groups, and the viruses effectively replicated in the nasal cavity and lung tissue. Based on the results, it is possible that the new genotype of the swine H1N2 virus that emerged in China may become widespread in the swine population and pose a potential threat to public health.

Introduction

Swine influenza (SI) is a respiratory disease affecting pigs and is caused by the influenza A virus, which is responsible for regular outbreaks of influenza in these animals. Influenza viruses of the H1N1, H3N2, and H1N2 subtypes have circulated in pig populations all over the world since the classic swine (CS) H1N1 influenza virus was first isolated from pigs in 1930 (Brown, 2000, Webby et al., 2000). Pigs can be easily infected by avian and human influenza viruses in addition to SI viruses (SIVs) because the cells in their respiratory tracts contain receptors for both avian and human influenza viruses (Ito et al., 1998a). Furthermore, if influenza viruses of different subtypes were to simultaneously co-infect a pig, the genes of the two viruses might reassort, resulting in a virus that is readily transmissible between pigs.

The first detection of the H1N2 influenza A virus in pigs was in Japan in 1978. The reassortant virus was a classical swine H1N1 virus that had gained the neuraminidase (NA) gene from human H3N2 viruses (Nerome et al., 1985), and this virus was re-isolated in subsequent studies in 1989–1992, 2000–2001, and 2006 (Ito et al., 1998b, Ouchi et al., 1996). Since 1987, H1N2 influenza A viruses have caused widespread outbreaks of respiratory disease among pigs in major swine-producing regions in Europe (Brown et al., 1998, Gourreau et al., 1994, Maldonado et al., 2006, Schrader and Suss, 2003, Trebbien et al., 2013), North America (Ali et al., 2012, Karasin et al., 2006, Karasin et al., 2000), and Asia (Chutinimitkul et al., 2008, Jo et al., 2007, Jung and Chae, 2004, Takemae et al., 2013). In China, an H1N2 outbreak in pigs was reported in 2006 (Qi and Lu, 2006). The outbreak was caused by a double-reassortant virus with the NA gene most closely related to the corresponding gene from the human H3N2 virus circulating in 1995. The remaining seven genes were most closely related to those from CS H1N1 viruses. H1N2 viruses were also reported in China in 2009 and 2011 (Kong et al., 2011, Xu et al., 2009). Efficient surveillance of SIVs is urgently needed to understand their evolution and epidemiology and to address the challenges of monitoring, understanding, and controlling influenza in pigs after the emergence of a novel reassortant pandemic H1N1 (pH1N1/2009) virus in 2009 (Vincent et al., 2014).

During SIV surveillance conducted between 2006 and 2007 in China, two H1N2 viruses were isolated from pigs in northern China. In this study, we examined the genetic composition, antigenicity, and pathogenicity of the H1N2 subtypes isolated from Chinese pigs in 2006 and 2007. We examined the antigenic characteristics of the hemagglutinin (HA) protein of the H1N2 viruses. To investigate these relationships in detail, the sequences of genes were compared with the existing data to determine their origins and establish the likely mechanisms for genetic reassortment. Furthermore, we evaluated the replication and pathogenicity of these viruses in mice and pigs.

Section snippets

Virus isolation and identification

In October 2006, 200 samples were collected from apparently healthy pigs in a slaughterhouse in the Hebei province in China as part of a routine virological surveillance project. In November 2007, approximately 3-month-old pigs and sows at a swine farm in Tianjin were reported to have the typical signs and symptoms of a respiratory disease. In total, 60 swab samples were collected into the transport medium containing antibiotics (Yang et al., 2012) and kept at a temperature of 4 °C until they

Virus isolation and identification

MDCK cell cultures demonstrated cytopathic effects and HA positivity, and the HA agent was characterized using hemagglutination- and NA-inhibition assays. Based on the results, the isolates were identified as type A SIV belonging to the H1N2 subtype. The isolates were designated as A/swine/Hebei/10/2006 (H1N2) and A/swine/Tianjin/1/2007 (H1N2), respectively.

Antigenic analysis of swine influenza isolates

The HI assay, performed with the antigen and antiserum, was used to analyze the swine viruses. The two isolates were antigenically similar

Discussion

In Southern China, frequent contact between wild birds, human, poultry, and pigs creates an opportunity for interspecies transmission and generation of new reassortment influenza viruses. Moreover, the cold and dry weather in northern China may contribute to rapid transmission of SIVs (Lowen et al., 2007). In the present study, A/swine/Hebei/10/2006 appeared to be derived from North American-lineage triple-reassortant H1N2 strains. The isolation of A/swine/Tianjin/1/2007 provides further

Conflict of interest

None of the authors have any personal or financial conflicts of interest relating to the outcomes of this study.

Acknowledgements

This study was supported by the 973 Program (2011CB505001, 2010CB534001), the Chinese National Science Fund for Distinguished Young Scholars (30825032), the National Natural Science Foundation of China (31302108), and the Scientific Research Program of the State Key Laboratory of Veterinary Biotechnology (NKLVSP201209).

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