Genetic characterization of Duck Hepatitis A Viruses isolated in China

Highlights

• The 16 reported DHAV isolates (15 DHAV-1 strains and one DHAV-3) were isolated from infected ducks with clinical symptoms in China between 2009 and 2012.

• The various DHAV-1 strains have different lethal doses, and duck eggs were more susceptible to DHAV than chicken eggs.

• We report the genetic characterization of the DHAVs isolated in clinical samples in China, which will be helpful for understanding the epidemiology and evolution of DHAVs.

Abstract

In recent years, the spread of Duck Hepatitis A Viruses (DHAVs) has represented a serious threat and significant economic impact in duck industry of China. The sixteen reported DHAV isolates (15 DHAV-1 strains and one DHAV-3) were identified from infected ducks with clinical symptoms in China between 2009 and 2012. In the present study, the virulence of these viruses and complete sequences of the virion protein 1 (VP1) genes of the 16 DHAVs were characterized. The median embryonic lethal doses (ELD₅₀) of the second generation duck embryo allantoic fluid of the 16 DHAV isolates were calculated on duck and chicken embryos. The results demonstrated that the various DHAV-1 strains have shown different pathogenic ability in embryos, and duck eggs were more susceptible to DHAV than chicken eggs. The histopathological examination revealed significant signs of virus infection, severe vacuolation, and hepatocyte necrosis. Phylogenetic analyses indicated that the 15 DHAV-1 viruses display significant correlation in their geographic distribution. The DHAV-1 strains isolated from Shandong Province were more evolutionarily divergent than the JX strains. There were two hypervariable regions in the VP1 protein, which may determine the virulence of DHAV-1 isolates in chicken eggs but not virulence in duck eggs. These results demonstrate the genetic and biological diversity of DHAVs in China and aid in understanding the epidemiology and evolution of DHAVs.

Introduction

Duck virus hepatitis (DVH) is an acute and fatal disease of young ducklings characterized by rapid transmission and primary hepatitis (Kaleta, 1988). In 1949, DVH was first described on Long Island, New York (Macpherson and Avery, 1957). Subsequently, outbreaks were reported in England, Canada, Germany, Japan, and elsewhere. In 1963, the virus reached Mainland China, though it was not identified until 1984 (Guo and Pan, 1984, Liu et al., 2008). The disease is caused by three different DHV types (1, 2, and 3), and there is no cross-neutralization among these serotypes (Ding and Zhang, 2007, Kim et al., 2006, Tseng et al., 2007). Of the three DHV serotypes, the most common and virulent is DHV-1, which can cause >80% mortality in infected ducklings younger within 3 weeks. In addition, the disease has also been diagnosed in some duck-raising areas (Anchun et al., 2009).

DHV-1 was originally classified as an enterovirus based on the observed morphology and physicochemical properties of the virion. However, it was recently found that DHV-1 was more closely related to members of the genus Parechovirus than to other picornaviruses (Kim et al., 2006, Liu et al., 2008, Wang et al., 2008). In the Virus Taxonomy ninth Report of the International Committee on Taxonomy of Viruses (ICTV), DHV-1 was classified as a member of a novel genus Avihepatovirus in the family Picornaviridae and renamed Duck Hepatitis A Virus (DHAV) (Fu et al., 2008). DHAV is genetically divided into three different types: the original type DHAV-1, a type recently isolated in Taiwan (DHAV-2), and a recently described type isolated in South Korea and China (DHAV-3) (Kim et al., 2007, Kim et al., 2008, Tseng and Tsai, 2007).

The complete genomes of DHV-1 strains isolated from Korea and Taiwan have been sequenced and contain a single-stranded, 7691-nt, positive sense RNA with a 3′-ploy(A) tail. The genome is organized similar to that of other Picornaviridae family members, i.e., as a single, large open reading frame (ORF) flanked by 5′- and 3′-untranslated regions (UTRs). The ORF can be translated into 12 mature proteins, including structural (VP0, VP3, and VP1) and nonstructural proteins (2A1, 2A2, 2A3, 2B, 2C, 3A, 3B, 3C, and 3D) (Wang et al., 2008, Yang et al., 2008). Based on results from other picornaviruses, DHV-1 VP1 is expected to have the highest genetic diversity among isolates.

DHAVs cause an acute, highly lethal infection of young ducklings usually less than three weeks of age (Kim et al., 2009, Liu et al., 2010, Liu et al., 2011a). In China, outbreaks of DVH have occurred in many ducklings that were already vaccinated with the traditional DHAV-1 attenuated vaccine. Thus, a major concern is whether variation of virulence or variation in the external capsid proteins of the pandemic DHAV isolates are responsible for this immune failure (Gao et al., 2012). To learn more about the overall prevalence of DHAVs in China and provide useful data for DVH control, we utilized the DHAV sequences available in GenBank from 1999 to 2011 and sequenced Chinese DHAV viruses isolated between 2009 and 2012 to analyze their genetic evolution.