Molecular Characterization of Animal Strains of Hepatitis E Virus (HEV): Avian HEV and Swine HEV
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Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. It mainly infects young adults and has a mortality of up to 25% in pregnant women. Although hepatitis E is only sporadic in industrialized countries including the United States, a relative high seroprevalence rate has been reported in healthy individuals. Evidence suggests that there exist animal reservoirs for HEV and HEV transmission is zoonotic. Animal strains of HEV, swine HEV and avian HEV have been identified from a pig and a chicken, respectively, in the United States. Studies showed that swine HEV and avian HEV are genetically and antigenically related to human HEV, and that pigs and chickens are useful animal models to study HEV replication, pathogenesis and cross-species infection. The objectives of this dissertation were to genetically characterize both avian HEV and swine HEV, to determine their serological and molecular epidemiology in the United States, to assess the ability of avian HEV cross-species infection in non-human primates, to determine the full-length genomic sequence and genome organization, and to construct an infectious cDNA clone of avian HEV. The prevalence of swine HEV infections in US swine herds and the heterogeneity of swine HEV isolates from different geographic regions of the United States were determined. We found that 35% pigs and 54% swine herds were positive for swine HEV RNA. Partial capsid gene region of twenty-seven US swine HEV isolates was sequenced and was showed to share 88%-100% nucleotide sequence identity to each other and 89-98% identity with the prototype US swine HEV, but only <79% identity with Taiwanese swine HEV isolates and most known human strains of HEV worldwide. All US swine HEV isolates belong to the same genotype 3 with the prototype US swine HEV and the two US strains of human HEV. Similarly, the prevalence of avian HEV infections in US chicken flocks and the heterogeneity of avian HEV isolates were also determined. Helicase gene region of eleven field isolates of avian HEV from chickens with hepatitis-splenomegaly (HS) syndrome was sequenced and was found to share 78-100% nucleotide sequence identities with each other, 79-88% identities with the prototype avian HEV, 76-80% identities with Australian chicken big liver and spleen disease virus (BLSV), and 56-61% identities with other known strains of mammalian HEV. A relative high prevalence of anti-avian HEV antibodies was found in apparently healthy chicken flocks in 5 states. Like swine HEV, the seropositivity of avian HEV in adult chickens was higher than that in young chickens. To genetically characterize the avian HEV genome, we determined the full-length genomic sequence of avian HEV, which is 6,654 bp in length excluding the poly (A) tail, and 600 bp shorter than that of mammalian HEVs. Avian HEV has similar genomic organization with human and swine HEVs, but shared only about 50% nucleotide sequence identity with mammalian HEVs in the complete genome. Significant genetic variations such as deletions and insertions, particularly in the ORF1 of avian HEV, were observed, but motifs in the putative functional domains of the ORF1 were relatively conserved between avian HEV and mammalian HEVs. Phylogenetic analyses based on the full-length genomic sequence revealed that avian HEV represents a branch distinct from human and swine HEVs. Since swine HEV infects non-human primates and possibly humans, the ability of avian HEV cross-species infection in non-human primates was also assessed. However, unlike swine HEV, avian HEV failed to infect two rhesus monkeys under experimental conditions. With the availability of the complete genome sequence of avian HEV, we constructed three full-length cDNA clones of avian HEV and tested their infectivity by in vitro transfection of the LMH chicken liver cells and by in vivo intrahepatic inoculation of specific-pathogen-free (SPF) chickens. The results showed that all 3 cDNA clones of avian HEV were infectious both in vitro and in vivo, as the capped RNA transcripts from each of the clones were replication-competent in transfected LMH cells and developed active infection in inoculated SPF chickens. In summary, avian HEV and swine HEV infections are enzootic in chicken flocks and in swine herds in the United States, respectively. Like human HEV, swine HEV and avian HEV isolates from different geographic regions are also genetically heterogenic. Complete genomic sequence analyses showed that avian HEV is related to, but distinct from, human and swine HEVs. Unlike swine HEV, avian HEV is probably not transmissible to non-human primates. Infectious cDNA clones of avian HEV have been successfully constructed. The availability of the infectious clones for a chicken strain of HEV now affords us an opportunity to study the mechanisms of HEV replication, pathogenesis and cross-species infection.