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Short Communication |
Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0342, USA
Correspondence
X.-J. Meng
xjmeng{at}vt.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the avirulent HEV sequence obtained in this study is EF206691.
The GenBank accession numbers for all sequences and primers used in this study are available as supplementary material in JGV Online.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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; Purcell & Emerson, 2001). Hepatitis E is an important human disease of public health importance in many developing countries. Sporadic cases of acute hepatitis E have also been reported in industrialized countries including the USA (Harrison, 1999; Meng, 2000, 2003, 2005; Purcell & Emerson, 2001; Schlauder et al., 1998; Wang et al., 2001). The disease mostly occurs in young adults and the mortality is generally low, but can reach up to 25 % in infected pregnant women (Khuroo & Kamili, 2003). HEV is primarily transmitted by the faecal–oral route (Emerson & Purcell, 2003). Recently, zoonotic food-borne transmissions of HEV have been reported in Japan (Matsuda et al., 2003; Tamada et al., 2004; Tei et al., 2003).
HEV is a single-stranded, positive-sense, non-enveloped RNA virus belonging to the genus Hepevirus (Emerson et al., 2004). The genome size is 7.2 kb and has three open reading frames (ORFs) (Emerson et al., 2001; Purcell & Emerson, 2001). The largest of the three ORFs is ORF1, which encodes the non-structural proteins, including the methyltransferase, protease, helicase and RNA-dependent RNA polymerase (RdRp), while ORF2 encodes the capsid protein. The small ORF3 encodes a cytoskeleton-associated phosphoprotein (Purcell & Emerson, 2001).
The first animal strain of HEV, swine hepatitis E virus (swine HEV), was isolated from a pig in the USA in 1997 (Meng et al., 1997) and shown to be closely related to human HEV strains worldwide (Hsieh et al., 1999; Huang et al., 2002a; Nishizawa et al., 2003; Okamoto et al., 2001; Takahashi et al., 2003a, b; van der Poel et al., 2001; Wang et al., 2002). More recently, another animal strain of HEV, avian HEV, was identified from chickens with hepatitis–splenomegaly (HS) syndrome in the USA (Haqshenas et al., 2001). Avian HEV is genetically and antigenically related to human HEV, and is associated with a hepatic disease. The clinical and pathological findings associated with avian HEV infection in chickens under the natural route of infection have been reported (Billam et al., 2005). The complete genomic sequence of avian HEV was determined (Huang et al., 2004). In spite of an approximately 50 % nucleotide sequence identity with mammalian HEVs, avian HEV shares many significant structural and functional features with human and swine HEVs. Avian HEV also shares approximately 80 % nucleotide sequence identity with the Australian chicken big liver and spleen disease virus (Haqshenas et al., 2001; Payne et al., 1999).
Recently we found that antibodies to avian HEV were also prevalent in healthy chicken flocks in the USA (Huang et al., 2002b), suggesting that chickens are subclinically infected. From a prospective study, we isolated an apparently avirulent strain of avian HEV from healthy chickens without clinical disease (Sun et al., 2004). Chickens became infected at about 3–4 months of age under natural conditions; however, the clinical signs of HS syndrome were absent. Since the avian HEV strain from healthy chickens does not cause any clinical disease in the field, it is important to determine its complete sequence and compare it to the pathogenic strain from chickens with HS syndrome. Thus far, the complete sequence for avian HEV is reported only for a single pathogenic strain (Huang et al., 2004).
The original virus material used for the determination of the complete sequence of the apparently avirulent avian HEV was collected from healthy chickens in a prospective study (Sun et al., 2004). The original virus was further biologically amplified by infection of young specific-pathogen-free (SPF) chickens to produce a larger virus stock. Six out of seven inoculated chickens had seroconverted by 18 weeks post-inoculation. The faecal and bile suspension harvested from two of the SPF chickens at 3 weeks post-inoculation was positive for avian HEV RNA, and was pooled to generate a virus stock for use in this study.
Primers used for the amplification and sequencing of genomic fragments of the avirulent avian HEV strain in this study were designed on the basis of the prototype pathogenic avian HEV (primer sequences are shown in Supplementary Table S1, available in JGV Online). Briefly, RNA was extracted with TRI reagent (MRC) from 200 µl virus stock. Total RNA was resuspended in 12.25 µl DNase-, RNase- and proteinase-free water. Reverse transcription was performed at 42 °C for 60 min with respective reverse primer and SuperScript II reverse transcriptase (Invitrogen). Five microlitres of the resulting cDNA was amplified in a 50 µl reaction using Platinum PCR Supermix high fidelity (Invitrogen) by two-round nested PCR.
To identify the extreme 3' genomic sequence, we employed the 3' rapid amplification of cDNA ends (RACE) technique using a commercially available 3' adapter and outer and inner antisense primers (Ambion). Total RNA, extracted from 200 µl avirulent avian HEV material, was resuspended in 9 µl DNase-, RNase- and proteinase-free water (Invitrogen), and then reverse-transcribed at 55 °C using a thermostable reverse transcriptase (ThermoScript; Invitrogen) and a commercial 3' adapter. Outer 3' RACE antisense primer and avirulent avian HEV-specific forward primer F6227 were used in first-round PCR amplification with the following PCR conditions: initial incubation at 94 °C for 9 min, followed by 39 cycles of denaturation at 94 °C for 0.5 min, annealing at 46 °C for 0.5 min, extension at 72 °C for 1 min and a final extension at 72 °C for 7 min. Inner 3' RACE reverse primer and avirulent avian HEV-specific inner forward primer FF6307 were employed in a second-round nested PCR with essentially the same PCR conditions except for an annealing temperature of 48 °C.
The sequence at the extreme 5' end was determined by using the 5' RACE technique. First-strand cDNA synthesis was performed by reverse transcription at 55 °C using a thermostable reverse transcriptase and avirulent avian HEV-specific reverse primer R355. The cDNA was then purified using a S.N.A.P. column (Invitrogen). The 3' end of cDNA was tailed with homopolymeric dCTP using terminal deoxynucleotidyl transferase (TdT) by incubating at 37 °C for 10 min. The cDNA was denatured at 94 °C for 3 min before TdT tailing to disrupt potential secondary structure. Nested PCR was performed on dC-tailed cDNA. First-round PCR was done with avirulent avian HEV-specific reverse primer R339 and abridged anchor primer (Invitrogen) with the following PCR conditions: initial incubation at 94 °C for 9 min, followed by 10 cycles of denaturation at 94 °C for 0.5 min, annealing at 50 °C for 0.5 min, extension at 72 °C for 1 min, followed by 25 cycles of denaturation at 94 °C for 0.5 min, annealing at 55 °C for 0.5 min, extension at 72 °C for 1 min and a final extension at 72 °C for 7 min. Second-round nested PCR was done using reverse primer R252 and abridged universal amplification primer (Invitrogen) with the following PCR conditions: initial incubation at 94 °C for 9 min, followed by 39 cycles of denaturation at 94 °C for 0.5 min, annealing at 58 °C for 0.5 min, extension at 72 °C for 1 min and a final extension at 72 °C for 7 min.
The PCR products were excised from 0.8 % agarose gel, purified using a Geneclean III kit (Qbiogene) and sequenced at the Virginia Bioinformatics Institute (Blacksburg, VA, USA). The primer-walking strategy was employed to determine the complete genomic sequence of both DNA strands. The sequence was assembled and analysed using Lasergene version 6 (DNASTAR) and MacVector version 9.0 (MacVector, Inc.) computer programs. The consensus sequence was obtained by assembling approximately 19 overlapping sequences. Each sequence is based, in a majority of cases, on at least four sequence reads, two each from forward and reverse primers. Multiple nucleotide and amino acid alignments were analysed using CLUSTAL W of the MacVector program. Phylogenetic analyses were performed using the PAUP program version 4.0 (David Swofford, Smithsonian Institute, Washington, DC, USA).
The complete genomic sequence of the apparently avirulent strain of avian HEV has been deposited in the GenBank database with accession no. EF206691 [GenBank] . GenBank accession nos for other HEV strains used in the study are available in Supplementary Table S2 in JGV Online.
The complete genome of the avirulent strain of avian HEV, excluding the 3' poly(A) tail, is 6649 nt in length, 5 nt shorter than the prototype avian HEV genome and 605 nt shorter than mammalian HEV genomes. The 5' non-coding region (NCR) is 24 nt and is the same length as that of the prototype avian HEV genome. The ORF1 encodes a polyprotein of 1530 aa in length, 1 aa shorter than that of ORF1 of the prototype avian HEV. ORF2 is 1821 nt in length and encodes a putative capsid protein of 606 aa, identical in length to that of prototype avian HEV. ORF3 comprises 264 nt and encodes a small, 87 aa long protein. The 3' NCR, excluding the poly(A) tail, is 128 nt in length, 2 nt shorter than that of the prototype strain. The nucleotide sequence identities of the avirulent strain of avian HEV with mammalian HEVs and prototype pathogenic avian HEV are presented in Table 1.
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), with nucleotide sequence identity ranging from 89 to 92 % in different functional domains. Two non-silent mutations, V187I and S201T, were identified in the methyl transferase domain. Compared to the prototype strain, the papain-like cysteine protease domain of the avirulent strain had a single mutation, K498R, while the helicase gene region underwent three mutations, V854I, I936M and R950K. The RdRp domain encoded 483 aa and had six non-silent mutations (L1071S, V1106A, I1171V, G1179E, D1382E and R1402H). Multiple deletions and insertions were also noted in the avirulent strain. The hypervariable region (HVR) of the avirulent strain was positioned 8 aa ahead that of the prototype strain. The HVR contains 23 mutations.
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). The tetrapeptide APLT is conserved, as is the case in other strains of HEV. A 54 aa deletion present in the prototype avian HEV compared to mammalian HEVs was also observed in the avirulent strain. The ORF3 shared 97 % nucleotide sequence identity with the prototype avian HEV with four non-silent mutations (A45V, A48V, I54T and A59V).
The 5' NCR of the avirulent strain is identical in length to that of the prototype avian HEV, but the 3' NCR has two deletions at positions corresponding to nt 6558 and 6612, respectively, of the prototype strain. Phylogenetic analyses revealed that the avirulent avian HEV clustered together with the prototype avian HEV but was distantly related to mammalian HEVs (Fig. 2a). The avian HEV isolates from different geographical locations, the prototype avian HEV and the apparently avirulent avian HEV isolate are heterogeneic in the capsid gene (Fig. 2b).
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). Unfortunately, only a single strain of avian HEV has thus far been fully sequenced (Huang et al., 2004). The prototype avian HEV shares significant structural and functional motifs with mammalian HEVs (Haqshenas et al., 2001; Huang et al., 2004), indicating that it belongs to the same genus (Hepevirus) as human and swine HEVs (Emerson et al., 2004). Like the prototype avian HEV, the avirulent strain shares significant features with mammalian HEV strains. Apart from the deletions and mutations observed in the ORF1 of prototype avian HEV, an additional 3 nt deletion (coding for a glycine) with no change in the reading frame was also noted in the avirulent strain. The HVR was the most divergent region not only with the mammalian HEVs, but also with the prototype avian HEV strain.
ORF1 of the avirulent strain contains the majority of mutations compared to the prototype avian HEV. A total of 41 mutations occurred in ORF1, with nine of them in helicase and RdRp genes, the regions responsible for virus replication. It has been demonstrated that a single or a few amino acid changes can alter the virulence and attenuation phenotypes, tissue tropism and cell culture adaptation of numerous viruses. For examples, the Y73H mutation in the 3Dpol region of a temperature-resistant mutant S138C5 of Sabin type 1 poliovirus strain was found to contribute to the neurovirulence phenotype in monkeys. This mutation was also found in the neurovirulent wild-type Mahoney strain, but not in the attenuated phenotype Sabin type 1 (Christodoulou et al., 1990). Point mutations leading to G108V or G108D amino acid changes in the putative N-terminal cleavage site of the porcine transmissible gastroenteritis virus replicase resulted in a 3 log reduction in virus titre (Galán et al., 2005). The identified critical mutations in the functional domains of ORF1, encoding proteins and enzymes involved in virus replication of the avirulent avian HEV, may influence the replicative competence and may be responsible for naturally occurring attenuation phenotype of avian HEV. Clearly, additional studies are warranted to systematically determine which mutation(s) in the ORF1 are critical for virus attenuation.
Six unique non-silent mutations were identified in the capsid gene of the avirulent strain. One of the non-silent mutations (R600K) is located in the putative antigenic domain IV of the prototype avian HEV and another in the putative signal peptide (C4R) that is necessary for translocation of the peptide into the endoplasmic reticulum. The mutation from a hydrophobic cysteine to a highly charged hydrophilic arginine (C4R) could potentially alter the signal peptide’s secondary structure and function. The R600K mutation in the antigenic domain IV might have an effect on the virulence characteristics of this isolate. Virus capsid is the major determinant of virus virulence and it is well known that mutations in the capsid gene can attenuate viruses. It has been shown that the genetic basis of the attenuation phenotype of poliovirus, serotype 3, lies in an S91F amino acid substitution in the coat protein VP3, along with a point mutation in the non-coding region (Westrop et al., 1989). Another Sabin 3-specific amino acid mutation of capsid protein VP1 (I6T) was also found to be contributing to the attenuating phenotype of this vaccine strain (Tatem et al., 1992). It is interesting to note that just a few amino acid mutations drastically diminished the neurovirulence of this strain and made it safe enough to use as a vaccine. In foot-and-mouth disease virus, the amino acid arginine at codon 56 in the capsid protein VP3 was reported to be critical for cell tropism and plaque morphology, making the variant bearing the arginine residue attenuated in vivo in cattle (Sa-Carvalho et al., 1997). The identification of six unique non-silent mutations in the capsid gene of the avirulent strain suggests that they may play a potential role in determining the attenuation phenotype of avian HEV. Further experiments, beyond the scope of this study, to characterize these six non-silent mutations will be helpful in understanding the genetic basis of HEV attenuation. It will now be important to definitively assess the pathogenic nature of this apparently avirulent strain and compare its pathogenicity with the prototype avian HEV strain under laboratory conditions in SPF chickens. The availability of the complete genomic sequence of the apparently avirulent strain will now afford us the opportunity to study the genetic determinants of HEV virulence and aid HEV vaccine development efforts in the future.
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ACKNOWLEDGEMENTS |
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Received 4 December 2006;
accepted 5 January 2007.
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