Improved one-tube RT-PCR method for simultaneous detection and genotyping of duck hepatitis A virus subtypes 1 and 3.

BACKGROUND: The cocirculation of duck hepatitis A virus subtypes 1 (DHAV-1) and 3 (DHAV-3) in ducklings has resulted in significant economic losses. Ducklings with DHAV-1 or DHAV-3 infection show similar clinical signs and gross lesions; hence, it is important to identify the viral subtypes in infected ducklings as early as possible for better clinical management. METHODS AND RESULTS: Based on multiple 5' noncoding region (5'-NCR) sequences of DHAV-1 and DHAV-3 strain alignments, universal and type-specific primers were designed and synthesized. With three primers in one-tube reverse transcription-PCR (RT-PCR), reference DHAV-1 and DHAV-3 isolates ranging over 60 years and across many different countries were successfully amplified, indicating that the primer sequences were completely conserved. The sequence results and the sizes of amplicons from reference DHAV-1 and DHAV-3 isolates are completely correlated with their subtypes. Moreover, with this one-tube RT-PCR system, amplicon sizes from liver samples of reference DHAV-1- or DHAV-3-infected birds fit closely with their subtypes, which was determined by virus isolation and neutralization testing. No other duck-origin RNA viruses were detected. The sensitivity of viral RNA detection was 10 pg. With this system, 20% subtype 1, 45% subtype 3, and 9% coinfection of two subtypes were detected in 55 clinical samples. CONCLUSIONS AND SIGNIFICANCE: This novel approach could be used for rapidly typing DHAV-1 or DHAV-3 infection in routine clinical surveillance or epidemiological screening.

Comparative pathogenicity of two subtypes (hepatitis-type and pancreatitis-type) of duck hepatitis A virus type 1 in experimentally infected Muscovy ducklings.

Duck hepatitis A virus type 1 (DHAV-1) causes acute hepatitis with high morbidity and mortality in ducklings of the genera Cairina and Anas and is characterized by ecchymotic haemorrhage and necrosis of the liver surface. Since September 2011, a new subtype of DHAV-1 (named pancreatitis-type DHAV-1) has been isolated. This new subtype is characterized by yellowish or haemorrhagic pancreatitis, but with no significant pathological changes in the liver. To further investigate the difference in pathogenicity between hepatitis-type DHAV-1 and pancreatitis-type DHAV-1, we infected Muscovy ducklings with a hepatitis-type DHAV-1 strain, FZ86, or a pancreatitis-type DHAV-1 strain, MPZJ1206, and then compared the resulting gross lesions, histopathological changes, viral distribution and cellular apoptosis in the liver and pancreas of Muscovy ducklings. The results suggested that FZ86 induced a more efficient viral propagation in the liver than MPZJ1206, and the gross and histopathological lesions were also limited to the liver. However, MPZJ1206 induced more effective viral replication in the pancreas than FZ86. The MPZJ1206-infected Muscovy ducklings showed an obviously yellowed and haemorrhagic pancreas, but with no significant pathological changes in the liver. Furthermore, FZ86 induced notable hepatocyte apoptosis and increased the expression of caspase-3 in the liver, whereas MPZJ1206 caused apoptosis in a large number of acinar epithelial cells and elevated the expression of caspase-3 in the pancreas. Taken together, these results demonstrated that pancreatitis-type DHAV-1 has many new pathogenic features which distinguish it from the hepatitis-type DHAV-1. RESEARCH HIGHLIGHTS Pancreatitis-type DHAV-1 (MPZJ1206) was characterized by pancreatic haemorrhage and yellow discolouration, but with no obvious haemorrhage and necrosis in the liver. Pancreatitis-type DHAV-1 (MPZJ1206) exhibits many new pathogenic features which distinguish it from the hepatitis-type DHAV-1 (FZ86).

Molecular epidemiology and genetic diversity of duck hepatitis A virus type 3 in Shandong province of China, 2012-2014.

 The infections with duck hepatitis A virus type 3 (DHAV-3) become common in eastern Asia. To better understand the molecular evolution and genetic variation of DHAV-3, a total of 482 dead Cherry Valley duckling liver samples collected from Shandong province of China during 2012-2014 were tested, and the complete P1 coding sequences of 18 DHAV-3 strains were analyzed. The detection rate of DHAV-3 was 64.5% (311/482) in clinical liver samples and 73.0% (92/126) in duckling flocks. The P1 genes of the 18 DHAV-3 isolates shared 91.9%-99.0% nucleotide similarity and 95.2%-100% amino acid similarity with those of the other 26 reference strains. Based on the P1 and VP1 gene sequences, phylogenetic analysis results indicated that the genotyping of DHAV-3 strains presented a distinct geographical distribution. Except B63 strain, all Chinese strains isolated from different host species (duck or goose) at different time were classed into the CH genotype. All Korean and Vietnamese strains belonged to the KV genotype, and all the Korean strains were clustered into KV1 subgenotype, while B63 strain and the Vietnamese strains from different host species (duck or goose) were clustered into KV2 subgenotype. Ten variable amino acid residues were highly conserved within genotypes or subgenotypes in the VP0, VP3 and VP1, respectively, which were possibly the geographic molecular markers of DHAV-3. To the best of our knowledge, this is the first study about the genetic variation of the P1 gene of different DHAV-3 strains, which will be helpful for understanding of the molecular epidemiology of DHAV-3.

A one-step duplex rRT-PCR assay for the simultaneous detection of duck hepatitis A virus genotypes 1 and 3.

Duck hepatitis A virus (DHAV) is a highly infectious pathogen that causes significant bleeding lesions in the viscera of ducklings less than 3 weeks old. There are three serotypes of DHAV: serotype 1 (DHAV-1), serotype 2 (DHAV-2) and serotype 3 (DHAV-3). These serotypes have no cross-antigenicity with each other. To establish an rRT-PCR assay for the rapid detection of a mixed infection of DHAV-1 and DHAV-3, two pairs of primers and a pair of matching TaqMan probes were designed based on conserved regions of DHAV-1 VP0 and DHAV-3 VP3. Finally, we established a one-step duplex rRT-PCR assay with high specificity and sensitivity for the simultaneous detection of DHAV-1 and DHAV-3. This method showed no cross-antigenicity with the other pathogens tested, including duck plague virus, Muscovy duck parvovirus, Riemerella anatipestifer, and pathogenic E. coli from ducks. Sensitivity tests identified the minimum detection limits of this method as 98 (DHAV-1) and 10 (DHAV-3) copies/reaction. To validate the method, thirty-eight clinical samples and thirty artificially infected samples collected from dead duck embryos were studied. Thirty-seven samples were positive for DHAV-1, seventeen samples were positive for DHAV-3, and fourteen samples were positive for a mixed infection using the duplex rRT-PCR method. The method established in this study is specific, sensitive, convenient and timesaving and is a powerful tool for detecting DHAV-1, DHAV-3, and their mixed infection and for conducting surveys of pandemic virus strains.

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

OBJECTIVE: To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. METHODS: We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. RESULTS: DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. CONCLUSION: The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

Epidemiology and molecular characterisation of duck hepatitis A virus from different duck breeds in Egypt.

Duck hepatitis virus (DHV) is an acute highly contagious disease of ducklings caused by three distinct serotypes of duck hepatitis A virus (DHAV), a member of the RNA family Picornaviridae, where serotype 1 is the most widespread serotype worldwide. To date, little if any is known about the prevalence and genetic characterisation of DHAV outside Asia. The current study describes surveillance on DHV in 46 commercial duck farms in Egypt with a history of high mortality in young ducklings from 3 to 15 day-old from 2012 to 2014. Clinical samples were examined by generic RT-PCR assays followed by partial sequence analysis of the 5'UTR, VP1 and 3D genes of the vaccine strain and 15 field viruses. The overall positive rate was 37% (n=17/46). All duck breeds (Pekin, Muscovy, Mallard and Green Winged) were susceptible to the disease with mortality ranged from 15% to 96.7%. Sequence and phylogenetic analyses indicated that the Egyptian strains cluster in the DHAV serotype 1 with Asian viruses and distinguishable from the vaccine strains. So far, this is the first report on the genetic characterisation of DHAV in Egypt. This study may be useful to better understand the epidemiology and evolution of DHAV.

The prevalence of duck hepatitis A virus types 1 and 3 on Korean duck farms.

This study reports the prevalence of duck hepatitis A virus (DHAV) types 1 and 3 on Korean duck farms. By RT-nested PCR assays specific for DHAV-1 or DHAV-3, DHAV-1 was detected in 9 of 157 liver samples (5.7 %) from 2 of 30 farms (6.7 %), and DHAV-3 was positive in 104 of 157 liver samples (66.2 %) from 23 of 30 farms (76.7 %). Dual infections with DHAV-1 and DHAV-3 were detected in 23 of 157 samples (14.6 %) from 5 of 30 farms (16.7 %). The data indicate that DHAV-3 infections are prevalent and that DHAV-1 reemerged in Korea, resulting in dual infections on several farms. Our data will help to establish a vaccination policy against DHAV-1 and DHAV-3 in Korea.

[Molecular characteristic of duck hepatitis A virus type 1 causing pancreatitis ].

[OBJECTIVE] We studied the molecular characteristics of the full-length genome of duck hepatitis A virus type 1 causing pancreatitis in Muscovy ducklings. [METHODS] We determined the entire genomic sequence of duck hepatitis A virus type 1 strain MPZJ1206 using reverse transcription polymerase chain reaction assay and analyzed the bioinformatics of the viral genome sequence. [ RESULTS] The genome length of strain MPZJ1206 comprised 7703 bases, with a G + C content of 43.05%. The genome of MPZJ1206 contains a single, long open reading frame encoding a polypeptide of 2249 amino acids, with a genomic orgariization similar to those of other isolates of duck hepatitis A virus type 1. MPZJ1206 is identical with previously isolates by 93. 5% - 99. 6% in nucleotide sequence and 97. 9% - 99. 6% in amino acid sequence and shares genetic distance no more than 7%. Phylogenetic analysis based on genome sequence indicates that MPZJ1206 shares a close genetic relationship with two strains isolated in 2011. [CONCLUSION] Although pathotype caused by MPZJ1206 strain is significantly distinct from those induced by classical isolates of duck hepatitis A virus type 1, the genome of MPZJ1206 shares high homology with those of previous isolates. The change of pathotype may result from an alteration in viral tissue tropism of MPZJ1206.

Recombinant VP1 protein of duck hepatitis virus 1 expressed in Pichia pastoris and its immunogenicity in ducks.

The VP1 gene of duck hepatitis virus type 1 (DHV-1) strain VJ09 was amplified by reverse transcription PCR from the liver of a duckling with clinical symptoms of viral hepatitis. The resulting VP1 cDNA was 720 bp in length and encoded a 240-amino-acid protein. In VP1 gene-based phylogenetic analysis, the VJ09 strain grouped with DHV-1 genotype C. The VP1 gene was inserted into the expression vector pPICZαA and expressed in Pichia pastoris. The expressed VP1 protein was purified and identified by western blot analysis. To evaluate the recombinant VP1's immunogenic potential in ducklings, the antibodies raised in the immunized ducklings were titrated by ELISA, and lymphocyte proliferation and virus neutralization assays were performed. The results show that the recombinant VP1 protein induced a significant immune response in ducklings and this could be a candidate for the development of a subunit vaccine against DHV-1 genotype C.

Duck hepatitis A virus (DHAV) genotype definition: comment on the article by Cha et al.

Duck hepatitis A virus (DHAV) is genetically divided into three different genotypes: 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). Recently, Cha et al. (2013) concluded that the existence that both DHAV-1 and DHAV-2 had been classified into one branch, with DHAV genotype 3 (DHAV-3) in another, and that the phylogenetic distance unit showed was 0.5, a tremendous value. However, there might be some concerns on the methodology application to define the genotypes of DHAV. Based on 110 genomic and 100 amino acid sequences of DHAV which included all the sequences from Cha et al. (2013) respectively, phylogenetic analysis in the present study showed a distinct and proposed DHAV genotype definition, that both DHAV-2 and DHAV-3 were clustered in one branch while DHAV-1 in another branch only, and that the phylogenetic distance unit of 0.02 was confirmed, which was much smaller than the value 0.5. Taking into account the genotype definition of DHAV, we also conducted the pairwise sequence comparisons (PASC) analysis of 110 genomic sequences, and proposed that the distance genotype definition threshold was 0.045.

Molecular characterization of a duck hepatitis virus 3-like astrovirus.

Using an ORF1b-based astrovirus-specific reverse transcription (RT)-PCR assay, a duck hepatitis virus type 3 (DHV-3)-like astrovirus was detected from four intestinal samples collected from diseased ducks in China. Complete genome sequencing and comparative sequence analysis showed that the four duck astrovirus (DAstV) isolates were closely related and possessed a typical astrovirus genome organization. Genetic analysis of the complete ORF2 region revealed that mean amino acid genetic distances between the DHV-3-like isolates and previously known avastrovirus species were between 0.579 and 0.721, suggesting that the DHV-3-like isolates could be classified as an additional avastrovirus species. In the ORF1a and ORF1b regions, however, mean amino acid genetic distances between the DHV-3-like viruses and the turkey astrovirus 2 (TAstV-2)-like isolates were substantially less than those between TAstV-2-like isolates and DAstV/C-NGB-like astroviruses belonging to the same species. Pairwise comparisons and phylogenetic analyses demonstrated that the DHV-3-like isolates were most closely related to TAstV-2-like viruses in ORF1a and ORF1b, while showed highest similarity with the chicken astrovirus (CAstV) 612-like viruses in ORF2. These findings provide evidence that recombination events may have occurred during evolution of the avastroviruses and support the view that genomic analysis is required for classification of the avastroviruses.

Improved duplex RT-PCR assay for differential diagnosis of mixed infection of duck hepatitis A virus type 1 and type 3 in ducklings.

Infection with duck hepatitis A virus (DHAV) causes an acute, rapidly spreading, and fatal disease of young ducklings. DHAV type 1 (DHAV-1) and type 3 (DHAV-3) have been identified in China. In this study, a duplex RT-PCR assay was developed to identify DHAV-1 and DHAV-3 with mixed infection. The method was shown to be high specificity and sensitivity. The minimum detection limit of the method has been determined to be 10pg total RNA templates extracted from duck liver samples or 10² copies viral RNA of DHAV-1 and DHAV-3 respectively. Using the method, from 60 clinical liver samples of 26 duckling flocks in Shandong, Guangdong, Sichuan and Henan provinces of China, 15 (57.7%) flocks were identified as mixed infection of DHAV-1 and DHAV-3, and 9 (34.6%) flocks were DHAV-1 or DHAV-3 single infection. Among them, 38.3% (23/60) of duckling samples were detected as mixed infection of DHAV-1 and DHAV-3, and 48.3% (29/60) of samples were DHAV-1 or DHAV-3 single infection. These results indicated that the improved duplex RT-PCR method provides a rapid and cost-effective laboratory differential diagnosis for mixed infection of DHAV-1 and DHAV-3 in ducklings.

Isolation and characterization of a low pathogenic duck hepatitis A virus 3 from South Korea.

The D11-JW-018 strain of duck hepatitis A virus (DHAV) was isolated from 7-day-old ducklings in Kyeonggi province, South Korea with no clinical signs of typical hepatitis. Phylogenetic analyzed of whole genome showed that D11-JW-018 strain was belonged to DHAV-3 genotype. The pathogenicity of the D11-JW-018 strain in 1-, 7-, 14-, and 21-day-old ducklings was examined. Mortality of D11-JW-018 strain was lower than DRL-62 (DHAV-1) age-dependent but incubation period was longer in 1-day-old ducklings. Unlike DRL-62 strain infection, D11-JW-018 strain induced only liver discoloration without hemorrhagic mottling and lymphocyte infiltration and bile duct hyperplasia in histological lesion. The D11-JW-018 strain was detected only in the heart, liver, spleen, gall bladder, pancreas, and kidney among 12 organs in infected 1-day-old ducklings. Serum biochemical analyses revealed a significant difference in aspartate transaminase and alanine transaminase between the D11-JW-018 strain-infected ducklings and those infected with the DRL-62 strain (P<0.05). We identified the D11-JW-018 strain in South Korean ducklings and provide the characteristics of DHAV-3.

[Sequence analysis of VP1 gene of the duck hepatitis A virus type 3 strains isolated from Shandong Province of China in 2012].

To reveal the genetic variation of the viral protein 1 (VP1) gene of the duck hepatitis A virus type 3 (DHAV-3), the VP1 gene of 13 virulent DHAV-3 strains isolated from Shandong province of China in 2012 were amplified by RT-PCR, sequenced and analyzed. The results showed that all the VP1 genes of the 13 isolates contained 720 nucleotides encoding 240 amino acids, and shared with nucleotide identities of 94. 6%-99.9% and amino acid identities of 95.0%-100%. The nucleotide and amino acid sequence homologies between the 13 DHAV-3 isolates and other 31 DHAV-3 reference strains were 92.5%-100% and 90. 8%-100%, respectively. Phylogenetic analysis showed that the VP1 gene of DHAV-3 had distinct geographical characteristics. Distribution of genotypes of the 44 DHAV-3 strains was as follows: except the vaccine strain B63, all the other Chinese isolates belonged to genotype I (GI), Vietnamese wild isolates mainly belonged to subtype 1 (S1) of genotype II (GII), and all Korean isolates belonged to subtype 2 (S2) of GII.

[Overview on duck virus hepatitis A].

This article describes the nomenclature, history and genetic evolution of duck hepatitis A virus, and updates the epidemiology, clinical symptom and surveillances of duck virus hepatitis A. It also summarizes the present status and progress of duck virus hepatitis A and illustrated the necessity and urgency of its research, which provides rationale for the control of duck hepatitis A virus disease in China.

Complete genome sequence of a novel duck hepatitis A virus discovered in southern China.

We report here the complete genomic sequence of a novel duck hepatitis A virus (DHAV) isolated from mixed infections with DHAV type 1 (DHAV-1) and DHAV-3 in ducklings in Southern China. The whole nucleotide sequence had the highest homology with the sequence of DHAV-3 (GenBank accession number DQ812093) (96.2%). To our knowledge, this is the first report of gene rearrangement between DHAV-1 and DHAV-3, and it will help to understand the epidemiology and molecular characteristics of duck hepatitis A virus in Southern China.

Goose haemorrhagic hepatitis caused by a new subtype duck hepatitis type 1 virus.

Duck hepatitis type 1 virus (DHV-1) causes a fatal disease in ducklings but there is no report of DHV-1 isolation from goose. Recently, cases of a new disease in overfeeding geese were reported from China. The cases were characterized by haemorrhagic hepatitis lesions on liver after post mortem examinations. The flocks showed about 20-40% morbidity and less than 5% mortality. The histopathological lesions showed destroyed structure of hepatocytic tissue, severe vacuolation and necrosis of hepatocytes. Viral antigen could be detected by monoclonal antibody against duck hepatitis type 1 virus (DHV-1) in the cytoplasm of positive hepatocytes. PCR amplified viral sequences with primers specific for recent Korean-like duck hepatitis type 1 virus (DHV-1C). Alignment of the complete sequence demonstrated that the isolated JT strain from goose exhibiting 95.9% identity to DHV-1C AP-03337 strain, and only 75.3% to classical DHV-1 virus. 80% goslings developed haemorrhagic hepatitis after infection with JT strain. This is the first report on the involvement of a DHV-1 virus in goose.

Analysis of codon usage in type 1 and the new genotypes of duck hepatitis virus.

In this study, an abundant (A+U)% and low codon bias were revealed in duck hepatitis virus type 1 (DHV-1) and the new serotype strains isolated from Taiwan, South Korea and Mainland China (DHV-N). The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in these samples. By comparative analysis of the codon usage patterns of 40 ORFs of DHV, we found that all of DHV-1 strains grouped in genotype C; the DHV-N strains isolated in South Korea and China clustered into genotypes B; and the DHV-N strains isolated from Taiwan clustered into genotypes A. The findings revealed that more than one subtype of DHV-1 circulated in East Asia. Furthermore, the results of phylogenetic analyses based on RSCU values and Clustal W method indicated obvious phylogenetic congruities. This suggested that better genome consistency of DHV may exist in nature and phylogenetic analyses based on RSCU values maybe a good method in classifying genotypes of the virus. Our work might give some clues to the features and some evolutionary information of DHV.

[Genomic sequence of a new serotype duck hepatitis virus].

OBJECTIVE: A strain of highly pathogenic duck hepatitis virus was isolated in south China from a Pekin duck flock in 1999. No cross reaction with type 1 and 3 duck hepatitis virus was found by serum neutralization. We suggested the strain should be classified as a new serotype of duck hepatitis virus and named it as DHV-N G strain in our previous study. We wanted to reveal the evolution of this virus in molecular level and gene sequence differences between it and DHV-1 strains. METHODS: We used RT-PCR and 5'-/3'-RACE to amplify the complete genome sequence of DHV-N G strain and compared it with other picornaviruses. RESULTS: The genome of DHV-N G consists of 7774 nucleotides excluding a poly (A) tail of 12 nucleotides. It contains a single large open reading frame encoding a polypeptide of 2251 amino acid residues. The length of 3' UTR of DHV-N G is 366 nucleotides, which is 52 nucleotides longer than that of DHV-1 C80 strain. Significant amino acid variation was found in the protein VP1, especially at the position of 140 - 221 comparing with DHV-1. The DHV-N G genome shares 72.8% - 73.4%, 96.3% - 96.5% and 78.3% similarity with DHV-1 strains, Korea's and Taiwanese DHV-N strains, respectively. CONCLUSION: The genome structure of DHV-N G strain is obviously different with that of DHV-1 strains. The homologies of genome among the DHV-N group are variable, therein DHV-N G strain is more homologous with Korea's strains than with Taiwanese.

Identification of chicken enterovirus-like viruses, duck hepatitis virus type 2 and duck hepatitis virus type 3 as astroviruses.

Earlier work identified and biologically characterized antigenically distinct enterovirus-like viruses (ELVs) of chickens. Three of these ELVs can now be identified as astroviruses. Characterization involved the use of a hitherto undescribed, degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) to amplify astrovirus open reading frame (ORF) 1b-specific cDNA fragments followed by nucleotide sequence determination and analysis of the amplified fragments. ELV-1 was confirmed as an isolate of the astrovirus avian nephritis virus (ANV). ELV-4 (isolate 612) and ELV-3 (isolates FP3 and 11672) were antigenically and genetically related to the second characterized astrovirus of chickens, namely chicken astrovirus (CAstV). Using indirect immunofluorescence, the FP3 and 11672 ELV-3 isolates were very closely related to one another, and less closely related to ELV-4 and the previously described CAstV (P22 18.8.00 reference isolate). Comparative analyses based on the ORF 1b amplicon sequences showed that the FP3 and 11672 ELV-3 isolates shared high nucleotide (95%) and amino acid (98%) identities with one another, and lower nucleotide (76% to 79%) and amino acid (84% to 85%) identity levels with ELV-4 and the reference CAstV P22 18.8.00 isolates. The combined degenerate primer RT-PCR and sequencing methods also provided a nucleotide sequence specific to duck hepatitis virus type 2 (DHV-2) (renamed duck astrovirus) and duck hepatitis virus type 3 (DHV-3), which, for the first time, can also be identified as an astrovirus. Phylogenetic analyses based on the amplified ORF 1b sequences showed that ANV was the most distantly related avian astrovirus, with DHV-3 being more closely related to turkey astrovirus type 2 than DHV-2.