Avian Rotavirus Research Articles

Partial genome sequence analysis of parvoviruses associated with enteric disease in poultry

Poult enteritis mortality syndrome (PEMS) of turkeys and runting-stunting syndrome (RSS) of chickens are significant viral enteric diseases of poultry. Although a number of different viruses, including avian reoviruses, rotaviruses, astroviruses and coronaviruses, have been isolated from the intestinal contents of birds in affected poultry flocks, their role in PEMS and RSS is not yet understood. Here, we report the application of a molecular screening method to detection of novel viruses in intestinal samples of chickens and turkeys exhibiting characteristic signs of enteric disease. The technique is based on random amplification of particle-associated nucleic acids in clinical samples. Using this method we successfully identified parvovirus DNA sequences in intestinal homogenates of affected birds. This is the first time partial genomic sequences of autonomously replicating chicken and turkey parvoviruses have been described. Sequence analysis of the left end of the genome, including the complete non-structural gene, demonstrated that the chicken and turkey parvoviruses were closely related to each other and were representative of a novel member of the Parvovirus family. These parvoviruses may play a significant role in the aetiology of PEMS and RSS.

Full genome-based classification of rotaviruses reveals a common origin between human Wa-Like and porcine rotavirus strains and human DS-1-like and bovine rotavirus strains.

Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.

A Multiplex RT-PCR Test for the Differential Identification of Turkey Astrovirus Type 1, Turkey Astrovirus Type 2, Chicken Astrovirus, Avian Nephritis Virus, and Avian Rotavirus

Recent studies have revealed the presence ofastroviruses and rotavirus in numerous poorly performing and healthy chicken and turkey flocks in the United States. The phylogenetic analysis of the sequence data produced during these studies has identified four groups of avian astroviruses circulating in the United States: turkey astrovirus types 1 and 2 (TAstV-1 and TAstV-2), avian nephritis virus (ANV), and a chicken-origin astrovirus (CAstV). As the molecular epidemiology of poultry enteric disease is poorly understood, the development of updated diagnostic assays is crucial to the continued surveillance and management of enteric disease in affected as well as healthy flocks. This report details the development of a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for astroviruses and avian rotavirus in turkey-origin and chicken-origin samples. The assay consists of two multiplex tests, one for turkey-origin samples and one for chicken-origin samples. The turkey sample test differentially identifies TAstV-1, TAstV-2, ANV, and avian rotavirus. The test for chicken-origin samples differentially identifies CAstV, ANV, and avian rotavirus. Assay sensitivity varied by target sequence between approximately 10 copies for avian rotavirus alone and approximately 2 x 10(6) copies for TAstV-2 in the presence of a heterologous competitor RNA sequence. Each test was shown to be specific for the intended target by testing for cross-reaction with other common avian enteric viruses. The specificity was further shown by testing 109 chicken specimens and 32 turkey specimens from commercial flocks with the appropriate test and sequencing the RT-PCR amplicons to confirm amplification of the correct target.

Isolation, Identification and Characterization of Group A Rotavirus from a Chicken: the Inner Capsid Protein Sequence shows only a Distant Phylogenetic Relationship to Most Other Avian Group A Rotaviruses

Rotavirus particles were identified in the intestinal content of a 35-day-old stunted chicken. The virus was isolated, RNA pattern was analysed and the viral genome segment 6 was sequenced. In particular, the sequence data showed a very close similarity to the chicken rotavirus isolate Ch-1 (99.2% amino acid homology), this is distantly related to all known avian rotaviruses and supports the existence of different VP6 types amongst avian group A rotaviruses.

Segmented double-stranded genomic RNA viruses in fecal samples from broiler chicken

Segmented double-stranded RNA (dsRNA) viruses were identified by polyacrylamide gel electrophoresis (PAGE) technique in fecal samples from broiler chicken. A total of 378 fecal samples from 1-7 weeks old chickens were analyzed. dsRNA with migration profile characteristic of avian rotavirus (AvRV), reovirus (ARV) or picobirnavirus (PBV) was identified in 32 (8.5%), 7 (1.8%) and 13 (3.4%) samples, respectively. AvRV and ARV occurred more frequently in chickens up to 1 month old and were related with enteritis signs. Considering only fecal samples of chickens with diarrhea, the AvRV was detected in 37.8% (14/37) and the ARV in 13.5% (5/37) of analyzed samples. AvRV was identified in only 1.5% (4/274) and ARV was not detected in normal feces collected from assymptomatic chickens (controls). PBV dsRNA was detected in broiler chickens from two to seven weeks old, more frequently in feces with pasty consistency. The AvRV showed great electrophoretic profile variability in the dsRNA segments and nine different electropherotypes were identified. Variation in genome pattern was not observed in either ARV or PBV.

Roles of outer capsid proteins as determinants of pathogenicity and host range restriction of avian rotaviruses in a suckling mouse model

We previously demonstrated that a pigeon rotavirus, PO-13, but not turkey strains Ty-3 and Ty-1 and a chicken strain, Ch-1, induced diarrhea in heterologous suckling mice. In this study, it was suggested that these avirulent strains, but not PO-13, were inactivated immediately in gastrointestinal tracts of suckling mice when they were orally inoculated. To determine which viral proteins contribute to the differences between the pathogenicitiy and the inactivation of PO-13 and Ty-3 in suckling mice, six PO-13 × Ty-3 reassortant strains that had the genes of the outer capsid proteins, VP4 and VP7, derived from the opposite strain were prepared and were orally inoculated to suckling mice. A single strain that had both PO-13 VP4 and VP7 with the genetic background of Ty-3 had an intermediate virulence for suckling mice. Three strains with Ty-3 VP7, regardless of the origin of VP4, rapidly disappeared from gastrointestinal tracts of suckling mice. These results indicated that the difference between the pathogenicity of PO-13 and that of Ty-3 was mainly dependent on both their VP4 and VP7. In particular, VP7 was found to be related to the inactivation of Ty-3 in gastrointestinal tracts of suckling mice.

Antigenic analysis of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13.

In order to analyze the antigenic structure of nonstructural protein (NSP) 4 of group A avian rotavirus strain PO-13, 25 monoclonal antibodies (MAbs) against NSP4 expressed in Escherichia coli were produced. All MAbs reacted with NSP4 on Western blotting, indicating that they recognized sequential epitopes. To determine the antigenic sites (ASs) recognized by the produced MAbs, seven truncated NSP4s were expressed in E. coli. Western blotting analysis showed that there are at least four major ASs on PO-13 NSP4, designated as AS I located in amino acids (aa) 151 to 169, AS II (aa 136 to 150), AS III (aa 112 to 133) and AS IV (aa 1 to 24). Two MAbs reacted exclusively with AS III encompassing the region that has been reported to be an enterotoxin domain. MAbs against ASs II, III and IV reacted with all avian rotaviruses tested by indirect immunofluorescent antibody assays. MAbs against AS I reacted with turkey strains, Ty-1 and Ty-3, but not with a chicken strain, Ch-1. Nine of 11 MAbs against AS II cross-reacted with NSP4 of mammalian rotavirus strains with different NSP4 genotypes. These results suggest that AS II on NSP4 is widely conserved among a variety of rotaviruses.

Sequential analysis of nonstructural protein NSP4s derived from Group A avian rotaviruses

We determined the NSP4 sequences of turkey rotavirus strains Ty-1 and Ty-3 and a chicken rotavirus, strain Ch-1, and compared these sequences with those of a pigeon rotavirus, strain PO-13, and mammalian rotaviruses. The turkey strains and PO-13 were found to be closely related (90–97% homologies). Ch-1 NSP4 was distinctly different from other avian rotavirus NSP4s, with 78–79% homologies. The NSP4 sequences of avian rotaviruses were found to be 6–7 amino acids shorter than those of all mammalian strains and to have considerably low identities (31–37%) with them. Therefore, it seems highly likely that the NSP4 genes of avian rotaviruses are classified into two NSP4 genotypes distinct from those of mammalian rotaviruses. The enterotoxin domain in NSP4 is conserved in terms of its sequential and structural properties despite extremely low homologies in the full lengths of NSP4s in avian and mammalian rotaviruses.

Diarrhea-inducing activity of avian rotavirus NSP4 glycoproteins, which differ greatly from mammalian rotavirus NSP4 glycoproteins in deduced amino acid sequence in suckling mice.

Avian rotavirus NSP4 glycoproteins expressed in Escherichia coli acted as enterotoxins in suckling mice, as did mammalian rotavirus NSP4 glycoproteins, despite great differences in the amino acid sequences. The enterotoxin domain of PO-13 NSP4 exists in amino acid residues 109 to 135, a region similar to that reported in SA11 NSP4.

Avian-to-mammal transmission of an avian rotavirus: analysis of its pathogenicity in a heterologous mouse model.

It has been suggested that group A avian rotaviruses can be transmitted to mammals, but there is no direct evidence that such viruses induce disease in mammals. Suckling mice were orally inoculated with two avian rotaviruses. A pigeon rotavirus, PO-13, was found to induce diarrhea, but a turkey rotavirus, Ty-3, did not. The diarrhea induced by PO-13 was dependent on the age of the mouse. In histopathological examinations, antigens of PO-13 were sporadically detected in absorptive cells in the ileum, and lesions were observed as ballooning degenerations of absorptive cells in a region from the duodenum to the ileum. However, the rotavirus antigen was not detected in the majority of these degenerative cells. These results indicated that PO-13 could infect and induce diarrhea in suckling mice. This is the first evidence of an avian rotavirus being experimentally transmissible to a mammal.

Complete nucleotide sequence of a group A avian rotavirus genome and a comparison with its counterparts of mammalian rotaviruses

The nucleotide sequences encoding four structural proteins (VP1–4) and six nonstructural proteins (NSP1–6) of avian rotavirus PO-13 were determined. Based on the results of earlier sequencing studies [Ito et al., 1995, Sequence analysis of cDNA for the VP6 protein of group A avian rota viruses. Arch. Vriol. 140, 605–612; Rohwedder et al., 1997, Chicken rotavirus Ch-1 shows a second type of avian VP6 gene, Virus Genes 15, 65–71; Rohwedder et al., 1997, Bovine rotavirus 993/83 shows a third subtype of avian VP7 protein, Virus Genes 14, 147–151], determination of PO-13 genome sequence has been completed. The PO-13 genome is 18 845 nucleotides in length. It is 290 nucleotides longer than the genome of SA11. The amino acid sequence homology between PO-13 and mammalian rotaviruses ranged from 76–77% (VP1) to 16–18% (NSP1). The features of gene and amino acid sequence were compared with those of the corresponding protein of mammalian rotaviruses. Based on results of the phylogenetic analyses of NSP1, we speculate that an ancestral rotavirus could have separated into groups A, B and C rotaviruses at an early evolutionary stage and that group A rotavirus separated into mammalian and avian rotaviruses with host evolution.

Viral Agents Associated with Poult Enteritis and Mortality Syndrome: The Role of a Small Round Virus and a Turkey Coronavirus

Intestinal samples from turkey poults affected with poult enteritis and mortality syndrome (PEMS) were examined for viruses by immune electron microscopy and double-stranded RNA virus genome electropherotyping. Turkey coronavirus (TCV), avian rotaviruses, reovirus, and a yet undefined small round virus (SRV) were detected. The SRV and TCV were isolated and propagated in turkey embryos. Challenge of specific-pathogen-free turkey poults with SRV, TCV, or both resulted in mortality and clinical responses similar to those of natural PEMS. Our experiments indicate that SRV and TCV are possibly important agents in the etiology of PEMS and the combination of these infections might result in outbreaks with high mortality. The severity of clinical signs and mortality of PEMS are postulated to be partly related to the virus agents involved in individual outbreaks.

Serotypic characterization of outer capsid spike protein VP4 of vervet monkey rotavirus SA11 strain.

The vervet monkey rotavirus SA11, a prototype strain of group A rotaviruses, has been shown to possess VP7 serotype 3 specificity but its neutralization specificity with regard to the other outer capsid protein VP4 has not been elucidated. We thus determined its VP4 specificity by two-way cross-neutralization with guinea pig antiserum prepared with a single gene substitution reassortant that had only the VP4-encoding gene from the simian rotavirus SA11 strain and remaining ten genes from human rotavirus DS-1 strain (G serotype 2). The SA11 VP4 was related antigenically in a one-way fashion to rhesus monkey rotavirus MMU18006 VP4 (a P5B strain) and marginally to human and canine rotavirus VP4s with P serotype 5A specificity. In addition, the SA11 VP4 was shown to be distinct antigenically from those of other known P serotypes (1-4, and 6-11) as well as those of uncharacterized equine, lapine, and avian rotavirus strains. The SA11 VP4 is thus proposed for classification as a P5B serotype.

Isolation and identification of avian rotavirus from pheasant chicks with signs of clinical enteritis

Three rotaviruses were isolated from intestinal contents obtained from a flock of 6–8-day-old pheasant chicks showing diarrhoea and increased mortality. The isolates were characterized as avian group A rotavirus by immunoenzymatic technique (ELISA) and polyacrylamide gel electrophoresis (PAGE).

Sequence analysis of the VP6 gene in group A turkey and chicken rotaviruses

cDNAs corresponding to the VP6 gene of the turkey rotavirus strains Ty-1 and Ty-3, abd the chicken rotavirus strain Ch-1, were cloned and sequenced. The nucleotide and deduced amino acid sequence homology in the coding region of the VP6 gene in avian rotaviruses ranged from 78.1 to 93.9% and 86.1 to 98.7%, respectively. Both sequences of VP6 from avian rotaviruses exhibited a low degree of sequence homology (67.8–70.7% and 69.8–74.6%, respectively) compared with mammalian rotaviruses. Phylogenetic tree analysis showed that all avian rotaviruses were included in a single cluster and have separated early on from mammalian rotaviruses during evolution. The chicken rotavirus strain Ch-1 was a distant relative of other avian rotaviruses.

Bovine rotavirus 993/83 shows a third subtype of avian VP7 protein.

VP7 genes of rotavirus (RV) 993/83 isolated from a German calf with diarrhea and of RV PO-13 isolated from a Japanese pigeon were sequenced. Alignment of the deduced VP7 amino acid sequence showed 98.8% sequence identity, while only 70% and 84% identity was seen with VP7 from chicken RV Ch-2 and turkey RV Ty-1, respectively. Over the antigenic regions A, B, and C mammalian RV 993/83 showed more aa identity with mammalian G3 RVs than with chicken RV Ch-2, which could explain the strong one-way cross-neutralization observed between RV 993/83 and G3 RVs. Despite marked VP7 sequence diversity avian RVs could not be differentiated into distinct G types.

Mapping of antigenic sites on the major inner capsid protein of avian rotavirus using an Escherichia coli expression system.

The cDNA encoding the VP6 gene of avian rotavirus PO-13 strain was inserted into the bacterial expression vector pET-3a. Upon isopropyl-1-thio-beta-D-galactoside induction, the E. coli BL21 (DE3) harboring the vector containing cDNA of the VP6 gene produced an approximately 45-kDa polypeptide, which reacted with rabbit serum against PO-13 strain in Western blotting. To study the antigenic sites on VP6, various deletion mutants were constructed, expressed in E. coli and the reactivity with antigenic site I- and II-specific MAbs analyzed by Western blotting. Site I, which is shared with all group A mammalian and avian rotaviruses except for chicken rotavirus, was found to be located at amino acid positions 45 to 65, and site II, which probably contributes to an authentic group A antigen common to both mammalian and avian rotaviruses, at amino acid positions 134 to 142.

Expression of the major inner capsid protein, VP6, of avian rotavirus in mammalian cells

A gene encoding the major inner capsid protein, VP6, of avian rotavirus was inserted into the eukaryotic expression vector pAX-91 under the control of the SR α promoter and was expressed at a high level in simian COS7 cells. The expressed VP6 was indistinguishable in terms of electrophoretic mobility from the corresponding protein synthesized in simian MA104 cells infected with avian rotavirus. Binding assays with a series of monoclonal antibodies (mAbs) that corresponded to four antigenic sites on VP6 of avian rotavirus showed that the antigenic characteristics of the expressed product were identical to those of the native VP6 of avian rotavirus virions. Fiber-like structures that reacted strongly with antiserum against rotavirus were observed in VP6-expressing COS7 cells. Furthermore, an analysis of the tertiary structure of the expressed VP6 protein indicated that it adopts a trimeric configuration, similar to that of the major inner 0capsid protein of PO-13 virus. From these results, it appears that recombinant VP6 will facilitate studies of the structure and function of authentic VP6, an important protein in avian rotavirus.

Sequence Analysis of Pigeon, Turkey, and Chicken Rotavirus VP8* Identifies Rotavirus 993/83, Isolated from Calf Feces, as a Pigeon Rotavirus

Partial-length gene 4 cDNA encompassing the VP8* portion of VP4 from chicken rotavirus (RV) Ch-1, turkey RV Ty-1 and Ty-3, and pigeon RV PO-13 was sequenced and compared to RV 993/83 isolated from a calf with diarrhea. Ninety-six percent amino acid sequence identity was seen between VP8* from calf RV 993/83 and pigeon RV PO-13, while only 77 to 83% identity was seen in comparison with turkey and chicken RV VP8* sequences. Phylogenetic tree analysis places all avian RV (including calf isolate 993/83) on a branch at the bottom of the VP8* tree. When tested with three neutralizing monoclonal antibodies raised against pigeon RV PO-13, RV 993/83 and Ty-1 share three, Ch-1 shares two, and Ty-3 shares one neutralizing epitope(s) on VP4 with pigeon RV PO-13.

Recognition of rotavirus antigens by mouse L3T4-positive T helper cells

A lymphocyte proliferation assay was used to examine the helper T cell response to rotavirus in mice following parenteral immunization with the UK strain of bovine rotavirus. Mixed populations of lymphocytes prepared from spleen or peripheral lymph nodes were tested for proliferation in the presence of UK strain rotaviruses, prepared as cell culture lysates, ultracentrifuged (pelleted) lysates, sucrose-purified virus and caesium chloride-purified virus. Live rotavirus induced non-specific stimulation of lymphocytes, which was not observed in response to inactivated virus. Putative helper T cells of the L3T4+ phenotype were prepared as an enriched population from UK strain-immunized mice or grown in vitro as a polyclonal T cell line. The response of L3T4(+)-enriched cells from mice immunized with inactivated virus was dependent on antigen-presenting cells (APCs). Cells obtained following immunization with live virus did not require further addition of APCs. The response of the L3T4+ T cell line was wholly dependent on APCs. UK strain-specific L3T4+ cells responded to whole UK rotavirus and to isolated VP6 of both UK and C486 rotavirus strains. The results indicate that virus-specific L3T4+ T cells are induced following rotavirus immunization and can respond to epitopes on VP6. UK strain-primed L3T4+ cells also responded to an avian rotavirus strain, Ch2, which shares only minimal serological cross-reactivity with the UK strain. T cell recognition of rotavirus may thus be broadly cross-reactive.