key: cord-0720898-g2a9l3tv
authors: Oliver, S. L.; Asobayire, E.; Charpilienne, A.; Cohen, J.; Bridger, J. C.
title: Complete genomic characterization and antigenic relatedness of genogroup III, genotype 2 bovine noroviruses
date: 2006-10-25
journal: Arch Virol
DOI: 10.1007/s00705-006-0856-2
sha: 9046c95b831f07022b9212c09af9fd2232b02363
doc_id: 720898
cord_uid: g2a9l3tv

Bovine enteric noroviruses form a genogroup, III, distinct from the 2 human norovirus genogroups, I and II. Two genogroup III genotypes were suggested by partial genomic analyses. In the present study, analysis of the full-length genome sequence of Bo/Newbury2/76/UK and the more contemporary Newbury2-like virus, Bo/Dumfries/1994/UK, showed that both were 7311 nucleotides in length and had three open reading frames (ORFs), amino acids motifs typical of noroviruses, and 95% or greater amino acid identities to each other in all regions of their genome. Apart from the ORF1 NTPase region, their ORF1 regions had less than 90% identity to the genogroup III genotype 1 Bo/Jena/80/DE virus, confirming two genogroup III genotypes. A close antigenic relationship was demonstrated by ELISA between the genotype 2 viruses, which will allow their serological diagnosis.

Noroviruses are a well-studied, leading cause of non-bacterial gastroenteritis in human adults, classified in the family Caliciviridae [10] . Recently, bovine viruses identified some years ago were identified as noroviruses [5, 25] . Human noroviruses belong to genogroups I and II [10] , whereas the bovine noroviruses have been placed in a separate genogroup, III [1, 5, 25, 33] . Comparisons of partial genomic sequences of the two original bovine viruses identified two genetic clusters (genotypes) within genogroup III, represented by the Bo/Jena/80/DE virus (genogroup III/1), and the Bo/Newbury2/76/UK virus (genogroup III/2). as a homologous challenge control and was given a primary inoculation with Newbury2 plus primary and secondary challenge inoculations with Newbury2. Two calves (P137 and P142) were given a primary inoculation and primary challenge inoculation with Newbury2 and secondary challenge to Newbury1. The fourth calf (Q21) served as a Newbury2 challenge control and was inoculated for the first time with Newbury2 at the time of challenge. Preinoculation and post-primary, post-primary challenge and post-secondary challenge sera were taken (see Table 2 ) and had been stored at −20 • C. No evidence of antibody degradation was obtained during storage by monitoring antibody titres from calves infected with bovine rotavirus (unpublished) . Two gnotobiotic calves were orally inoculated with Bo/Dumfries/1994/UK, and sera were taken 14 days later, then again 8 and 15 days after homologous challenge. Bovine antisera from calves inoculated with bovine rotavirus UK, the bovine enteric coronavirus, bovine astrovirus and the Haden strain of parvovirus [3] were used in the Newbury2 ELISA to determine specificity.

Viral RNA extraction and reverse transcription were performed as described previously [30] using MMLV reverse transcriptase and random hexamers (Promega). PCR was performed using the Hi Fidelity PCR system (ROCHE). An amino acid alignment of polyproteins from the genus Norovirus (Bo/Jena/1980/DE, 17 human noroviruses from genogroup I or II plus the murine norovirus) was used to identify conserved sequences in the NTPase region of the polyprotein, DRIEK/NK and APQGGFD. Primers JENA01 and JENA02 were designed to the conserved region in the NTPase corresponding to the Jena virus nucleotide sequence ( Table 3 ). The subsequent nucleotide sequence of a 262-bp amplicon generated from the Dumfries genome that was cloned into a pCR2.1-TOPO vector was used to design additional primers, which were used to amplify two regions (JANA01/BoCVhelR 02 -1851 nucleotides; BoCVhel 01/BoCVproR 01 -1944 nucleotides) of the Newbury2 and Dumfries genomes. Amplicons cloned into pCR2.1 (Invitrogen) were sequenced in both directions using overlapping primers ( Table 3 ). The 5 end of the Newbury2 genome was deduced using 5 rapid amplification of cDNA ends (RACE). cDNA generated by reverse transcription using the primer BoCV5 RACE 05 was purified using a QIAquick gel extraction kit (Qiagen). Terminal deoxynucleotide transferase (Promega) was used to add a poly-C tail to the 5 end of the purified cDNA. The 5 end of the Newbury2 genome was amplified using the Hi Fidelity PCR system (ROCHE) with an abridged anchor primer (Invitrogen) and BoCV5 RACE 06 ( Table 3 ). The resulting 337-bp amplicon was cloned into pCR2.1, and 5 clones were sequenced in both directions to establish a consensus nucleotide sequence. The MRC gene service performed sequencing and the sequences were assembled using Staden [39] .

The Simplot computer program [27] was used to generate identity plots of the complete genomes and the complete ORF1 polyproteins of the bovine noroviruses Newbury2, Dumfries and Jena using a window size of 200 and step size of 20 with gap strip off and J-C correction on. A multiple alignment of the translated amino acid sequences of the complete polyprotein (ORF1) was generated using Clustal X version 1.8 [43] , which was used for phylogenetic analyses. PHYLIP (J. Felsenstein, Department of Genetics, University of Washington, Seattle; Phylogeny Inference Package, version 3.5c) was used for parsimony (DNApars and Protpars), UPGMA, Fitch-Margoliash and bootstrap analyses, plus TreePuzzle 5.2 [37] for additional maximum likelihood with quartet puzzling analyses.

The nucleotide sequence accession numbers for the noroviruses referred to in the text are: Genogroup I; Norwalk -NC 001959, Southampton -L07418, Chiba -AB042808, WUG1 -AB081723, SzUG1 -AB039774: Genogroup II; Snow Mountain -AY134748, Lordsdale -X86557, Langen1061 -AY485642, Gifu -AB045603, U1 -AB039775, U3 -AB039776, U4 -AB039777, U16 -AB039778, U17 -AB039779, U25 -AB039780, U18 -AB039781, U201 -AB039782: Genogroup III; Jena -AJ011099, Newbury2 -AF097917, Dumfries -AY126474: Unassigned; Murine -AY228235.

The primers BoCVCap5 EcoRI (5 -GAATTCATGAAGATGACTGACAGA-3 -the initiation codon is underlined) and NA2Capsid3 XhoI (5 -CTCGAGTCAGAAGCCATGAAG GCG-3 -the reverse complementary sequence of the termination codon is underlined) were designed using the genomic sequence at the 5 and the 3 end of the Newbury2 ORF2 gene that incorporated the restriction sites EcoRI and an XhoI. A 1581-bp RT-PCR amplicon of the expected size was cloned using pCR2.1-TOPO-TA cloning kit (Invitrogen) to create the construct pCR2.1-Newbury2-ORF2. A clone of pCR2.1-Newbury2-ORF2, which had been sequenced in both directions to confirm the integrity of ORF2 gene, and the pFastBac TM 1 transfer vector (Invitrogen) were digested with EcoRI and XhoI (Promega). The Newbury2-ORF2 gene and the pFastBac TM 1 transfer vector were ligated to create the construct pFastBac TM 1-Newbury2-ORF2 that was used to transform TOP10 E. coli from which the construct was amplified and purified.

Donor plasmid pFastBac TM 1-Newbury2-ORF2 was transformed into DH10Bac (Invitrogen) competent cells for transposition. After the recombinant bacmids were determined to be correct by two successive blue-white colonies analysis, the recombinant bacmid DNA was used to lipofect Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus was amplified twice, and expression of ORF2 was checked by Coomassie blue staining of SDS-PAGE of infected Sf9 cells lysate. For VLP production, Sf9 cell monolayers were infected with recombinant baculovirus at a multiplicity of infection of 5 PFU/cell and incubated in Hink's medium supplemented with 1% fetal calf serum for 5 days at 26 • C. VLPs were extracted from supernatant of freezed-thawed infected Sf9 cells as described previously [22] except that Freon 113 was substituted by Vertrel XF as described by Mendez et al. [28] . VLPs were purified by isopicnic centrifugation in cesium chloride gradients and quantified by the method of Bradford using bovine serum albumin as standard. Recombinant VLPs were negatively stained with 2% uranyl acetate and examined in a Philips CM12 electron microscope operated at 80kV. Micrographs were taken at 25,000 magnification on Kodak SO163 Image plates.

Bovine serum albumin (BSA) was used to establish the protein concentrations of double CsClpurified VLPs with a Micro-BCA assay (Pierce). The Newbury2 capsid protein (250 ng/lane) was blotted onto nitrocellulose membranes (Invitrogen) following electrophoresis with PAGEr Duramide 4-20% Tris-Glycine pre-cast gels (Cambrex) and western transfer. Prior to immunostaining, the nitrocellulose membranes were stained with Ponceau S (Sigma) to establish that the Newbury2 capsid protein had been successfully transferred. Membranes were blocked with 2% non-fat dried skimmed milk in Tris-buffered saline (TBS; 10mM Tris-HCl [pH 7.4], 0.15 M NaCl) and washing steps performed with TBST (TBS + 0.05% Tween 20) . Convalescent bovine antisera to Newbury2 and rotavirus UK (as a negative control serum) were diluted 1:100 in TBST + 1% non-fat dried skimmed milk powder and detected using a 1:5000 dilution of rabbit anti-bovine IgG horseradish-peroxidase conjugate (Sigma). HRP activity was detected using TMB (3,3 ,5, 5 -tetramethylbenzidine) stabilised substrate (Promega). The molecular mass of the Newbury agent-2 capsid protein was calculated from a standard curve produced using the Prosieve Color (Cambrex) molecular mass marker.

Maxisorb plates (Nunc) were coated overnight at 4 • C with 5 µg/ml of antigen in carbonate buffer pH 9.6. Antigens used were double CsCl-purified Newbury2 VLPs or rotavirus 2/6 VLPs produced from the bovine rotavirus RF strain, or the supernatant from wild-type baculovirus-infected Sf9 cells (mock antigen). Plates were blocked with 2% non-fat dried skimmed milk in PBS and washed with PBST (PBS + 0.05% Tween 20 [Sigma]) between steps. Antisera from experimental calves were serially diluted from 1:50 in a 2-fold dilution series in 1% non-fat dried skimmed milk in PBST, dispensed into wells previously coated with test or mock antigen, and incubated at room temperature for 1 h. Binding was detected by incubation with a 1:10,000 dilution of rabbit anti-bovine IgG horseradish peroxidase-conjugate (Sigma) in 1% non-fat dried skimmed milk in PBST at room temperature. TMB was used as substrate (Sigma). Serum titres were calculated by linear regression using Prism 4 (GraphPad Software). Endpoints were defined as the highest dilution at which the net absorbance value (absorbance units of the serum with the test antigen minus absorbance units with the mock antigen) was twice the absorbance value for antigen-coated wells without a test bovine serum.

The homology model of the Newbury2 capsid protein was generated using the translated amino acid sequence of the complete Newbury2 ORF2 gene, which was submitted to the Swiss-model server http://swissmodel.expasy.org/. The software programme VMD 1.8.2 [15] was used to visualise the co-ordinate data and generate the image, which was rendered using Pov-ray TM for Windows TM .

Completion of the sequence of the Newbury2 genome showed that it was 7311 nucleotides in length, excluding the poly-A tail, that is, 27 nucleotides shorter than the Jena virus genome. The shorter length was attributed to deletions in the Newbury2 ORF3 gene and the shorter 3 untranslated region reported previously [33] . The 5 untranslated region of Newbury2 was identical in length (21 nucleotides) and composition to that of Jena [25] except for a single transition substitution (U-C) at the 21 st nucleotide. The G + C content of the Newbury2 genome was 57%, similar to that of Jena (56%) [25] . The Newbury2 ORF1 gene was 5055 nucleotides in length and coded for a protein of 1684 amino acids with a predicted molecular mass of 186 kDa. It was 12 nucleotides longer than the Jena ORF1 gene. There were 2 insertions, of 3 and 21 nucleotides, towards the 5 end of the Newbury2 ORF1 gene at nucleotide positions 130-132 and 157-177. In contrast, compared with the Newbury2 genome, there were 2 insertions, of 9 and 3 nucleotides, in the Jena ORF1 gene, at nucleotide positions 2650-2658 and 2689-2691.

The Newbury2 and Jena ORF1 genes had, overall, 73% nucleotide identity (range 38-83%) (Fig. 1A) and 84% amino acid identity (range 71-92%) ( Table 1 ). The majority of the variation was within the first 500 nucleotides and between nucleotides 2000 and 3000, which included the 3A-B(VPg) region, with only 71% [33] amino acid identity. The NTPase regions of the two viruses showed a remarkably high level of identity (92% amino acid), higher than that of the 3D-polymerase regions. Extensive variation between the Newbury2 and Jena genomes was also seen for the ORF2 and 3 genes, but this was reported previously [30] . All regions of the Newbury2 genome, including the ORF2 and 3 genes reported previously [30] , had low levels of amino acid identities with genogroups I and II noroviruses and the murine norovirus ( Table 1) . The Newbury2 genome showed moderate nucleotide conservation (11 of 24 nucleotides) between its 5 terminus region and the region of the ORF1 and 2 overlap ( Fig. 1 panel A) . This is typical for all members of the family Caliciviridae, especially members of the genus Norovirus [10] and including the bovine Jena genome. This finding suggests that the transcription/translation mechanism of ORFs 1 and 2 of bovine noroviruses is similar to other caliciviruses. Consistent with Jena, the putative protease cleavage sites, LQGP and LQAP, on either side of the NTPase, plus FQAP and LEGG, on either side of the 3C protease, were conserved for Newbury2 (Fig. 1B) . A cleavage site could not be identified for the 3A-B(VPg) region because the conserved human noroviruses dipeptides used to predict the cleavage sites were not present for the bovine noroviruses. The amino acid motifs typical of caliciviruses were present in the Newbury2 ORF1 polyprotein in the NTPase (GPPGIGKT), the 3C protease (GDCG) and the 3D polymerase (GLPSG and YGDD), as previously described for Jena [25] .

Phylogenetic analyses of the Newbury2 complete ORF1 protein confirmed that bovine noroviruses formed two distinct clusters in genogroup III, most closely related to, but distinct from, genogroup I (Fig. 2) . The internal branch node that 

The complete Dumfries genome was identical to that of Newbury2 in its overall length (7311 nucleotides), organization, and the length of the 3 ORFs. ORF1 was 5055 nucleotides in length, ORF2 was 1569 nucleotides and ORF3 was 849 nucleotides in length. The genome had the same G + C content (57%) as Newbury2. The amino acids motifs in the 2C NTPase, the 3C protease and 3D polymerase, typical of the caliciviruses, were present in the Dumfries genome. The putative protease cleavage sites LQGP and LQAP on either side of the NTPase, plus FQAP and LEGG on either side of the 3C protease, were also conserved. The nucleotide and amino acid variation between the Dumfries and Newbury2 ORF1 genes was surprisingly low for two RNA viruses isolated 18 years apart and from disparate geographical locations. Nucleotide identities across the ORF1 gene ranged from 82 to 95% (Fig. 1A) . Amino acid identities of the N-terminal, NTPase, 3A-3B (VPg), 3C-protease and 3D-polymerase proteins were high, ranging from 95 to 99% (Table 1) . Similar levels of identity were found between the Dumfries and Newbury2 ORF2 and 3 proteins, including the hypervariable capsid P2 region [33] . Because of the high similarity of the Newbury2 and Dumfries genomes, the variation seen between the Dumfries and Jena genomes was almost identical to that between the Newbury2 and Jena genomes. The N-terminal region of Dumfries and Jena had 75% amino acid identity, the NTPase 92%, 3A-3B(VPg) 72%, the 3C-protease 88% and the 3D-polymerase 90% amino acid identity. Extensive variation between the Dumfries and Jena genomes was also seen for the ORF2 and 3 genes (33) . Phylogenetic analysis of the complete ORF1 gene (Fig. 2 ) and the ORF2 capsid and ORF3 genes [33] confirmed that the Dumfries and Newbury2 genomes were closely related but distinct from that of Jena, thus forming two genotypes in genogroup III.

Newbury2 VLPs, similar in morphology to native virions, were produced in milligram quantities when the Newbury2 ORF2 capsid gene was expressed using (Fig. 3A) . The molecular mass of the capsid protein monomer from denatured VLPs was approximately 56 kDa by SDS-PAGE (Fig. 3B ), in agreement with the molecular mass predicted from translated amino acids of the capsid gene [33] . Denatured Newbury2 VLPs reacted weakly in western blots with a Newbury2 convalescent calf serum but non-denatured Newbury2 VLPs reacted strongly with a high-molecular-weight band (not shown). Good antibody responses were detected by ELISA using the VLPs and sera from four gnotobiotic calves inoculated with Newbury2 virus as a primary or challenge inoculation (mean IgG titre log 10 3.9, range 3.1-4.4) ( (Fig. 4A ). A homology model of the Newbury2 capsid protein showed that the amino acid substitutions in the Dumfries capsid protein formed two clusters in the P-domain, one tight cluster in the P2 domain and a less well-defined cluster in the P1 domain (Fig. 4B) . Thus, these two clusters of amino acid substitutions did not prevent the development of cross-reactive antibodies.

Complete genomic characterization of the bovine noroviruses, Newbury2 and Dumfries, showed that they had the genomic organization and conserved motifs expected of noroviruses and suggested that Newbury2 was genomically and antigenically representative of genogroup III, genotype 2 bovine noroviruses. The levels of genomic identity between the Newbury2 and Dumfries ORF1 genes reported in the present study, and between the ORF2 and 3 genes of Newbury2, Dumfries and the Newbury2-like viruses [14, 32, 33, 38, 44, 45] , was surprisingly high for RNA viruses identified over almost 3 decades. The tight clustering of the Newbury2 viruses resembled the tight clustering seen, for example, for human genogroup II/4 Lordsdale-type viruses. It remains to be seen whether this tight clustering will persist and whether the number of bovine genotypes will increase as more studies are conducted. The levels of identity observed between the Newbury2 and Dumfries genomes was not a consequence of selection by the RT-PCR primers used to amplify these viruses. Both Newbury2 and Dumfries were originally identified by electron microscopy, from disparate geographical locations and 18 years apart [46; D. Snodgrass, personal communication] . The apparent stability of the genotype 2 bovine norovirus genome will simplify diagnostic tests based on RT-PCR. Most recent studies have used primers designed to the polymerase region that might favour detection of Newbury2-like viruses [33, 38, 44, 45] but the finding that the NTPase was also highly conserved between genotype 1 and 2 bovine noroviruses (92% amino acid identity) offers an opportunity to use RT-PCR primers based on the NTPase region which might identify a wider range of bovine noroviruses, if they exist. The high amino acid identities in the capsid regions of Newbury2 and Dumfries genomes correlated with the ability of Newbury2 VLPs to detect Dumfries virus antibodies. The results of the present paper, combined with observations that other genotype 2 bovine noroviruses have 94% or greater amino acid identity with Newbury2 [14, 33, 45] , suggested that serological assays based on Newbury2 will be suitable to detect genogroup III/2 bovine noroviruses and their antibodies. This view is endorsed by studies with human noroviruses with similar identities in their capsid proteins and which show antigenic cross-reactivity [12, 19] . In contrast, a recent study of the antigenic relationship of Newbury2 and the genotype 1 Jena virus, which have 68% amino acid identify in their capsid proteins, showed that they were poorly related antigenically and that independent ELISAs are required to detect both [31] . Similarly, studies of human norovirus capsid proteins with 73% amino acid identity or less showed little or no cross reactivity in ELISA [12, 19] . Although not tested by ELISA, the capsid amino acid identities of Newbury2 with human genogroup I and II noroviruses of 48 and 43% make it unlikely that the Newbury2 viruses cross-react with human genogroup I and II noroviruses in ELISA. However, it should be noted that a cross-reactive epitope between some genogroup II human noroviruses and the Newbury2 and Jena viruses has been identified using a monoclonal antibody [31] . The lack of reactivity of Newbury1 antiserum in the Newbury2 ELISA reported in the present study, and the lack of cross-protection between the two viruses [3] is explained by the recent finding that Newbury1 belongs to a new genus in the family Caliciviridae [30] .

The Newbury2 VLPs were composed of a protein of similar molecular mass to VLPs expressed from the Newbury2-like virus Bo/CV186-OH/00/US [14] . They appeared antigenically authentic. Antibody titres in experimental calf sera were similar to those obtained previously [14] and similar to those obtained with human patients [9] . Homologous protection to Newbury2 disease and virus shedding was reported in calves [3] , thus allowing calves to be used as an experimental model for immunity to disease. Except for the study by Subekti et al. [42] , norovirus disease models using chimpanzees and non-human primates [36, 47] have not produced clinical signs, although serological responses and virus excretion were demonstrated. The calf model allows pre-exposure to norovirus infection to be controlled, but it does have the limitations of production of few offspring and animal size.

Rotaviruses and coronaviruses are recognised as the major viral pathogens in outbreaks of neonatal calf diarrhoea [2, 35, 39] . However, in the UK, up to 30% of calf diarrhoea outbreaks are without an identifiable pathogen [2, 35, 39] and the dam vaccines are said not to be totally effective. The presence of noroviruses might explain some of these undiagnosed outbreaks. The results from the present study will allow the role of bovine noroviruses to be established and vaccines to be developed, if necessary.

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Author's address: Dr. J. C. Bridger, Department of Pathology and Infectious Diseases

We thank Jean Lepault, CNRS-INRA, for conducting the electron microscopy and Didier Poncet, CNRS-INRA, for his support.