pubmed.ncbi.nlm.nih.gov

Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses - PubMed

Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses

J R Smiley et al. J Clin Microbiol. 2003 Jul.

Abstract

Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3'-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.

Sequence alignments of selected human-NLV and NLV-BEC with BEC-universal primers. (A) Sequence identity of selected human NLVs and NLV-BECs with the CBECU-F and CBECU-R primers. Virus bases sharing identity at degenerate positions of the CBECU-F primer are indicated by underlining. Note that the sequence given for the CBECU-R (reverse primer) is the complementary sequence. (B) Comparison of the sequence identity of different SLV, NLV, and BECs with the NBU-F/R primer set. Underlined virus bases found at degenerate locations of the NBU-F primer indicate positive sequence identities. Note that the sequence given for the NBU-R (reverse primer) is the complementary sequence.

FIG. 2.
FIG. 2.

Nucleotide (upper) and amino acid (lower) identities of various BECs and human Norwalk virus for a 274-bp sequence segment of the RdRp gene. The GenBank accession numbers for NA-2, Jena, and human Norwalk virus are given in Fig. 5.

FIG. 3.
FIG. 3.

Diagrammatic representation of the predicted ORFs and overlap regions (including stop codons) of the Jena 117/80/GD and CV95-OH and CV186-OH BECs. Sequence positions indicated for Jena virus correspond to actual positions in the known complete genome sequence (GenBank accession no. AJ011099). The potential extended ORF-3 of the CV95-OH (nt 2736 to 3584, 282 aa) and CV186-OH (nt 2736 to 3587, 283 aa) viruses are shown, along with short ORF-3 gene sequences with potential initiation codons in positions analogous to those of the Jena virus.

FIG. 4.
FIG. 4.

Distance (A) and maximum-likelihood (B) consensus trees of the 3′-terminal (155-aa) RdRp gene of prototype viruses representing all four calicivirus genera. Trees were prepared from the sequence alignment by using the PHYLIP and Treeview programs and are based on 100 bootstrapped data sets. GenBank accession numbers for most viruses can be found in Fig. 5, while accession numbers of the CV95/186/500/514/521-OH viruses are noted in Materials and Methods.

FIG. 5.
FIG. 5.

Comparison of P289/290 RT-PCR assay products for different caliciviruses. (A) Conserved amino acid sequence motifs found in the nonstructural proteins of calicivirus ORF-1. The position of the DYSKWDST and YGDD motifs found in the RdRp are shown with their corresponding P289/290 target primer underlying. (B) Comparison of the nucleotide identities of various NLV, SLV, and BECs with sequences of the P290(F) and P289(R) primers. Note that the sequences given for the P289(R) primer is the complementary sequence. (C) Comparison of corresponding amino acid sequence identities of different caliciviruses with the P290 (DYSKWDST) and P289 (YGDDGIY) translated primer sequences. The expected product size of amplified products and the GenBank accession numbers of the different caliciviruses are also given in the right-hand columns.

Similar articles

Cited by

References

    1. Ando, T., S. S. Monroe, J. S. Noel, and R. I. Glass. 1997. A one-tube method of reverse transcription-PCR to efficiently amplify a 3-kilobase region from the RNA polymerase gene to the poly(A) tail of small round-structured viruses (Norwalk-like viruses). J. Clin. Microbiol. 35:570-577. - PMC - PubMed
    1. Ando, T., J. S. Noel, and R. L. Fankhauser. 2000. Genetic classification of “Norwalk-like viruses.” J. Infect. Dis. 181(Suppl. 2):S336-S348. - PubMed
    1. Berke, T., B. Golding, X. Jiang, D. W. Cubitt, M. Wolfaardt, A. W. Smith, and D. O. Matson. 1997. Phylogenetic analysis of the caliciviruses. J. Med. Virol. 52:419-424. - PubMed
    1. Dastjerdi, A. M., J. Green, C. I. Gallimore, D. W. Brown, and J. C. Bridger. 1999. The bovine Newbury Agent-2 is genetically more closely related to human SRSVs than to animal caliciviruses. Virology 254:1-5. - PubMed
    1. Dastjerdi, A. M., D. R. Snodgrass, and J. C. Bridger. 2000. Characterization of the bovine enteric calici-like virus, Newbury Agent 1. FEMS Microbiol. Lett. 192:125-131. - PMC - PubMed

Publication types

MeSH terms

Substances