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REVERSETRANSCIPTION-PCR ASSAYS FOR THE DETECTION OF BOVINE ENTERIC CALICIVIRUSES (BEC) AND ANALYSIS OF THE GENETIC RELATIONSHIPS AMONG BEC AND HUMAN CALICIVIRUSES
Citation:
Smiley, J. R., A. E. Hoet, M. Traven, H. Tsunemitsu, AND L. J. Saif. REVERSETRANSCIPTION-PCR ASSAYS FOR THE DETECTION OF BOVINE ENTERIC CALICIVIRUSES (BEC) AND ANALYSIS OF THE GENETIC RELATIONSHIPS AMONG BEC AND HUMAN CALICIVIRUSES. JOURNAL OF CLINICAL MICROBIOLOGY 41(7):3089-3099, (2003).
Impact/Purpose:
Overarching Objectives and Links to Multi-Year Planning
This task directly supports the 2003 Drinking Water Research Program Multi-Year Plan's long term goal 1 for "regulated contaminants" and long term goal 2 for "unregulated contaminants and innovative approaches" under GRPA Goal 2 (Clean and Safe Water). The overarching objective is to provide the Office of Water, Agency risk assessors and managers, academics, the scientific community, state regulators, water industry and industry spokes-groups the methods they need to measure occurrence of waterborne viral pathogens. The methods developed will improve the quality of risk-based assessments and tools used by the Agency to set regulations, policies and priorities for protecting human health and will allow the Agency to assure the public that the appropriate methods are being used to demonstrate that drinking water is safe from pathogenic agents.
Specific Subtask Objectives:
o Evaluate techniques for enhancement of growth of human enteric viruses in support of CCL #2 and #3 and for use in the UCMR (Subtask A; to be completed by 9/05 in support of LTG 2)
o Develop a multiplex RT-PCR method that incorporates internal controls for use in the UCMR (Subtask B; completed 9/03 in support of LTG 2)
o Develop and evaluate new molecular technologies for use in the UCMR. Included will be real-time RT-PCR methods for Norwalk virus and astroviruses, and integrated cell culture/molecular procedures for detection of infectious viruses (Subtask B; to be completed by 9/05 in support of LTG 2)
Description:
Two genetically distinct bovine enteric caliciviruses (BEC) have been identified: the Norwalk-like-viruses (NLV), which are genetically related to human NLV, and the distinct NB-like BEC, which is most closely related to Sapporo-like viruses and lagoviruses, but potentially may represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Also, although 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 this study, veal calf fecal samples were assayed for enteric caliciviruses using six RT/PCR primer sets designed for the detection of human NLV or BEC. Caliciviruses genetically related to the NLV-BEC Jena and Newbury-agent-2 (NA-2) strains, or the recently characterized NB BEC strain, were identified in 3 of 4, and 4 of 4 sampled veal herds, respectively. Extended 3' terminal genome sequences of two NLV-BECs, designed CV95-OH and CV186-OH, encoding the RdRp (ORF-1), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome regoin demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289/290 primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positvie fecal pools (using other assays) led us to design two new primer sets, CBEC-UFP/URP and NB-UFP/URP, for the sensifitive 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 using the new assays identified 72% (54/75) of veal calf fecal samples 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.