PubMed · 210136
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1978. Picornaviridae: second report.. https://doi.org/10.1159/000148981
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We developed a conceptually new subtraction strategy for the detection and isolation of target DNA and/or RNA from complex nucleic acid mixtures, called Primer Extension Enrichment Reaction (PEER). PEER uses adapters and class IIS restriction enzymes to generate tagged oligonucleotides from dsDNA fragments derived from specimens containing an unknown target ('tester'). Subtraction is achieved by selectively disabling these oligonucleotides by extension reaction using ddNTPs and a double stranded DNA template generated from a pool of normal specimens ('driver'). Primers that do not acquire ddNTP are used to capture and amplify the unique target DNA from the original tester dsDNA. We successfully applied PEER to specimens containing known infectious agents (Hepatitis B Virus and Walrus Calicivirus) and demonstrated that it has higher efficiency than the best comparable technique. The strategy used for PEER is versatile and can be adapted for the identification of known and unknown pathogens and mutations, differential expression studies and other applications that allow the use of subtractive strategies.
OBJECTIVE: To investigate the epidemiological features of human calicivirus( HuCV) infection in children with diarrhea in a hospital in Guangzhou. METHODS: Stool specimens were collected from children with viral diarrhea diagnosed between October, 2003 and January, 2004 and between October, 2004 and January, 2005. HuCV was detected by means of RT-PCR and sequence analysis of the PCR products. RESULTS: Eighty specimens positive for Norwalk-like viruses (NLVs) were identified from 648 stool specimens, with a positivity rate of 12.35%, and sapporo-like viruses (SLVs) were identified in 2 specimens (0.31%). The monthly NLV positivity rate was 11.74% (Oct.), 14.16% (Nov.), 9. 09% (Dec.) and 13.95% (Jan.), respectively, showing no significant variation in these months. NLVs mainly infected children below 2 years old. Twenty-two strains of NLVs were sequenced and analyzed, and 21 of them were identified as GII and the genotype of 1 strain could not be determined. The prevalent viral population were GII-3 and GII-4 in 2003 and was GII-4 in 2004, and both of the SLVs belong to GI-1. CONCLUSION: NLVs is one of the important pathogens causing sporadic acute gastroenteritis in children admitted in the hospital in Guangzhou, and the prevalent strains are GII-3 and GII-4 , but different prevalent strains are possible in different periods.
Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 5' end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF 4 E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF 4 E by 4 E-BP 1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF 4 E and the cap structure or 4 E-BP 1, suggesting that VPg binds to eIF 4 E at a different site from both cap and 4 E-BP 1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA.