RESUMO
In this study, a new multiplex RT-PCR method for detecting various viral genes in patients with rash and fever illnesses (RFIs) was constructed. New primer sets were designed for detection of herpes simplex viruses 1 and 2 (HSV1 and 2), and Epstein-Barr virus (EBV). The newly designed and previously reported primer sets were used to detect 13 types of RFI-associated viruses by multiplex RT-PCR assay systems. Moreover, to eliminate non-specific PCR products, a double-stranded specific DNase was used to digest double-stranded DNA derived from the templates in clinical specimens. RFI-associated viruses were detected in 77.0% of the patients (97/126 cases) by the presented method, multiple viruses being identified in 27.8% of the described cases (35/126 cases). Detected viruses and clinical diagnoses were compatible in 32.5% of the patients (41/126 cases). Sensitivity limits for these viruses were estimated to be 101 -103 copies/assay. Furthermore, non-specific PCR products were eliminated by a double-stranded specific DNase with no influence on sensitivity. These results suggest that this method can detect various RFI-associated viruses in clinical specimens with high sensitivity and specificity.
Assuntos
Exantema/diagnóstico , Febre/diagnóstico , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/genética , Herpesvirus Humano 4/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Primers do DNA/genética , DNA Viral/genética , Infecções por Vírus Epstein-Barr/diagnóstico , Exantema/virologia , Febre/virologia , Genes Virais/genética , Herpes Genital/diagnóstico , Humanos , Sensibilidade e EspecificidadeRESUMO
A recombinant norovirus, GII.P16-GII.4_Sydney2012, was first detected from nine patients with gastroenteritis in Kawasaki City, Japan, in 2016. The viral genome showed nucleotide sequence identities of 95.1% and 97.2% to the closest strains in the regions of 5' terminus to ORF1 and ORF2 to 3' terminus, respectively.
RESUMO
A number of novel recombinant human adenoviruses (HAdVs) have recently been identified through sequencing of the complete genomes. The recombinant HAdV sequences share similarity with other types in the major capsid genes, namely the hexon, penton base, and fiber genes, implying recombination events, which may result in escape from the immune response and the acquisition of different organotropisms. Therefore, a surveillance system of HAdVs that considers the effect of frequent recombination on genetic evolution in these genes must be constructed. In this study, we designed new primer sets that can amplify the partial penton base and fiber genes from species HAdV-A to HAdV-F and proteotype HAdVs on the basis of sequence analyses, including previously reported primers that amplify loop 1 of the hexon. Phylogenetic analysis through sequencing with these primers correctly classified clinical HAdV isolates in loop 1 of the hexon gene, the Arg-Gly-Asp (RGD) loop of the penton base gene, and the knob of the fiber gene, which contain neutralizing, hemagglutination, and receptor binding epitopes associated with immunogenicity and tissue tropisms of HAdVs. This study contributes to the accumulation of correct information regarding genetic diversity and evolution in the worldwide HAdV surveillance.
