Detection of WU polyomavirus in children with low respiratory tract infections using real-time fluorescent quantitative PCR / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>Development and application of a real time fluorescent quantitative PCR (FQ-PCR) assay for detecting WU polyomavirus in children with low respiratory tract infections.</p><p><b>METHODS</b>The VP2 gene of WU polyomavirus was selected as the detection target, from which the real time primers and probes were designed. The standard curve was established by using recombinant plasmid as template. And the FQ-PCR assay for specific detection of WU polyomavirus was established. The specificity, sensitivity and reproducibility of the method were evaluated. Furthermore, the clinical specimens from children with respiratory tract infections collected in Wenling First People's Hospital were quantitatively detected using this method.</p><p><b>RESULTS</b>In this study, the FQ-PCR method was established to detect a specific fragment in VP2gene of WU polyomavirus. The standard curve coefficient R2 was 0.998. And this method can detect as low as 50 copies recombinant plasmid. The clinical specimens of sputum and throat swab from children with respiratory tract infections were quantitatively detected using this method. 7 sputum specimens were detected as WU polyomavirus positive in 700 sputum specimens, the positive ratio was 1.00%. No positive specimens were detected in 146 specimens of throat swabs and 846 blood samples from same patient population.</p><p><b>CONCLUSION</b>The results indicated that the FQ-PCR assay method established in this study was specific, rapid and sensitive for detecting WU polyomavirus in children with lower respiratory tract infections. The sputum specimen is more suitable to be used for gene detection of WU polyomavirus than throat swab or blood.</p>

Detection of BK virus infection in renal transplant recipients and clinical application / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the detection methods of BK virus infection in kidney transplant recipients, and to explore the clinical application.</p><p><b>METHODS</b>132 cases of renal transplant recipients were undertaken BK virus detection including presence of decoy cells in urinary sediment, urine and serum BKV-DNA to demonstrate the BK virus replication.</p><p><b>RESULT</b>Among 132 cases of renal transplant recipients, urinary decoy cell was found in 37 (28.0%) patients and the median time was 12 months after surgery. 32 (24.2%) patients were diagnosed as BK viruria at a median of 11 months after surgery, and 16 (12.1%) recipients were diagnosed as BK viremia at a median of 15 months after surgery, 5 patients with BK viruria were diagnosed as BK virus associated nephropathy according to allograft biopsy.</p><p><b>CONCLUSION</b>To make early diagnosis of BK virus infection, detection of urine decoy cells and BKV-DNA in urine and plasma sample is important,which provides an important basis for the prevention of BK virus associated nephropathy.</p>

Expression of recombinant VP2 gene in insect sf9 cells and screening of clinical specimens / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone and express VP, gene from HBoV, and the expressed VP, protein was as the antigen in order to detect serum from children in Wenling area with lower respiratory tract infections.</p><p><b>METHODS</b>The VP, gene was recombined with the genome of Baculovirus, which infected the insect cell. The fusion protein with HA tag was applied to confirm the specificity of expressed protein. Furthermore, the recombinant protein was observed using electron microscopy. The 176 serum from children in Wenling area with lower respiratory tract infections was screened using Western blot.</p><p><b>RESULTS</b>The expressed VP2 protein was more than 60% in total proteins from insect cell, and MWt about 60 x 10(3). The virus-like particle (VLP) was observed using electron microscopy, and size about 20 nm. The 176 serum from children in wenling area with lower respiratory tract infections was screened using Western blot. The HBoV positive rate was 2.28% (4/176).</p><p><b>CONCLUSION</b>The VP2 protein from human bocavirus was expressed in insect cell successfully. Through HA tag the VP2 protein was specific, and then the assay using SDS-PAGE with Western blot could detect and screen the antibody in serum from children with lower respiratory tract infections rapidly and accurately.</p>

Discovery and identification of WU polyomavirus in children from Zhejing region / 病毒学报

WU polyomavirus, which was firstly discovered in 2007, is a new human polyomavirus belonging to Polyomaviridae and containing circular double-stranded genomic DNA. In this study, the 278 clinical sputum specimens from children under 5 years old were collected from Wenzhou Medical College affiliated Wenling First Hospital, Zhejiang Province. Based on identification assay of WU polyomavirus previously reported, a WU polyomavirus was identified from clinical samples successfully, the positive rate was 0.4%. The sequences of PCR products were identical to that of VP2 gene and large T antigen gene derived from WU polyomavirus reported. The above results strongly suggested that the WU polyomavirus isolated was firstly found in Chinese children with acute lower respiratory tract infections. This study provides a firm basis for further research of WU polyomavirus.

Identification of KI polyomavirus in children with lower respiratory tract infections from Zhejiang region of China / 病毒学报

KI polyomavirus, which was firstly discovered in 2007, is a new human polyomavirus belonging to Polyomaviridae and containing circular double-strand genomic DNA. This study was based on identification assay of KI polyomavirus reported. Total 2293 clinical sputum specimens from children under 3-years-old were collected and screened from Wenzhou Medical College affiliated Wenling Hospital, Zhejiang Province. A KI polyomavirus was detected and identified, the positive rate was 0.04%. The sequences of PCR products was identical to that of the viral capsid protein (VP1) gene derived from KI polyomavirus. The results strongly suggested that the KI polyomavirus was found firstly in Chinese children with acute lower respiratory tract infections from Zhejiang region. This study provided new information for further investigation of etiopathogenisis and diagnosis in children with lower respiratory tract infections.

Characterization of the cytopathic effect in human bronchial epithelial cell after Human Bocavirus Infection (HBoV) / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>In this study, human bronchial epithelial cells were inoculated with positive sputum specimens of HBoV. After four days' infection, cytopathic effects (CPE) were observed by inverted microscopy. These viruses all cause typical cell damages such as rounded and shrivelled, fusion and fallout. These damages got quick following increased future degenerations. The other assay result of CPE within the infected cells were observed by inverted microscopy, have typical "owl's eye" plaque and above 90 percent hemadsorption within the infected cells by erythrocytes for hemadsorption technique. The typical fluorescence lump of nucleus within the infected cells was found by indirect immunofluorescence technique.</p><p><b>CONCLUSION</b>Isolation and identification of HBoV could be done in the human bronchial epithelial cell, and we found some characterizing CPE in the human bronchial epithelial cell after HBoV infection. The above studies pave a way for studying pathogenicity of human bocavirus.</p>

Study on relationship between hepatitis B virus DNA load and genotype with large envelope protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore the relation between hepatitis B virus DNA load and genotype with the level of large envelope protein.</p><p><b>METHODS</b>Serum HBV DNA was quantitively detected by using real time polymerase chain reaction (RT-PCR). The LHBs were detected by using enzyme linked immuno sorbent assay (ELISA) and HBV markers were detected by time differentiate immunofluorescence assay in 140 serum samples collected from chronic hepatitis B patients.The genotypes of HBV were identified by DNA sequencing; and analyze their relationship.</p><p><b>RESULTS</b>There was no significant difference between positive rate of LHBs and that of HBV DNA in HBeAg negative and positive group (P > 0.05); The HBV LHBs absorbency was markedly correlated with the HBV DNA load ( R2 = 0.9267). The difference of HBV LHBs absorbency between HBV genotype B and C was not significant.</p><p><b>CONCLUSIONS</b>The close correlation between HBV LHBs absorbence and HBV DNA load illustrated that he level of serum LHBs can be used to estimate the state of HBV replication; and there is no relationship between HBV LHBs absorbency and genotypes. So HBV LHBs may be used as a new serological marker to detect HBV replication.</p>

Clinical prospective study on maternal-fetal transmission of human bocavirus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate maternal-fetal transmission at human bocavirus (HBoV).</p><p><b>METHODS</b>IgG antibody to HBoV in serum samples of 316 mothers were determined with ELISA and HBoV DNA was determined with real time PCR in the sera of the mothers and their infants.</p><p><b>RESULTS</b>HBoV-IgG was positive in 40.20 percent (127/316) of the mothers, while it was positive in 29.43 percent (93/316) of the cord blood specimens of the infants. The difference between the two groups was significant (X2=8.12, P less than 0.005); 93 samples of both the mothers and the infants were positive for HBoV-IgG.</p><p><b>CONCLUSION</b>HBoV-IgG can cross the placenta to the fetuses through placenta. Further study is needed to answer the question whether vertical maternal-fetal transmission occurs.</p>