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Wu S.,Centers for Disease Control and Prevention | Wu S.,Fujian Province Key Laboratory of Zoonosis Research | Wu S.,Fujian Medical University | Yan P.,Centers for Disease Control and Prevention | And 9 more authors.
Archives of Virology | Year: 2015

HIV/AIDS is a leading public health concern throughout the world. Currently, treatment of HIV/AIDS still depends on highly active antiretroviral therapy (HAART); however, there is increasing evidence showing the emergence of resistance to antiretroviral drugs in HIV-1 strains, making ART less effective over time. Intensive monitoring of HIV-1 drug resistance is therefore of great importance to evaluate the current sensitivity of antiretroviral agents and is urgently needed. The aim of this study was to develop a single-loop recombinant pseudotyped-virus-based assay to detect phenotypic resistance in clinical HIV-1 strains. HIV-1 RNA was extracted from HIV-1-infected human plasma samples, and an approximately 3-kb fragment containing p7/p1/p6 cleavage sites and full-length protease (PR), reverse transcriptase (RT), thermonuclease (TNase), and integrase (1–280 aa) genes was amplified by nested RT-PCR. A retroviral vector was constructed using the HIV-1 infectious molecular clone pLWJ to test antiretroviral drug susceptibility. pLWJ-SV40-Luc contained a luciferase expression cassette inserted within a deleted region of the envelope (env) gene as an indicator gene. Resistance test vectors (RTVs) were constructed by incorporating amplified target genes into pLWJ-SV40-Luc by using ApaI or AgeI and AarI restriction sites and conventional cloning methods. The virus stocks used for drug susceptibility test were produced by co-transfecting 293T cells with RTVs and a plasmid that provided vesicular stomatitis virus glycoprotein (VSV-G). Viral replication was monitored by measuring luciferase activity in infected target cells at approximately 48 h postinfection. A total of 35 clinical plasma samples from HIV-1-infected humans were tested, and target fragments were successfully amplified from 34 samples (97.1 %) and 33 RTVs were successfully constructed by directional cloning, with an overall success rate of 94.3 %. A clear-cut dose-dependent relationship was detected between virus production and luciferase activity in the constructed phenotypic resistance testing system. The highest coefficient of determination (R2) was found between luciferase activity and drug concentration and viral inhibition at 293T cell concentrations of 5 × 104 cells per well. The phenotypic profiles of the viruses from 29 clinical samples almost completely matched the observed genotypes. The results demonstrate that a single-loop recombinant pseudotyped-virus-based assay was successfully developed, and this testing system has high stability and appears to be applicable for testing phenotypic resistance of clinical HIV-1 strains to commonly used antiretroviral agents. © 2015, Springer-Verlag Wien.


Yang X.-H.,Fujian Medical University | Yang X.-H.,Fujian Province Key Laboratory of Zoonosis Research | Yan Y.-S.,Fujian Medical University | Yan Y.-S.,Fujian Province Key Laboratory of Zoonosis Research | And 5 more authors.
Journal of Medical Virology | Year: 2013

Echovirus 30 (E-30) was responsible for an outbreak of aseptic meningitis between April 1 and June 2, 2011 in Fujian Province, China. A molecular epidemiology study of 115 E-30 strains was performed to characterize the genetic features of the etiologic agent of the 2011 aseptic meningitis outbreak. The phylogenetic trees of the complete VP1 gene (876bp) from 74 of 115 isolates and 50 reference sequences were analyzed. Three lineages (E-30_h, i, and j) were detected that had co-circulated in Fujian in the last decade, of which E-30_j was new. The other 72 Fujian strains and 16 representative strains from other provinces of China all belong to E-30_h and E-30_i. Two distinct E-30 clusters including virus isolates obtained during adult surveillance were associated with the 2011 outbreak and differed from Fujian isolates prior to 2011, suggesting that the viruses may vary and adult infections play an important role in viral transmission. Thus, the multiple lineages of E-30 in Fujian and variant viruses enhanced transmissibility, which may be related to the epidemic activity of E-30. © 2013 Wiley Periodicals, Inc.

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