Rapid Generation of Recombinant Flaviviruses Using Circular Polymerase Extension Reaction.

The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

Development of Miniaturized Water Quality Monitoring System Using Wireless Communication.

Recently, environmental pollution resulting from industrial waste has been emerging in an endless stream. The industrial waste contains chemical materials, heavy metal ions, and other toxic materials. Once the industrial waste is discharged without standards, it might lead to water or environmental pollution. Hence, it has become more important to provide evidence-based water quality monitoring. The use of a multifunctional miniaturized water quality monitoring system (WQMS), that contains continuous monitoring, water quality monitoring, and wireless communication applications, simultaneously, is infrequent. Thus, electrodes integrated with polydimethylsiloxane flow channels were presented in this study to be a compound sensor, and the sensor can be adopted concurrently to measure temperature, pH, electrical conductivity, and copper ion concentration, whose sensitivities are determined as 0.0193 °C/mV, -0.0642 pH/mV, 1.1008 mS/V·cm (from 0 mS/cm to 2 mS/cm) and 1.1975 mS/V·cm (from 2 mS/cm to 5.07 mS/cm), and 0.0111 ppm/mV, respectively. A LoRa shield connected into the system could provide support as a node of long range wide area network (LoRaWAN) for wireless communication application. As mentioned above, the sensors, LoRa, and circuit have been integrated in this study to a continuous monitoring system, WQMS. The advantages of the multifunctional miniaturized WQMS are low cost, small size, easy maintenance, continuous sampling and long-term monitoring for many days. Every tested period is 180 min, and the measured rate is 5 times per 20 min. The feedback signals of the miniaturized WQMS and measured values of the instrument were obtained to compare the difference. In the measured results at three different place-to-place locations the errors of electrical conductivity are 0.051 mS/cm, 0.106 mS/cm, and 0.092 mS/cm, respectively. The errors of pH are 0.68, 0.87, and 0.56, respectively. The errors of temperature are 0.311 °C, 0.252 °C, and 0.304 °C, respectively. The errors of copper ion concentration are 0.051 ppm, 0.058 ppm, 0.050 ppm, respectively.

[Genetic characteristics of viral quasispecies of HIV-1 CRF07_BC among intravenous drug users].

To explore the genetic characteristics of viral quasispecies in HIV-1 CRF07_BC infections among intravenous drug users (IDU), the gp120 fragments of HIV-1 env gene were amplified from plasma samples collected from 6 CRF07_BC infected persons using single genome amplification and sequencing (SGA/ SGS) method, and 11 to 28 sequences were obtained from these samples, respectively, A neighbor-joining phylogenetic tree was reconstructed to describe the genetic characteristics of viral quasispecies. The Simplot, segments' phylogenetic trees and diversity plots based on average pairwise distance (APD) were used to identify the recombination events between quasispecies. The SGA sequences derived from single specimen formed a large monophyletic cluster in the neighbor-joining phylogenetic tree and showed the complex topologic structures of viral quasispecies. Of the 6 CRF07_BC infected patients, only one possessed the high genetic homogeneity, whereas the other five individuals showed high heterogeneity, with two to four subclusters inside the monophyletic cluster for each specimen. In addition, the recombinant events were identified among viral quasispecies from 3 cases. The results show SGA technique and phylogenetic analyses are useful tool to investigate the intrahost CRF07_BC gp120 complex quasispecies variation and high genetic diversity.

[Distribution of HIV-1 subtypes among different populations in Dehong prefecture, Yunnan province, in 2011].

OBJECTIVE: To investigate the distribution of HIV-1 subtypes in Dehong prefecture, Yunnan province, in 2011. METHODS: 300 HIV-1 positive plasma samples were collected from Jan. 2011 to May 2011 in Dehong prefecture. HIV-1 gag genes and env genes were amplified by nested-polymerase chain reaction (PCR) from viral RNA. After sequencing, the HIV-1 subtypes were determined by phylogenetic analysis. RESULTS: Based on the phylogenetic trees of gag gene and env gene fragments, a total of 222 samples were genotyped. Subtype C was the predominant strain in Dehong (43.2%, 96/222), followed by unique recombinant forms (URFs, 27.0%, 60/222), CRF01_AE (21.2%, 47/222), CRF08_BC (5.0%, 11/222), B' (2.3%, 5/222) and CRF07_BC (1.4%, 3/222). Subtype C strains were predominant in both heterosexually transmitted population and intravenous drug users (IDUs), but different subtype distribution patterns were found in these two populations. All 6 genotypes including subtype C (40.7%, 70/172), CRF01_AE (25.0%, 43/172), and URFs (25.0%, 43/172) found in this area among heterosexually transmitted population, which showed the diversity of genotypes in this population. Except subtype B' and CRF07_BC, the other 3 subtypes and URFs were detected among IDUs, mainly including subtype C (54.8%, 23/42) and URFs (38.1%, 16/42), which showed the concentration trend of genotypes distribution among IDUs. The proportion of URFs increased significantly in this area, including the new BC recombinants (41.7%, 25/60) and CRF01_AE relative URFs (58.3%, 35/60). However, the distributions of these two URFs among heterosexually transmitted population and IDUs showed no statistical significance. CONCLUSION: The distribution of HIV-1 strains prevailing in Dehong prefecture was diversity, including 5 subtypes and a variety of URFs, of which subtype C was the predominant strain. The distribution patterns of subtype were different among different populations.

Preparation of a porous composite film for the fabrication of a hydrogen peroxide sensor.

A series of dopant-type polyaniline-polyacrylic acid composite (PAn-PAA) films with porous structures were prepared and developed for an enzyme-free hydrogen peroxide (H(2)O(2)) sensor. The composite films were highly electroactive in a neutral environment as compared to polyaniline (PAn). In addition, the carboxyl group of the PAA was found to react with H(2)O(2) to form peroxy acid groups, and the peroxy acid could further oxidize the imine structure of PAn to form N-oxides. The N-oxides reverted to their original form via electrochemical reduction and increased the reduction current. Based on this result, PAn-PAA was used to modify a gold electrode (PAn-PAA/Au) as a working electrode for the non-enzymatic detection of H(2)O(2). The characteristics of the proposed sensors could be tuned by the PAA/PAn molar ratio. Blending PAA with PAn enhanced the surface area, electrocatalytic activity, and conductivity of these sensors. Under optimal conditions, the linear concentration range of the H(2)O(2) sensor was 0.04 to 12 mM with a sensitivity of 417.5 µA/mM-cm(2). This enzyme-free H(2)O(2) sensor also exhibited a rapid response time, excellent stability, and high selectivity.

[Distribution of HIV-1 subtype and its relationship with HIV/AIDS epidemic in Guizhou province].

OBJECTIVE: To study the HIV-1 diversity and how did it promote the rapid spread of AIDS, in Guizhou province. METHODS: A total of 190 HIV-1 positive subjects were collected in different years and regions from Guizhou province. The env and gag genes were amplified with nested PCR and their sequences were determined. The subtypes were identified by the MEGA 4.0 software and the relationships between subtypes and AIDS epidemic were analyzed. RESULTS: The number of HIV/AIDS reported cases was increased from 66 in 1998 up to 8435 in 2009, a 16.38 time increase in 7 years. Subtypes B (9), B' (4), C (2), CRF07_BC (75), CRF08_BC (17), CRF01_AE (64) were identified in Guizhou province among the samples collected in various periods of time. The genetic diversities in env gene of CRF07_BC and CRF08_BC increased along with the spreading of HIV (from 0.035 ± 0.006 to 0.092 ± 0.011). Subtype B' (4/11) appeared the main subtype prevailed in Guizhou in 1998 as well as CRF07_BC (26/41) in 2002 and CRF01_AE (62/119) in 2007. The HIV/AIDS epidemic in Guizhou province showed an rapidly upward trend, with the main risk factors of HIV transmission as 2610 cases through injecting drug users (IDUs). and 176 cases due to sexually transmitted infections (STIs), from year 2001 to 2006. However, STIs began to increase rapidly, after 2006, with 1713 cases of IDUs and 1833 cases of STIs. Data indicated that the change of composition of different HIV-1 subtypes was correlated with the mode of transmission in Guizhou province (χ(2) = 41.253, P = 0.000). CONCLUSION: The types of HIV strains changed over time as well the turnover of the main risk factors. Sexual transmission, including both hetero- and homo-sexual became the main risk factors, suggesting the development of related prevention and control programs, on HIV/AIDS should be considered accordingly in the future.

[Genetic characteristics of HIV-1 CRF01_AE strains in four provinces, southern China].

OBJECTIVE: To analyze the genetic characteristics of HIV-1 CRF01_AE strains prevailing in the four provinces of southern China. METHODS: Plasma samples were collected from the newly diagnosed HIV-1 individuals reported in 2006 in Guangdong, Guangxi, Jiangxi and Hunan province. The gag and env gene fragments were amplified from RNA template extracted from plasma using RT and nested PCR methods. CRF01_AE sequences were analyzed by phylogenetic methods and characterized by calculating the genetic distance and Entropy analysis. RESULTS: Two main epidemic clusters were found to exist in the CRF01_AE strains from 210 HIV-1 CRF01_ AE infected individuals collected in the 4 provinces, southern China. It was found that no international reference strain was closely correlated with cluster I, which including 123 samples. The strains in cluster II, consisting 57 cases of samples, were closely related with the strains identified in Vietnam. Genetic distance analysis of gag and env genes showed that the diversity of cluster I was obviously less than that of cluster II. Data on nucleotide polymorphism showed that nucleotides compositions of 42 sites in gag and 40 sites in env were significantly different between the two clusters. When compared with cluster II, the polymorphism decreased at 61 nucleotide sites but increased at 21 sites in cluster I. CONCLUSION: This was the first report describing that two main epidemic clusters were existed in CRF01_AE strains prevailing in the 4 provinces, Southern China. The virus in cluster I was the dominant strain in this region, with shorter period of circulation and higher proportion seen in the HIV-infected population, which might belong to CRF01_AE strain with certain features facilitating the spread of the virus. The virus in cluster II was highly homology with the CRF01_AE strains from Vietnam, and seemed to have had several events of epidemics in populations in border regions of China and Vietnam.

[Primers of gag gene for HIV-1 subtyping in China and application thereof in practice].

OBJECTIVE: To design the appropriate primers of gag gene for HIV-1 subtyping in molecular epidemiology survey based on the genetic characteristics derived from the main HIV-1 strains prevailing in China. METHODS: The gag genes of HIV-1 CRF07_BC, CRF08_BC, and subtype C, together with subtyping reference sequences, were obtained from HIV sequence database. Referring to the alignments and genetic characteristics of HIV-1 full gag sequences, new primers of gag gene for amplification and subtyping were designed. The target fragment was used to construct neighbor-joining phylogenetic tree and evaluate its reliability. The newly designed primers (GUX/GDX) were used to amplify the plasma samples to evaluate their efficiency. RESULTS: The phylogenetic tree of 306/c-gag fragments (positions 836-1507 of HIV-1 strain HXB2) showed that CRF07_BC and subtype C strains formed clusters with low bootstrap values (59% for CRF07_BC and 70% for subtype C), and the phylogenetic tree could not distinguish the sequences of CRF07_BC, CRF08_BC, and subtype C very well. Whereas the sequences of CRF07_BC, CRF08_BC, and subtype C from GUX/GDX (positions 781-1861) were clustered separately with higher bootstrap values (99%, 99%, and 77% respectively). In practice, a very good amplification and sequencing efficiency with over 90% positive results on average were obtained with GUX/GDX. Five clusters of subtype B, C, CRF01_AE, CRF07_BC, and CRF08_BC were formed with higher confidence (Bootstrap values all above 80%). The reliable phylogenetic tree could be constructed based on the fragments sequenced only with antisense primer (GDX). CONCLUSION: Fragments obtained with GUX/GDX primers of gag gene can be used to reconstruct phylogenetic tree with high reliability to distinguish the HIV-1 strains circulating in China, especially for the major BC recombinant and subtype C strains, which provides a useful tool in HIV molecular epidemiologic research.