Complete genome analysis of coxsackievirus A2 and A5 strains in Changsha / 中国热带医学

@#Abstract: Objective　To investigate the molecular characteristic and evolutionary trends of full-genome sequences of coxsackievirus A2 (CV-A2) and A5 (CV-A5) in Changsha City. Methods　The CV-A2 and CV-A5 strains were isolated and detected from patients with hand, foot and mouth disease (HFMD) cases. The full-genome sequences of CV-A2 and CV-A5 strains were obtained using NGS sequencing. Homology and phylogenetic tree analysis were performed, and the recombination regions of the strains were examined by SimPlot software. Results　The full-genome sequences of CV-A2 and CV-A5 strains were obtained from routine surveillance cases of HFMD in Changsha in 2019. The CV-A2 strain was named S281/Changsha/CHN/2019 with the full-genome sequence of 7 422 bp long; the CV-A5 strain was named S272/Changsha/CHN/2019 with the full-genome sequence of 7 425 bp long. Homology analysis of the isolates by comparison with the nucleic acid sequences of CV-A2 and other CV-A2 strains in China showed that the non-structural protein region shared lower similarity than that of structural protein region. The CV-A2 showed 79.20% similarity with Fleetwood strain (NC038306), showed the highest similarity 95.60% with MN419014 strain from Hubei Province. The non-structural protein 3C and 3D region shared the lowest similarity with MN419014, 90.51 and 92.06%, respectively. Phylogenetic tree analysis showed that 3C and 3D regions were located in the CV-A4 branch. Amino acid mutation sites were found in non-structural protein region, and the amino acid sequence in structural protein region was conserved. SimPlot analysis showed that genetic recombination was found in the 3C and 3D region of CV-A2 strains. The full-genome sequence of CV-A5 showed 80.7% similarity with the Swartz (AY421763) and 97.43% similarity with the strain (MH111030) from Australian. Homology analysis showed that the non-structural protein region shared lower similarity than that of structural protein region, based on full-genome of CV-A5. Phylogenetic tree analysis showed that CV-A5 and MH111030 were in the same branch, indicating that CV-A5 strain not from local. The amino acid sequence of CV-A5 strain was conserved. Conclusions　The CV-A2 strain in Changsha City shared genome sequence information with CV-A4, and the CV-A5 strain was imported from abroad. Our findings are expected to understand the molecular and recombination characteristics of CV-A2 and CV-A5, provided the data of evolution and genetic features of the coxsackievirus, and interrupt disease transmission in a timely and effective manner.

The role of enterovirus 71 and coxsackievirus A strains in a large outbreak of hand, foot, and mouth disease in 2012 in Changsha, China.

BACKGROUND: During 2012, Changsha experienced a large outbreak of hand, foot, and mouth disease (HFMD), resulting in 25,438 cases, including 42 severe cases and eight deaths. METHODS: Seven hundred and forty-six clinical specimens were collected from hospital-based surveillance for HFMD in 2012. The detection and genotyping of enterovirus were performed by real-time RT-PCR and sequencing of the VP1 regions; phylogenetic analysis was performed based on the VP1 sequences. RESULTS: A total of 545 (73.1%) enterovirus-positive samples were identified, with the most frequently presenting serotype being enterovirus 71 (EV-71; n=364, 66.8%), followed by coxsackievirus A16 (CV-A16; n=84, 15.4%), CV-A6 (n=22, 4.0%), and CV-A10 (n=19, 3.5%). Most of the affected patients were children aged ≤5 years (n=524, 96.1%). EV-71 was the major pathogen in the severe and fatal cases (n=22, 78.6%). Phylogenetic analysis of VP1 gene sequences showed the EV-71 isolates to belong to subgenotype C4a, and the CV-A16 isolates to belong to subgenotype B1. The Changsha CV-A6 and CV-A10 circulating strains were homologous to strains circulating in other areas of mainland China. CONCLUSIONS: Our results demonstrate that EV-71 was the primary causative agent responsible for the HFMD outbreak in Changsha in 2012, and the co-circulation of other coxsackievirus A strains posed a potential risk to public health.

Surveillance of influenza virus during 2010-2012 in Changsha, China.

The objective of this study was to examine the circulating influenza viruses in Changsha, China, during 2010-2012. Nasopharyngeal specimens were collected from persons with influenza-like illness (ILI) who presented for care at two hospitals. Of 2,955 patients tested, 278/(9.4%) were positive for influenza virus: 116/(41.7%) were influenza type A(H3N2), 79/(28.4%) were type A(H1N1) pandemic 2009 (pdm09) and 83/(29.9%) were influenza type B. The rates of virus detection varied by age and sex. The highest rate was in the 5-14 year old age group and females were infected more than males. After the initial 2009 A(H1N1) pdm09 outbreak, the number of cases of this virus declined and the season become shorter. Influenza A(H3N2) and B viruses occurred mainly during the spring and summer, while influenza A(H1N1)pdm09 occurred mainly during the winter and spring. Influenza A(H1N1)pdm09 replaced the usual seasonal H1N1 virus during 2010-2012. Continuing epidemiological surveillance of influenza virus is important to monitor trends in influenza infections and to develop prevention and control measures.

[Risk related to the transmission of H5N1 subtype avian influenza virus in the environment of poultry markets in Changsha, China].

OBJECTIVE: To investigate the risk of H5N1 subtype avian influenza virus (AIV) transmission in the poultry market environment in Changsha city. H5N1 antibody levels among the groups related occupational exposure and AIV nucleic acid in the environment of poultry markets were detected. The characteristics of haemagglutinin (HA) genes of H5N1 AIV in the environment were analyzed. METHODS: One district and one county from Changsha city were selected randomly and two poultry markets at inner city or township levels were selected in the same district or county respectively. H5N1 antibody of the occupational exposure groups in the poultry market was tested and AIV nucleic acid in the poultry market environment monitored. One hundred and two blood samples of the occupational exposure groups were tested for H5N1 antibody with single radioimmunoassay diffusion hemolysis (SRH) while 160 environment samples (from sewage, birds stools, feathers and smearing samples of poultry cages) in the poultry market were also detected for AIV nucleic acid with real-time PCR method. Four sewage samples of H5N1 subtype AIV were collected from poultry markets in Changsha, and the HA genes of H5N1 subtype AIV amplified by RT-PCR and then sequenced with TA cloning. Amino acid sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega 5.0 software. RESULTS: The results through H5N1 antibody monitoring program showed that H5N1 antibody positive rates from workers were 25.5% (26/102), 50.0% (9/18) and 25.4% (17/67) respectively in the poultry markets of township and inner cities. H5N1 antibody positive rate in the township poultry markets was higher than in the inner cities poultry markets. RESULTS: from the surveillance on AIV nucleic acid showed that the overall H5 subtype positive rate in Changsha poultry markets was 31.3% (50/160), and the positive rate of townships poultry markets was 37.3% (31/83), which were both higher than those from the inner cities poultry markets (24.7%, 19/77). H5 subtype AIV positive rate was different in the tested specimens, with ranking of positive rates were sewage (50.0%, 24/48), feathers (44.5%, 4/9), birds stools (29.8%, 14/47) and smearing samples of poultry cages (14.3%, 8/56), with statistically significant differences (P < 0.01). Four H5N1 HA genes TA cloning were successfully constructed and identified as Eurasian branch, similar to viruses isolated in mainland China and Hong Kong in the same group, according to genetic analysis. Sequence data of the four HA genes showed the same feature of high pathogenicity, compared to the H5N1 AIV from mainland China of human origin. The receptor specificities were still with avian influenza origin (QSG) and the connecting peptide between HA1 and HA2 possessing the polybasic motif (RERRRKK or RERRGKK). CONCLUSION: One of the reasons for H5N1 antibody positive rate of 25.5% among poultry markets workers was that there were large numbers of H5N1 subtype AIV detected in the environment of poultry markets and HA genes of H5N1 subtype AIV in the poultry markets environment carried molecular characteristics of highly pathogenic which could increase the risk for H5N1 subtype AIV transmission in the environment of poultry markets.

[Genetic analysis of the NS genes of H5N1 avian influenza viruses isolated from sewage in poultry markets].

In order to investigate the transmission risk of H5N1 avian influenza viruses (AIV) from sewage in Changsha poultry markets, the evolution relationship and molecular characteristics of non-structural (NS) genes of H5N1 AIV from sewage were analyzed. Nine H5N1 AIV environmental sewage specimens were collected from Changsha poultry markets. The NS genes were amplifyed by PCR and then sequenced with TA cloning. Amino acid(aa) sequence alignment and phylogenetic tree analysis were conducted by Lasergene and Mega5 software. Eight NS genes TA cloning were constructed successfully. Phylogenetic tree indicated that they were belonged to the allele A subgroup. Aa homology analysis showed 90.1% 92.5% identity in NS1 proteins and 91.0% - 92.6% identity in NS2 proteins compared with reference viruses of the allele A (A/chicken/ Hubei/ w h/ 1999). The homologies of the amino sequences of NS1 and NS2 in this study were 93.8%-100.0% and 98.4%-100.0%, respectively. The C terminal of all eight H5N1 NS1 proteins from sewage in poultry markets carried a ESEV of PL motif and the 92 amino acids were E, furthermore, the 80 to 84aa were missed which were the characteristics of highly pathogenic AIV. The NS genes of H5N1 AIV from sewage in poultry markets have molecular characteristics of highly pathogenic and have the potential risk of H5N1 virus spreading.

[Rapid detection of norwalk GII with reverse transcription loop-mediated isothermal amplification].

To develop a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for rapid and sensitive detection of Norwalk GII. 4 primers which recognized 6 distinct regions on the RNA-dependent RNA polymerase gene of Norwalk GII were designed and used for LAMP assay. Norwalk GII RNA was amplified under isothermal conditions (65 degrees C) for 120 min, and LAMP results were then judged with naked eye, SYBR Green I staining, electrophoretic analysis and restriction digestion. To evaluate the specificity of the RT-LAMP, 48 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses were tested. To compare the sensitivity of the RT-LAMP with that of conventional RT-PCR, Norwalk GII RNA was serially diluted and amplified by RT-LAMP and RT-PCR, respectively. With 46 fecal specimens of Norwalk GII, observation with naked eyes, SYBR Green I staining and electrophoretic analysis were able to detect the PCR products in the RT-LAMP assay. The specificity of RT-LAMP products was also confirmed by digestion of the RT-LAMP products with restriction enzymes. No RNA amplification was observed in 2 fecal specimens of Norwalk GII and 12 fecal specimens of group A rotaviruses. The specificity of the RT-LAMP assay with regard to RT-PCR were 100% for Norwalk GII. The detection limits of RT-LAMP was 15.6 pg/tube for Norwalk GII and similar to that of a RT-PCR assay. Compared to RT-PCR, the RT-LAMP assay has been proven to be a rapid, sensitive, specific and accurate method for detection of the Norwalk GII in fecal specimens, and that RT-LAMP assay is potentially useful for the rapid detection of Norwalk GII from fecal specimens in outbreaks of infectious diarrhea.

[Pathogenic diagnosis of an unexplained epidemic event of infectious diarrhea].

OBJECTIVE: To determine the pathogen of an unexplained epidemic event of infectious diarrhea by laboratory diagnosis of suspected cases samples. METHODS: 28 samples from 28 suspected cases (22 fecal samples, 3 vomitus samples, 3 anus swab samples) were tested for Norovirus by RT-PCR. Sequencing and phylogenetic analysis were acomplished of 5 positive samples. RESULTS: 160 of 5694 population were ill with an attack rate of 2.81%. The peak period was 7-9, March. 14 of 28 samples were tested Norovirus positive.Sequencing and phylogenetic analysis showed Norovirus type GII/4 was the causative agent and it had highest identity (97. 9%) with epidemic strain 2006b. CONCLUSION: The epidemic event ofinfectious diarrhea were caused by GII/4 Norovirus strains.