Rusheng Zhang

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.

Clinical, epidemiological and virological characteristics of the first detected human case of avian influenza A(H5N6) virus

A human infection with novel avian influenza A H5N6 virus emerged in Changsha city, China in February, 2014. This is the first detected human case among all human cases identified from 2014 to early 2016. We obtained and summarized clinical, epidemiological, and virological data from this patient. Complete genome of the virus was determined and compared to other avian influenza viruses via the construction of phylogenetic trees using the neighbor-joining approach. A girl aged five and half years developed fever and mild respiratory symptoms on Feb. 16, 2014 and visited hospital on Feb. 17. Throat swab specimens were obtained from the patient and a novel reassortant avian influenza A H5N6 virus was detected. All eight viral gene segments were of avian origin. The hemagglutinin (HA) and neuraminidase (NA) gene segments were closely related to A/duck/Sichuan/NCXN11/2014(H5N1) and A/chicken/Jiangxi/12782/2014(H10N6) viruses, respectively. The six internal genes were homologous to avian influenza A (H5N2) viruses isolated in duck from Jiangxi in China. This H5N6 virus has not gained genetic mutations necessary for human infection and was suggested to be sensitive to neuraminidase inhibitors, but resistant to adamantanes. Epidemiological investigation of the exposure history of the patient found that a live poultry market could be the source place of infection and the incubation period was 2-5days. This novel reassortant Avian influenza A(H5N6) virus could be low pathogenic in humans. The prevalence and genetic evolution of this virus should be closely monitored.

Isolation and characteristic analysis of a novel strain H7N9 of avian influenza virus A from a patient with influenza-like symptoms in China

A novel H7N9 virus (A/Changsha/1/2013(H7N9)) identified through routine examination in the influenza network laboratory was analyzed retrospectively. The gene sequences of A/Changsha/1/2013(H7N9) were highly homologous to other viruses isolated in mainland China. Mutations of Q226L and G186V were found in the hemagglutinin protein (HA). Amino acid deletions were found at positions 69-73 of the neuraminidase protein (NA) and 218-230 of the non-structural protein (NS1). All viral genes except PB1 were essentially identical to the sequences of other Chinese influenza A H7N9 isolates. Overall, A/Changsha/1/2013(H7N9) is highly homologous to other H7N9 avian influenza viruses isolated in mainland China.

Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay

A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found.

Analysis of the full-length genome of a novel strain of the H7N9 avian influenza virus

The aim of the present study was to analyze the evolution and variation of a novel strain of the avian influenza virus. The virus-positive specimens [A/Changsha/2/2013 (H7N9)] from a patient infected with the novel avian influenza A (H7N9) virus was amplified by reverse transcription-PCR and the full genome was sequenced. The sequencing results were submitted to GenBank and then analyzed by phylogenetic tree analysis using BioEdit and Mega5 software. The phylogenetic tree of the hemagglutinin (HA) and neuraminidase genes revealed that A/Changsha/2/2013 (H7N9) and all the new H7N9 viruses in 2013 were in a large cluster, and their nucleotide evolutionary distances were closely associated. Phylogenetic tree analyses of the nucleoprotein and nonstructural genes demonstrated two main branches. One branch contained novel H7N9 viruses isolated from avian, human and environmental sources in different regions. The other branch contained three novel H7N9 virus strains isolated from environmental sources in Shanghai. All the phylogenetic trees of the matrix protein, polymerase acidic, polymerase basic protein 1 and polymerase basic protein 2 genes also showed two branches, with each branch including the novel H7N9 virus strains isolated from avian, human and environmental sources in different regions. Molecular characterization demonstrated that 52 novel H7N9 viruses sequenced to date contain the G228S and G186V mutations in the receptor binding site of the HA protein. The full-genome sequences of A/Changsha/2/2013 and analyses of its molecular characteristics suggest that the A/Changsha/2/2013 H7N9 virus strain has molecular characteristics that may facilitate adaptation of the virus to mammalian hosts and may even bind to human receptors.

Development and Evaluation of a Real-Time RT-PCR Assay for Detection of a Novel Avian Influenza A (H5N6) Virus

As of Aug 25, 2017, 17 incidences of human infection and 6 deaths due to the novel H5N6 virus have been reported in China. Genetic analysis of the viral genome revealed that this reassortant virus is highly pathogenic to poultry, and that the virus has a risk of transmission to humans. Accordingly, the development of a rapid, sensitive, and specific molecular diagnostic assay is critical for public health. In this study, a real-time reverse-transcription PCR (RT-PCR) assay was developed to specifically detect the novel H5N6 virus, with primer pairs targeting the hemagglutinin and neuraminidase gene sequences of this virus. RNA was extracted from throat swab specimens from patients with influenza-like illness (ILIs), and environmental samples were collected from live poultry markets (LPMs) for H5N6 virus detection by real-time RT-PCR. The method was demonstrated to enable specific detection of the avian H5N6 virus, with no cross-reactivity with seasonal influenza viruses (H1N1, H1N1 pdm09, H3N2 or B); H5N1, H7N9, H9N2 viruses; or other human respiratory viruses. The detection limit of the assay was 1.0 × 101 copies per reaction for N6 and 1.0 × 102 copies per reaction for H5 assays. The assay is a powerful tool for rapid, sensitive, and specific detection of H5N6 virus infection in specimens derived from humans, animals, and the environment.

Epidemiological and Genetic Characteristics of Rabies Virus Transmitted Through Organ Transplantation

In January 2016, two patients died of rabies after receiving kidney transplants from a common organ donor at a hospital in Changsha, Hunan, China. The medical records, epidemiological data of the organ donor, two kidney and a liver recipients were reviewed. Intravitam saliva samples of the two kidney recipients were tested for rabies virus (RABV) using real-time RT-PCR, and the nucleoprotein (N) gene was amplified and sequenced by Sanger sequencing. Whole genome sequences were analyzed using next-generation sequencing. The N genes of the two kidney recipients showed 100% nucleic acid identity. Phylogenetic analysis of the complete genome, N and glycoprotein (G) genes indicated that the RABV was homologous with dog isolates from the Hunan province and belong to the China I lineage, which is widespread in China. The organ donor was a 22-month-old boy who died from unknown acute progressive encephalitis. After undergoing sub-hypothermia hibernation therapy, rabies-associated symptoms were atypical, and rabies was neglected because serum RABV-specific antibodies were negative. An unknown wound on the forehead of the donor was found 2 months before the onset of symptoms. Based on the clinical, epidemiological, and molecular findings, we speculated that the RABV initially originated in the donor from a dog bite, and was then transmitted to the recipients by organ transplantation. An uncertain exposure history and misdiagnosis played important roles in the spread of the RABV. Rabies should be considered in patients with acute progressive encephalitis of unexplained etiology, especially in potential organ donors.

Primers and probe design and precision assessment of the real time RT-PCR assay in Coxsackievirus A10 and enterovirus detection

This data article contains data related to the research article entitled “Rapid detection of enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay” (Chen at al., 2017) [1]. Primers and probe sequence design are among the most critical factors in real-time polymerase chain reaction (PCR) assay optimization. Linearity, sensitivity, specificity and precision are the crucial criteria which are used to evaluate the performance of a new method. This data article report the primers and probe design and precision assessment of the new assay. VP1 gene of Coxsackievirus A10 (CV-A10) and 5′-NCR of different enterovirus (EV) serotypes were retrieved from GenBank database and aligned. The intra- and inter-assay variation were assessed using high, medium and low concentration of control plasmid DNA and viral RNA samples.