Haiyan Mao

Rapid detection of H5 avian influenza virus by TaqMan‐MGB real‐time RT‐PCR

Aims:  Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) assay based on a TaqMan‐minor groove binder (MGB) probe was developed for the rapid detection of avian influenza virus subtype H5.

Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

Background Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus. Methodology and Principal findings We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses. Conclusions These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.

The first avian influenza A (H7N9) viral infection in humans in Zhejiang Province, China: a death report.

This study reports the first death caused by a novel avian influenza A (H7N9) virus in Zhejiang Province, China. The patient had chronic hepatitis B and history of exposure to poultry. The patient initially complained of diarrhea and influenza-like symptoms on March 7 and 14 respectively. The disease progressed to severe pneumonia, sustained hypoxia, and coagulation abnormalities. The patient died on March 27 because of respiratory failure, multiple organ failure, and disseminated intravascular coagulation without oseltamivir treatment. This H7N9 virus from Zhejiang is highly similar to isolates obtained from Shanghai, Jiangsu, Anhui, etc. Analysis of hemagglutinin, neuramidinase, and matrix genes indicated that the isolates share the same avian origin, have low virulence, and are sensitive to oseltamivir, but are resistant to adamantine. Only the isolate that caused the fatality exhibited substitution of Q226I in the HA gene, which indicates a potentially enhanced human affinity. The secondary transmission rate was 1.6% (2/125). Only two health workers presented with influenza-like symptoms, and they subsequently tested negative for H7N9 RNA. In conclusion, underlying disease, late diagnosis, and untimely antiviral treatment are possible high-risk factors for infections and death caused by the lowpathogenicity avian influenza A (H7N9). Person-to-person transmission of the H7N9 virus was not detected among close contacts, but such transmission should be investigated in the future. Expanding and enhancing surveillance will help in the early discovery and diagnosis of suspected cases, which will reduce the number of severe cases and deaths.

Surveillance of Avian H7N9 Virus in Various Environments of Zhejiang Province, China before and after Live Poultry Markets Were Closed in 2013-2014.

Background To date, there have been a total of 637 laboratory-confirmed cases of human infection with avian influenza A (H7N9) virus across mainland China, with 28% (179/637) of these reported in Zhejiang Province. Surveillance of avian H7N9 virus was conducted to investigate environmental contamination during H7N9 outbreaks. We sought to evaluate the prevalence of H7N9 in the environment, and the effects of poultry market closures on the incidence of human H7N9 cases. Methods We collected 6740 environmental samples from 751 sampling sites across 11 cities of Zhejiang Province (China) between January 2013 and March 2014. The presence of H7N9 was determined by reverse transcription polymerase chain reaction, with prevalence compared between sites and over time. The relationship between environmental contamination and human cases of H7N9 infection were analyzed using Spearman’s ranked correlation coefficient. Results Of the 6740 samples, 10.09% (680/6740) were H7N9-positive. The virus was found to circulate seasonally, and peaked during the spring and winter of 2013–2014. The prevalence of the virus decreased from the north to the southeast of the province, coinciding with the geographical distribution of human H7N9 cases. Compared with other sampling sites, live poultry markets (LPMs) had the highest prevalence of H7N9 virus at 13.94% (667/4784). Of the various sample types analyzed, virus prevalence was highest for chopping board swabs at 15.49% (110/710). The prevalence of the virus in the environment positively correlated with the incidence of human H7N9 cases (r2 = 0.498; P < 0.01). Cities with a higher incidence of human H7N9 cases also had a higher prevalence of H7N9 among samples and at sampling sites. Following the closure of LPMs at the end of January 2014, the prevalence of H7N9 decreased from 19.18% (487/2539) to 6.92% (79/1141). This corresponded with a decrease in the number of human H7N9 cases reported. Conclusions The prevalence of H7N9 virus in environmental samples oscillated seasonally, regardless of whether LPMs were open. The presence of H7N9 in environmental samples positively correlated with the number of human H7N9 cases, indicating that eradication of the virus from the environment is essential in reducing the numbers of H7N9 cases and halting the spread of the virus.

Rapid and sensitive identification of RNA from the emerging pathogen, coxsackievirus A6

BackgroundHand, foot and mouth disease (HFMD) is caused by members of the family Picornaviridae in the genus Enterovirus. It has been reported that coxsackievirus A6 (CVA6) infections are emerging as a new and major cause of epidemic HFMD. Sporadic HFMD cases positive for CVA6 were detected in the mainland of China in recent years. To strengthen the surveillance of CVA6 infections and outbreak control, the clinical diagnosis is urgently needed to distinguish the CVA6 infection disease from other infections.MethodsIn order to develop a sensitive quantitative real-time RT-PCR assay for rapid detection of CVA6 RNA, primers and probe were designed to target the VP1 gene segment of CVA6. The conservation of the target segment was firstly analyzed by bioinformatic technology. The specificity of the real-time RT-PCR was further confirmed by detecting other related viruses and standard curves were established for the sensitivity evaluation. The pharyngeal swab samples from the EV71 and CVA16 unrelated HFMD patients were applied for CVA6 detection through the established method.ResultsBased on the primer–probe set to detect the target VP1 gene segment of CVA6, the quantitative real-time RT-PCR assay could discriminate CVA6 infection from other resemble viral diseases with a potential detection limit of 10 viral copies/ml. The specificity of the assay was determined by sequence alignment and experimentally tested on various related viruses. The standard curve showed that the amplification efficiency of templates with different concentrations of templates was almost the same (R2 >0.99). Evaluation of the established method with pharyngeal swabs samples showed good accordance with the results from serology diagnosis.ConclusionThis study is the first report developing a VP1 gene-based quantitative real-time RT-PCR for rapid, stable and specific detection of CVA6 virus. The real-time RT-PCR established in this study can be used as a reliable method for early diagnosis of CVA6 infection.

Nosocomial Co-Transmission of Avian Influenza A(H7N9) and A(H1N1)pdm09 Viruses between 2 Patients with Hematologic Disorders

Transmission of these viruses was limited to 2 immunocompromised patients in the same ward.

A family cluster of three confirmed cases infected with avian influenza A (H7N9) virus in Zhejiang Province of China

BackgroundA total of 453 laboratory-confirmed cases infected with avian influenza A (H7N9) virus (including 175 deaths) have been reported till October 2,2014, of which 30.68% (139/453) of the cases were identified from Zhejiang Province. We describe the largest reported cluster of virologically confirmed H7N9 cases, comprised by a fatal Index case and two mild secondary cases.MethodsA retrospective investigation was conducted in January of 2014. Three confirmed cases, their close contacts, and relevant environments samples were tested by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), viral culture, and sequencing. Serum samples were tested by haemagglutination inhibition (HI) assay.ResultsThe Index case, a 49-year-old farmer with type II diabetes, who lived with his daughter (Case 2, aged 24) and wife (Case 3, aged 43) and his son-in-law (H7N9 negative). The Index case and Case 3 worked daily in a live bird market. Onset of illness in Index case occurred in January 13, 2014 and subsequently, he died of multi-organ failure on January 20. Case 2 presented with mild symptoms on January 20 following frequent unprotected bed-side care of the Index case between January 14 to 19, and exposed to live bird market on January 17. Case 3 became unwell on January 23 after providing bedside care to the Index case on January 17 to 18, and following the contact with Case 2 during January 21 to 22 at the funeral of the Index case. The two secondary cases were discharged on February 2 and 5 separately after early treatment with antiviral medication. Four virus strains were isolated and genome analyses showed 99.6 ~100% genetic homology, with two amino mutations (V192I in NS and V280A in NP). 42% (11/26) of environmental samples collected in January were H7N9 positive. Twenty-five close contacts remained well and were negative for H7N9 infection by RT-PCR and HI assay.ConclusionsIn the present study, the Index case was infected from a live bird market while the two secondary cases were infected by the Index case during unprotected exposure. This family cluster is, therefore, compatible with non-sustained person-to-person transmission of avian influenza A/H7N9.

A study of family clustering in two young girls with novel avian influenza A (H7N9) in Dongyang, Zhejiang Province, in 2014

BACKGROUND The avian influenza A H7N9 virus, previously unknown in humans, has infected humans in many areas of China since February 2013. Here we report on a clustering case of H7N9 in two little girls in one family in Dongyang city, Jinhua area, Zhejiang Province. OBJECTIVES To determine (1) whether the infections were due to person-to-person transmission or to co-exposure to poultry and (2) the prevalence of this novel H7N9 virus in Dongyang inferred by this family clustering case. STUDY DESIGN Samples were collected from patients and environment. We undertook detailed epidemiological investigations and laboratory work. Phylogenetic analyses were done based on the sequenced genomes. The concentration of cytokines and chemokines in the serum was detected by cytometric bead array analyses. RESULTS A mixture of H7 and H9 was detected from the environmental sample. The three H7N9 viruses shared one infection source. The index patient who had significantly higher levels of IL-4, IL-8 and IL-10 suffered severe infection. CONCLUSIONS Based on a comparison with previous isolations of the virus in 2013, H7N9 has evolved different lineages through recombination with local H9N2 viruses. Determining whether it was human-to-human transmission or exposure to the same live poultry, since both patients had identical exposure histories, was ambiguous. The results from the cytokine analyses agreed with the conclusion that H7N9 severity is associated with a higher level of cytokines/chemokines. Long term influenza surveillance remains essential to allow for early warning of potential transmission events.

Characterization of the complete genome of chikungunya in Zhejiang, China, using a modified virus discovery method based on cDNA-AFLP.

Background Chikungunya (CHIK) virus is a mosquito-borne emerging pathogen presenting great health challenges worldwide, particularly in tropical zones. Here we report a newly detected strain of CHIK, Zhejiang/chik-sy/2012, in China, a nonindigenous region for CHIK, using a modified approach based on the classic cDNA-AFLP. We then performed etiological and phylogenetic analyses to better understand its molecular characterization and phylogenetic pattern, and also to aid in further evaluating its persistence in Southeast Asia. Methods By using this modified procedure, we determined for the first time the complete genome sequence of the chikungunya virus strain, Zhejiang/chik-sy/2012, isolated in 2012 from a patient in Zhejiang, China. Sequence analyses revealed that this positive single strand of RNA is 12,017 bp long. We found no single amino acid mutation in A226V, D284E and A316V. Phylogenetic analysis showed that our strain shared the greatest homology with a strain isolated in Taiwan, which was derived from a strain from Indonesia. Chik-sy/2012 is in a different clade from other CHIK viruses found in China previously. Conclusions A modified cDNA-AFLP in virus discovery was used to isolate the first CHIK and the first complete genome sequence of virus strain chik-sy/2012 in 2012 from a patient with CHIK fever in Zhejiang, China. The infection displayed great phylogenetic distance from viruses detected in Guangdong, China, in 2008 and 2010, since they were derived from another evolutionary lineage. Additional molecular epidemiology data are needed to further understand, monitor and evaluate CHIK in China.

Inapparent Infection During an Outbreak of Dengue Fever in Southeastern China

Dengue fever (DF) is often asymptomatic in endemic areas. Asymptomatic infection during a DF outbreak in China, where DF is not endemic, has not been reported until now. In this study a total of 365 subjects from 6 villages were recruited from October 4-7, 2009. Overall, 102 subjects (27.95%) were positive for dengue virus (DENV) IgM, and 14 subjects (3.84%) were positive for DENV IgG and IgM. In different age groups, seropositive rates varied from 12.50% to 50.00% for DENV IgM, and from 0% to 11.76% for DENV IgG. Seroprevalence of DENV IgM was significantly higher than that of DENV IgG. Seroprevalence rates of DENV IgM differed among different villages. However, the seroprevalence of DENV IgM was not statistically significantly different among gender and age groups. Asymptomatic DF infection is prevalent in non-endemic areas.