Yefei Zhu

Dynamic reassortments and genetic heterogeneity of the human-infecting influenza A (H7N9) virus

Influenza A (H7N9) virus has been causing human infections in China since February 2013, raising serious concerns of potential pandemics. Previous studies demonstrate that human infection is directly linked to live animal markets, and that the internal genes of the virus are derived from H9N2 viruses circulating in the Yangtze River Delta area in Eastern China. Here following analysis of 109 viruses, we show a much higher genetic heterogeneity of the H7N9 viruses than previously reported, with a total of 27 newly designated genotypes. Phylogenetic and genealogical inferences reveal that genotypes G0 and G2.6 dominantly co-circulate within poultry, with most human isolates belonging to the genotype G0. G0 viruses are also responsible for the inter- and intra-province transmissions, leading to the genesis of novel genotypes. These observations suggest the province-specific H9N2 virus gene pools increase the genetic diversity of H7N9 via dynamic reassortments and also imply that G0 has not gained overwhelming fitness and the virus continues to undergo reassortment.

Cytokine and Chemokine Levels in Patients Infected with the Novel Avian Influenza A (H7N9) Virus in China

H7N9 avian influenza is an emerging viral disease in China caused by avian influenza A (H7N9) virus. We investigated host cytokine and chemokine profiles in serum samples of H7N9 patients by multiplex-microbead immunoassays. Statistical analysis showed that IP-10, IL-6, IL-17, and IL-2 were increased in H7N9 infected patients. Furthermore, IL-6 and the chemokine IP-10 were significantly higher in severe H7N9 patients compared to nonsevere H7N9 cases. We suggest that proinflammatory cytokine responses, characterized by a combined Th1/Th17 cytokine induction, are partially responsible for the disease progression of patients with H7N9 infection.

Case-control study of risk factors for human infection with influenza A(H7N9) virus in Jiangsu Province, China, 2013

We describe a case-control study performed in Jiangsu, China, to evaluate risk factors for human infection with novel avian influenza A(H7N9) virus. Twenty-five cases and 93 controls matched by age, sex, and location were included in the study. Direct contact with poultry or birds in the two weeks before illness onset, chronic medical conditions (hypertension excluded), and environment-related exposures were significantly associated with A(H7N9) infection.

Rapid and Sensitive Detection of Novel Avian-Origin Influenza A (H7N9) Virus by Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Lateral-Flow Device

A severe disease in humans caused by a novel avian-origin influenza A (H7N9) virus emerged in China recently, which has caused at least 128 cases and 26 deaths. Rapid detection of the novel H7N9 virus is urgently needed to differentiate the disease from other infections, and to facilitate infection control as well as epidemiologic investigations. In this study, a reverse transcription loop-mediated isothermal amplification combined with a lateral flow device (RT-LAMP-LFD) assay to rapidly detect H7N9 virus was developed and evaluated. The RT-LAMP primers were designed to target the haemagglutinin (HA) and neuraminidase (NA) genes of H7N9 virus. Results of 10-fold dilution series assays showed that analysis of RT-LAMP products by the LFD method was as sensitive as real-time turbidity detection, and that the analytic sensitivities of the HA and NA RT-LAMP assays were both 10 copies of synthetic RNA. Furthermore, both the assays showed 100% clinical specificity for identification of H7N9 virus. The performance characteristics of the RT-LAMP-LFD assay were evaluated with 80 clinical specimens collected from suspected H7N9 patients. The NA RT-LAMP-LFD assay was more sensitive than real time RT-PCR assay. Compared with a combination of virus culture and real-time RT-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the RT-LAMP-LFD assay were all 100%. Overall, The RT-LAMP-LFD assay established in this study can be used as a reliable method for early diagnosis of the avian-origin influenza A (H7N9) virus infection.

Seroprevalence of antibodies against SFTS virus infection in farmers and animals, Jiangsu, China

BACKGROUND Severe fever with thrombocytopenia syndrome (SFTS) is a newly identified viral zoonosis caused by a phlebovirus. Most reported SFTS cases are farmers living in rural areas. The seroprevalence of SFTS virus in farmers has not been investigated. The current knowledge of SFTS virus seroprevalence in animals, especially in wild animals, is still poor. OBJECTIVES To investigate SFTS virus seroprevalence among farmers and a variety of animal species. STUDY DESIGN SFTS virus antibodies in sera were determined using a double-antigen sandwich ELISA. Serum samples were collected from 2547 farmers and 2741 animals in 6 SFTS-endemic counties from March 2012 to February 2013 in Jiangsu province. The farmer participants aged from 15 to 90 years. All of them were interviewed using a structured questionnaire. The animals sampled included 6 domesticated animal species and 2 wild animal species. RESULTS SFTSV antibodies were found in a total of 33 farmers (1.30%) and was more prevalent in males than in females (respectively 1.87% and 0.71%, P<0.01). The mean age of seropositive farmers was 56.5 years and seroprevalence increased gradually with age. Seroprevalence in animal species were: goats (66.8%), cattle (28.2%), dogs (7.4%), pigs (4.7%), chickens (1.2%), geese (1.7%), rodents (4.4%) and hedgehogs (2.7%). Multiple variable logistic regression analysis showed that grazing, grass mowing, raising cattle, age, farm work time and tick bites were risk factors for SFTS virus infection among farmers. CONCLUSIONS SFTSV readily infects humans with farming-related exposures as well as numerous domestic and wild animals. Serological results further suggest that the virus circulates widely in Jiangsu province.

Human co-infection with novel avian influenza A H7N9 and influenza A H3N2 viruses in Jiangsu province, China

In February, 2013, a novel avian infl uenza A H7N9 virus emerged in east China and quickly spread to other areas. By May 27, 130 human infections had been confi rmed, with 37 deaths. Transmission can occur through direct or close contact with poultry or through exposure to environments that are contaminated with poultry. No human-to-human trans mission has been reported. Coinfection of viruses in human beings, birds, or other animals provides the possibility for the emergence of a new reassortant virus. Here, we report human co-infection with novel H7N9 and seasonal infl uenza A H3N2 viruses. On April 25, 2013, a previously healthy 15-year-old male student began to have infl uenza-like symptoms of fever, mild cough, headache, and sore throat. The patient did not report a history of contact with sick persons or animals. Retrospective investigation showed that none of his close contacts developed infl uenza-like symptoms. The patient went to a local hospital on April 26 when he felt that his symptoms were getting worse. On physical examination, his temperature was 39·0°C. The white blood cell count was 5·63 cells × 109/L with 65% neutrophils and 21% lymphocytes. The patient was admitted to hospital and received oseltamivir 75 mg twice daily to alleviate symptoms. He recovered on May 2. Two throat swabs were collected on his fi rst visit to the hospital on April 26. One specimen was used for the detection of infl uenza A and B virus by a rapid, point-ofcare colloid gold assay (ABON, Alere Inc, Hangzhou, China), which was positive for infl uenza A. The other specimen was sent to the Nanjing Municipal Center for Disease Control and Prevention (NJCDC), where subtypespecifi c primers provided by the China CDC were used to detect seasonal infl uenza viruses (H1 and H3), pdm2009 H1N1, and avian infl uenza virus subtypes (H5N1, H9N2, and H7N9) on an ABI 7500 real-time PCR system. The Ct values of general infl uenza A, H7, and H3 were 18, 30, and 18 respectively. The remainder of the sample following this analysis was then sent to the Jiangsu Provincial CDC for confi rmation of these fi ndings. We isolated virus from this specimen using Madin-Darby canine kidney cells. Further sequence analysis of RNA extracted from the cell culture supernatants on MiSeq platform (Illumina inc., San Diego, CA, USA) and ABI 3130 automatic DNA analyzer (Life Technologies, Applied Biosystems, Foster City, CA, USA) showed that H3, N2, H7, and N9 genes coexisted in the sample. Nucleoid acid sequence comparison of the H7 and N9 genes of the H7N9 viruses isolated from the patient (A/Nanjing/M2/2013, GISAID accession number EPI450526 and EPI450527) and em erging H7N9 virus (A/Zhejiang/DTID-ZJU01/2013[H7N9]) showed that simi larity was 99·9% and 99·8%, respectively. The similarity of H3 and N2 genes of the H3N2 viruses isolated from the patient (A/Nanjing/M1/2013, GISAID accession number EPI450524 and EPI450525) and currently circulating seasonal H3N2 virus (A/Texas/ JMM_21/ 2012[H3N2]) was 99·5% and 99·8%, respectively. Since mid-April, 2013 (fi gure, appendix), pdm2009 H1N1 has replaced H3N2 to become the dominant strain in Jiangsu Province, China. So far no co-infection of H7N9 and H3N2 viruses has been reported. However, dual infl uenza virus infec tions are a potential source for virus reassort ment between a human and an avian viral strain. The fi nding of human co-infection with H7N9 and H3N2 viruses shows that human beings could act as mixing vessels for virus reassortment, which might facilitate human-to-human transmission. The public health and scientifi c com mun ities should enhance surveillance for virus evolution.

Comprehensive Characterization of Serum MicroRNA Profile in Response to the Emerging Avian Influenza A (H7N9) Virus Infection in Humans

A novel avian-origin influenza A (H7N9) virus recently occurred in China and caused 137 human infection cases with a 32.8% mortality rate. Although various detection procedures have been developed, the pathogenesis of this emerging virus in humans remains largely unknown. In this study, we characterized serum microRNA (miRNA) profile in response to H7N9 virus infection using TaqMan Low Density Arrays. Upon infection, a total of 395 miRNAs were expressed in the serum pool of patients, far beyond the 221 in healthy controls. Among the 187 commonly expressed miRNAs, 146 were up-regulated and only 7 down-regulated in patients. Further analysis by quantitative RT-PCR revealed that the serum levels of miR-17, miR-20a, miR-106a and miR-376c were significantly elevated in patients compared with healthy individuals (p < 0.05). Receiver operating characteristic (ROC) curves were constructed to show that each miRNA could discriminate H7N9 patients from controls with area under the curve (AUC) values ranging from 0.622 to 0.898, whereas a combination of miR-17, miR-20a, miR-106a and miR-376c obtained a higher discriminating ability with an AUC value of 0.96. Our findings unravel the significant alterations in serum miRNA expression following virus infection and manifest great potential of circulating miRNAs for the diagnosis of viral diseases.

Spatiotemporal Dynamics of Hand-Foot-Mouth Disease and Its Relationship with Meteorological Factors in Jiangsu Province, China.

Hand, foot and mouth disease (HFMD) is an important public health issue in mainland China, including Jiangsu Province. The main purpose of this study was to depict the epidemiological characteristics of HFMD and evaluate the effects of meteorological variables on its dynamics via spatiotemporal analytic methods, which is essential for formulating scientific and effective prevention and control strategies and measures. In total, 497,910 cases of HFMD occurred in the 2009-2013 period, with an average annual incidence of 126.3 per 100,000 in Jiangsu. Out of these, 87.7% were under 5 years old with a male-to-female incidence ratio of 1.4. The dominant pathogens of the laboratory-confirmed cases were EV71 and CoxA16, accounting for 44.8% and 30.6% of all cases, respectively. Two incidence peaks were observed in each year, the higher occurring between April and June, the lower between November and December. The incidence ranged between 16.8 and 233.5 per 100,000 at the county level. The incidence in the South of the province was generally higher than that in the northern regions. The most likely spatiotemporal cluster detected by space–time scan analysis occurred in May-June of 2012 in the southern region. Average temperature and rainfall were positively correlated with HFMD incidence, while the number of days with rainfall ≥ 0.1mm, low temperature, high temperature and hours of sunshine were negatively related. Particularly, relative humidity had no relationship. In conclusion, the prevalence of HFMD in Jiangsu Province has an obvious feature of seasonality. The etiological composition changed dynamically and might be a latent driving force for the temporal variation of the incidence of HFMD. A moderately warm environment promotes the transmission of the HFMD viruses, while particularly cold and hot climate conditions restrain their transmission.

Epidemiological and Clinical Characteristics and Risk Factors for Death of Patients with Avian Influenza A H7N9 Virus Infection from Jiangsu Province, Eastern China

Background A novel avian influenza A (H7N9) virus has caused great morbidity as well as mortality since its emergence in Eastern China in February 2013. However, the possible risk factors for death are not yet fully known. Methods and Findings Patients with H7N9 virus infection between March 1 and August 14, 2013 in Jiangsu province were enrolled. Data were collected with a standard form. Mean or percentage was used to describe the features, and Fishers exact test or t-test test was used to compare the differences between fatal and nonfatal cases with H7N9 virus infection. A total of 28 patients with H7N9 virus infection were identified among whom, nine (32.1%) died. The median age of fatal cases was significant higher than nonfatal cases (P<0.05). Patients with older age were more strongly associated with increased odds of death (OR = 30.0; 95% CI, 2.85–315.62). Co-morbidity with chronic lung disease and hypertension were risk factors for mortality (OR = 14.40; 95% CI, 1.30–159.52, OR = 6.67; 95% CI, 1.09–40.43, respectively). Moreover, the presence of either bilateral lung inflammation or pulmonary consolidation on chest imaging on admission was related with fatal outcome (OR = 7.00; 95%CI, 1.10–44.61). Finally, dynamic monitoring showed that lymphopenia was more significant in fatal group than in nonfatal group from day 11 to week five (P<0.05). The decrease in oxygenation indexes were observed in most cases and more significantly in fatal cases after week three (P<0.05), and the value of nearly all fatal cases were below 200 mmHg during our evaluation period. Conclusions Among cases with H7N9 virus infection, increased age accompanied by co-morbidities was the risk of death. The severity of lung infection at admission, the persistence of lymphocytopenia, and the extended duration of lower oxygenation index all contributed to worsened outcomes of patients with H7N9 virus infection.

Ecology of the Tick-Borne Phlebovirus Causing Severe Fever with Thrombocytopenia Syndrome in an Endemic Area of China

Background Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a tick-borne phlebovirus in family Bunyaviridae. Studies have found that humans, domestic and wildlife animals can be infected by SFTSV. However, the viral ecology, circulation, and transmission remain largely unknown. Methodology/Principal Findings Sixty seven human SFTS cases were reported and confirmed by virus isolation or immunofluorescence assay between 2011 and 2014. In 2013–2014 we collected 9,984 ticks from either vegetation or small wild mammals in the endemic area in Jiangsu, China, and detected SFTSV-RNA by real-time RT-PCR in both questing and feeding Haemaphysalis longicornis and H. flava. Viral RNA was identified in larvae of H. longicornis prior to a first blood meal, which has never been confirmed previously in nature. SFTSV-RNA and antibodies were also detected by RT-PCR and ELISA, respectively, in wild mammals including Erinaceus europaeus and Sorex araneus. A live SFTSV was isolated from Erinaceus europaeus captured during the off tick-feeding season and with a high SFTSV antibody titer. Furthermore, SFTSV antibodies were detected in the migratory birds Anser cygnoides and Streptopelia chinensis using ELISA. Conclusions/Significance The detection of SFTSV-RNA in non-engorged larvae indicated that vertical transmission of SFTSV in H. longicornis might occur in nature, which suggests that H. longicornis is a putative reservoir host of SFTSV. Small wild mammals such as Erinaceus europaeus and Sorex araneus could be infected by SFTSV and may serve as natural amplifying hosts. Our data unveiled that wild birds could be infected with SFTSV or carry SFTSV-infected ticks and thus might contribute to the long-distance spread of SFTSV via migratory flyways. These findings provide novel insights for understanding SFTSV ecology, reservoir hosts, and transmission in nature and will help develop new measures in preventing its rapid spread both regionally and globally.