Lunbiao Cui

Probable person to person transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological investigation

Objective To determine whether the novel avian influenza H7N9 virus can transmit from person to person and its efficiency. Design Epidemiological investigations conducted after a family cluster of two patients with avian H7N9 in March 2013. Setting Wuxi, Eastern China. Participants Two patients, their close contacts, and relevant environments. Samples from the patients and environments were collected and tested by real time reverse transcriptase-polymerase chain reaction (rRT-PCR), viral culture, and haemagglutination inhibition assay. Any contacts who became ill had samples tested for avian H7N9 by rRT-PCR. Paired serum samples were obtained from contacts for serological testing by haemagglutination inhibition assays. Main outcomes measures Clinical data, history of exposure before the onset of illnesses, and results of laboratory testing of pathogens and further analysis of sequences and phylogenetic tree to isolated strains. Results The index patient became ill five to six days after his last exposure to poultry. The second patient, his daughter aged 32, who provided unprotected bedside care in the hospital, had no known exposure to poultry. She developed symptoms six days after her last contact with her father. Two strains were isolated successfully from the two patients. Genome sequence and analyses of phylogenetic trees showed that both viruses were almost genetically identical. Forty three close contacts of both patients were identified. One had mild illness but had negative results for avian H7N9 by rRT-PCR. All 43 close contacts tested negative for haemagglutination inhibition antibodies specific for avian H7N9. Conclusions The infection of the daughter probably resulted from contact with her father (the index patient) during unprotected exposure, suggesting that in this cluster the virus was able to transmit from person to person. The transmissibility was limited and non-sustainable.

A Family Cluster of Infections by a Newly Recognized Bunyavirus in Eastern China, 2007: Further Evidence of Person-to-Person Transmission

BACKGROUND Seven persons in one family living in eastern China developed fever and thrombocytopenia during May 2007, but the initial investigation failed to identify an infectious etiology. In December 2009, a novel bunyavirus (designated severe fever with thrombocytopenia syndrome bunyavirus [SFTSV]) was identified as the cause of illness in patients with similar clinical manifestations in China. We reexamined this family cluster for SFTSV infection. METHODS We analyzed epidemiological and clinical data for the index patient and 6 secondary patients. We tested stored blood specimens from the 6 secondary patients using real time reverse transcription polymerase chain reaction (RT-PCR), viral culture, genetic sequencing, micro-neutralization assay (MNA), and indirect immunofluorescence assay (IFA). RESULTS An 80-year-old woman with fever, leucopenia, and thrombocytopenia died on 27 April 2007. Between 3 and 7 May 2007, another 6 patients from her family were admitted to a local county hospital with fever and other similar symptoms. Serum specimens collected in 2007 from these 6 patients were positive for SFTS viral RNA through RT-PCR and for antibody to SFTSV through MNA and IFA. SFTSV was isolated from 1 preserved serum specimen. The only shared characteristic between secondary patients was personal contact with the index patient; none reported exposure to suspected animals or vectors. CONCLUSIONS Clinical and laboratory evidence confirmed that the patients of fever and thrombocytopenia occurring in a family cluster in eastern China in 2007 were caused by a newly recognized bunyavirus, SFTSV. Epidemiological investigation strongly suggests that infection of secondary patients was transmitted to family members by personal contact.

Live-Animal Markets and Influenza A (H7N9) Virus Infection

A novel avian influenza A (H7N9) virus has shown the ability to infect humans. As described in this letter, human infection may be occurring because of multiple cross-species jumps from birds to humans.

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.

Whole-Genome Sequence of a Reassortant H5N6 Avian Influenza Virus Isolated from a Live Poultry Market in China, 2013.

ABSTRACT An avian influenza virus, A/environment/Zhenjiang/C13/2013(H5N6), was isolated from a live poultry market in eastern China. Phylogenetic analysis showed that the isolate was a novel reassortant virus with a neuraminidase (NA) gene from H6N6 viruses and the other seven genes from H5N1 viruses, which may pose a potential threat to human and animal health.

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.

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.

Development and Evaluation of a SYBR Green-Based Real Time RT-PCR Assay for Detection of the Emerging Avian Influenza A (H7N9) Virus

Most recently a novel avian-origin influenza A (H7N9) virus emerged in China and has been associated with lots of human infection and fatal cases. Genetic analysis of the viral genome revealed that this reassortant virus might be better adapted to humans than other avian influenza viruses. Molecular diagnostic methods are thus urgently needed in public health laboratories. In this study, a SYBR green-based one-step real time reverse transcription-PCR (RT-PCR) was developed to detect the novel H7N9 virus. The primer pairs on the basis of the hemagglutinin and neuraminidase gene sequences of H7N9 viruses amplified subtype-specific fragments with Tm values of 80.77±0.06°C for H7 and 81.20±0.17°C for N9 respectively. The standard curves showed a dynamic linear range across 6 log units of RNA copy number (106 to 101 copies/ µl) with a detection limit of 10 copies per reaction for both H7 and N9 assays by using serial ten-fold diluted in-vitro transcribed viral RNA. In addition, no cross-reactivity was observed with seasonal H1N1, H1N1 pdm09, H3N2, H5N1 and H9N2 viruses as well as other human respiratory viruses. When the assay was further evaluated in H7N9 virus infected clinical samples, positive amplification signals were obtained in all of the specimens with the accordance between H7 and N9 assays. Therefore, the established SYBR green-based real time RT-PCR assay could provide a rapid, sensitive, specific and reliable alternative approach with lower costs for high throughput screening of suspected samples from humans, animals and environments in first line public health laboratories.

Antigenic and genetic characterization of a European avian-like H1N1 swine influenza virus from a boy in China in 2011.

Cross-species transmission of influenza A viruses from swine to human occurs occasionally. In 2011, an influenza A H1N1 virus, A/Jiangsu/ALS1/2011 (JS/ALS1/2011), was isolated from a boy who suffered from severe pneumonia in China. The virus is closely related antigenically and genetically to avian-like swine H1N1 viruses that have recently been circulating in pigs in China and that were initially detected in European pig populations in 1979. The isolation of JS/ALS1/2011 provides additional evidence that swine influenza viruses can occasionally infect humans and emphasizes the importance of reinforcing influenza virus surveillance in both pigs and humans.