Zheng Teng

Intensive Cytokine induction in Pandemic H1N1 Influenza Virus Infection Accompanied by Robust Production of IL-10 and IL-6

Background The innate immune system is the first line of defense against viruses by inducing expression of cytokines and chemokines. Many pandemic influenza H1N1 virus [P(H1N1)] infected severe cases occur in young adults under 18 years old who were rarely seriously affected by seasonal influenza. Results regarding host cytokine profiles of P(H1N1) are ambivalent. In the present study we investigated host cytokine profiles in P(H1N1) patients and identified cytokines related to disease severity. Methods and Principal Findings We retrieved 77, 59, 26 and 26 sera samples from P(H1N1) and non-flu influenza like illness (non-ILIs) cases with mild symptoms (mild patients), P(H1N1) vaccinees and healthy individuals, respectively. Nine and 16 sera were from hospitalized P(H1N1) and non-ILIs patients with severe symptoms (severe patients). Cytokines of IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IFN-γ and TNF-α were assayed by cytokine bead array, IL-17 and IL-23 measured with ELISA. Mild P(H1N1) patients produced significantly elevated IL-2, IL-12, IFN-γ, IL-6, TNF-α, IL-5, IL-10, IL-17 and IL-23 versus to healthy controls. While an overwhelming IL-6 and IL-10 production were observed in severe P(H1N1) patients. Higher IL-10 secretion in P(H1N1) vaccinees confirmed our observation that highly increased level of sera IL-6 and IL-10 in P(H1N1) patients may lead to disease progression. Conclusion and Significance A comprehensive innate immune response was activated at the early stage of P(H1N1) infection with a combine Th1/Th2/Th3 cytokines production. As disease progression, a systemic production of IL-6 and IL-10 were observed in severe P(H1N1) patients. Further analysis found a strong correlation between IL-6 and IL-10 production in the severe P(H1N1) patients. IL-6 may be served as a mediator to induce IL-10 production. Highly elevated level of sera IL-6 and IL-10 in P(H1N1) patients may lead to disease progression, but the underlying mechanism awaits further detailed investigations.

High human bocavirus viral load is associated with disease severity in children under five years of age.

Human bocavirus (HBoV) is a parvovirus and detected worldwide in lower respiratory tract infections (LRTIs), but its pathogenic role in respiratory illness is still debatable due to high incidence of co-infection with other respiratory viruses. To determine the prevalence of HBoV infection in patients with LRTI in Shanghai and its correlation with disease severity, we performed a 3-year prospective study of HBoV in healthy controls, outpatients and inpatients under five years of age with X-ray diagnosed LRTIs. Nasopharyngeal aspirates were tested by PCR for common respiratory viruses and by real time PCR for HBoV subtypes 1–4. Nasopharyngeal swabs from healthy controls and serum samples and stools from inpatients were also tested for HBoV1-4 by real time PCR. Viral loads were determined by quantitative real time PCR in all HBoV positive samples. HBoV1 was detected in 7.0% of inpatients, with annual rates of 5.1%, 8.0% and 4.8% in 2010, 2011 and 2012, respectively. Respiratory syncytial virus (RSV) subtype A was the most frequent co-infection detected; HBoV1 and RSVA appeared to co-circulate with similar seasonal variations. High HBoV viral loads (>106 copies/ml) were significantly more frequent in inpatients and outpatients than in healthy controls. There was a direct correlation of high viral load with increasing disease severity in patients co-infected with HBoV1 and at least one other respiratory virus. In summary, our data suggest that HBoV1 can cause LRTIs, but symptomatic HBoV infection is only observed in the context of high viral load.

Human Antibody Responses to Avian Influenza A(H7N9) Virus, 2013

Understanding host antibody response is crucial for predicting disease severity and for vaccine development. We investigated antibody responses against influenza A(H7N9) virus in 48 serum samples from 21 patients, including paired samples from 15 patients. IgG against subtype H7 and neutralizing antibodies (NAbs) were not detected in acute-phase samples, but ELISA geometric mean titers increased in convalescent-phase samples; NAb titers were 20–80 (geometric mean titer 40). Avidity to IgG against subtype H7 was significantly lower than that against H1 and H3. IgG against H3 was boosted after infection with influenza A(H7N9) virus, and its level in acute-phase samples correlated with that against H7 in convalescent-phase samples. A correlation was also found between hemagglutinin inhibition and NAb titers and between hemagglutinin inhibition and IgG titers against H7. Because of the relatively weak protective antibody response to influenza A(H7N9), multiple vaccinations might be needed to achieve protective immunity.

Limited human-to-human transmission of avian influenza A(H7N9) virus, Shanghai, China, March to April 2013

In April 2013, two members of one family were successively confirmed as cases of avian influenza A(H7N9) virus infection in Shanghai, China. Respiratory specimens from the two cases and their close contacts were tested using real-time reverse-transcription (RT)-PCR. Paired serum specimens from contacts were tested by haemagglutination inhibition assay and microneutralisation test. The index patient developed severe pneumonia. Her husband presented with pneumonia shortly thereafter. Both cases had highly similar clinical features and infection with A(H7N9) virus was confirmed in both cases by genetic analysis. Phylogenetic analysis revealed a high level of similarity between the sequences from the two patients and environmental samples collected from wet markets in Minhang and Changning districts. Six samples from the Changning wet market were confirmed as A(H7N9) positive. Of 27 close contacts, one developed mild respiratory symptoms and another tested positive for A(H7N9) antibodies, but both were negative by real-time RT-PCR. The other 25 close contacts of both cases were A(H7N9) negative. Limited human-to-human transmission of the virus most likely occurred in the family cluster. However, other close contacts did not test positive for the virus, suggesting limited potential for extensive human-to-human transmission of the virus.

Novel Avian Influenza A(H7N9) Virus in Tree Sparrow, Shanghai, China, 2013

In spring 2013, influenza A(H7N9) virus was isolated from an apparently healthy tree sparrow in Chongming Dongping National Forest Park, Shanghai City, China. The entire gene constellation of the virus is similar to that of isolates from humans, highlighting the need to monitor influenza A(H7N9) viruses in different species.

Real-time cell analysis – A new method for dynamic, quantitative measurement of infectious viruses and antiserum neutralizing activity

A newly developed electronic cell sensor array--the xCELLigence real-time cell analysis (RTCA) system is tested currently for dynamic monitoring of cell attachment, proliferation, damage, and death. In this study, human enterovirus (HEV71) infection of human rhabdomyosarcoma (RD) was used as an in vitro model to validate the application of this novel system as a straightforward and efficient assay for quantitative measurement of infectious viruses based on virus-induced cytopathic effect (CPE). Several experimental tests were performed including the determination of optimal seeding density of the RD cells in 96-well E-plates, RTCA real-time monitoring of the virus induced CPE and virus titer calculation, and viral neutralization test to determine HEV71 antibody titer. Traditional 50% tissue culture infective dose (TCID50) assay was also conducted for methodology comparison and validation, which indicated a consistent result between the two assays. These findings indicate that the xCELLigence RTCA system can be a valuable addition to current viral assays for quantitative measurement of infectious viruses and quantitation of neutralization antibody titer in real-time, warranting for future research and exploration of applications to many other animal and human viruses.

Epidemiological study of influenza B in Shanghai during the 2009–2014 seasons: implications for influenza vaccination strategy

A new quadrivalent influenza vaccine has been available for influenza B, which can pose a significant global health burden. Shanghai has the highest GDP and largest metropolitan population in China. To understand the impact of influenza B in Shanghai in terms of age-related incidence and relative prevalence compared with other subtypes, we conducted this retrospective epidemiological study of influenza B in the 2009-2014 seasons. A total of 71 354 outpatients with influenza-like illness were included, and both lineages of influenza B and subtypes of influenza A were identified using real-time RT-PCR. The antigenic characteristics of influenza B isolates were analysed by sequencing and reciprocal haemagglutinin inhibition assay. On average, 33.45% of influenza strains were influenza B, and 40.20% of strains isolated from children were influenza B. The incidence of influenza B was highest (12.52 per 100 people with influenza-like illness) in children ages 6-17 years and usually peaked in this age group at the early stage of an influenza B epidemic. Overall, both matched and mismatched influenza B strains co-circulated in Shanghai annually, and 44.57% of the circulating influenza B belonged to the opposite lineage of the vaccine strains. We concluded that influenza B has caused a substantial impact in Shanghai and that school-aged children play a key role in the transmission of influenza B. Hence, it may be beneficial to prioritize influenza vaccination for school-aged children to mitigate the outbreaks of influenza B.

Epidemiological and serological surveillance of hand-foot-and-mouth disease in Shanghai, China, 2012–2016

Aside from enterovirus 71 (EV71) and coxsackie virus A16 (CV-A16), viruses that are known to cause hand-foot-and-mouth disease (HFMD), epidemiological profiles of other enteroviruses that induce HFMD are limited. We collected 9949 laboratory surveillance HFMD cases and 1230 serum samples from infants and children in Shanghai from 2012–2016. Since 2013, CV-A6 has displaced EV71 and CV-A16 to become the predominant serotype. Interestingly, novel epidemiological patterns in EV71 and CV-A16 infections were observed, with one large peak in both 2012 and 2014, followed by two smaller peaks in the respective following years (2013 and 2015). Through sequencing, we found that C4a, B1b, D-Cluster-1 and B constituted the major subgenotypes of EV71, CV-A16, CV-A6 and CV-A10, respectively. Among healthy individuals, 50.49% and 54.23% had positive neutralising antibodies (NtAbs) against EV71 and CV-A16, respectively, indicating that EV71 and CV-A16 silent infections were common. These populations may be an important potential source of infection. The overall seropositive rate of EV71 NtAbs showed a fluctuating, markedly downward trend, indicating the potential risk of a future EV71 epidemic. High CV-A16 NtAb seroprevalence corroborated a documented CV-A16 ‘silent’ epidemic. Children aged 1–5 years had the lowest EV71 NtAb seropositive rate, whereas those aged 1–2 years exhibited the lowest CV-A16 NtAb seropositive rate. This is the first comprehensive investigation of the epidemiology and aetiology, as well as the seroprevalence, of HFMD in Shanghai between 2012 and 2016. This study provides the latest insights into developing a more efficient HMFD vaccination programme.

The emergence and spread of one Coxsackievirus A16 Genogroup D novel recombinant strain that caused a clustering HFMD outbreak in Shanghai, China, 2016

Dear Editors, We report the first outbreak and spread of one Coxsackievirus A16 (CV-A16) novel Genogroup D recombinant strain in Shanghai, China, in 2016, 2 years after an initial report in France. CV-A16 belongs to the Enterovirus (EV) A species and is one of the major serotypes that causes hand, foot, and mouth disease (HFMD). CV-A16 can be classified into three genogroups, A–C, based on the VP4 and VP1 gene sequences. Genogroup B can be further divided into B1 (B1a–B1c) and B2. CV-A16 Genogroup D is a novel recombinant genogroup. The epidemiological origin can be traced to Peru. Hassel et al. reported its first emergence and circulation in France during 2010–2014 and provided further detailed evidence of an intertype recombinant origin of CV-A16 genogroup D. To the best of our knowledge, no more report of the novel genogroup D could be found in the PubMed database. The genogroup D Shanghai strain, designated SH-HP16-51, was isolated from a mild HFMD case collected by our laboratory surveillance system on 5 October 2016. The patient was a 3-year-old girl. The epidemiological origin was traced to an aggregated case of 14 suspected HFMD patients reported in Pudong CDC. Between 2 and 5 October, 11 and 3 students among these 14 patients belonged to two adjacent classes in one kindergarten, respectively. The first case occurred on 2 October with fever (high temperature 38.9 °C), sore throat, and oral ulcers, and the child was diagnosed with HFMD on the next day. Unfortunately, clinical samples of only two patients were successfully collected in this outbreak because the kindergarten was quickly closed before we could collect other patient samples. However, no EV RNA was detected in the first case sample. In addition, although the guardians of both the first and second cases denied any history of contact with foreigners or travel abroad 2 weeks before the onset, we cannot exclude the possibility that the SH-HP-16-51 strain originated from other geographic locations, like France, because of increasingly frequent international business and communication. The EV genome of strain SH-HP-16-51 was detected in a throat swab after RNA extraction. Pan EV and CV-A16 were confirmed with a commercial real-time RT-PCR Kit (BioPerfectus Technologies, Jiangsu, China). A human rhabdomyosarcoma cell line was then used for virus isolation. When typical cytopathic effects occurred (data not shown), culture medium and cells were harvested for purification. The complete genome was determined as described and submitted to GenBank (accession number: MG948605). The SH-HP-16-51 genome was 7231 bp long. Phylogenetic trees were constructed by using nucleotide sequences of the VP1, P1, P2, and P3 regions of the reference strains representing all genogroups with complete genome sequences. Phylogenetic analyses based on the complete VP1 sequence (891 bp) provided evidence that the isolate SH-HP-16-51 belonged to CV-A16 genogroup D and was most closely related to the strains isolated from France and Peru (Fig. 1a). These strains

Epidemiology, aetiology and seasonality of infectious diarrhoea in adult outpatients through active surveillance in Shanghai, China, 2012–2016: a cross-sectional study

Objectives This study aimed to identify the epidemiology, clinical characteristics, aetiology and seasonality of sporadic infectious diarrhoea in adults in Shanghai. Setting This study was based on a city-wide, active continuous hospital-based diarrhoea surveillance network established by Shanghai Municipal Center for Disease Control and Prevention. There were 22 sentinel hospitals in all 16 districts (9 primary-level hospitals, 6 secondary-level hospitals and 7 tertiary-level hospitals) which were selected using probability proportionate to size sampling method. Participants From 1 May 2012 to 31 May 2016, 90 713 patients were included in this study. Among 8797 patients whose stool samples were collected and detected, 4392 patients were male. Results The positive rate was 47.96%. Bacterial and viral infections accounted for 27.19% and 69.07% separately. Norovirus was the most common pathogen (43.10%), followed by rotavirus, Vibrio parahaemolyticus, diarrhoeagenic Escherichia coli (DEC) and Salmonella spp. Patients between 30–44 and 45–59 years were more likely to have infectious diarrhoea and viral diarrhoea. Those aged 30–44 years were the most likely to get infected with V. parahaemolyticus (adjusted OR, aOR vs 60+ years: 2.04, 95% CI 1.47 to 2.78) and norovirus (aOR vs 60+ years: 1.32, 95% CI 1.12 to 1.56). Bacterial (except V. parahaemolyticus) diarrhoea was characterised by fever, abdominal pain and loose stool; while viral diarrhoea was characterised by nausea, vomiting and watery stool. A seasonal distribution of infectious diarrhoea was observed with larger peaks in winter and smaller peaks in summer. Winter peaks were mainly due to norovirus and rotavirus, and summer peaks were due to bacterial infections. An emerging spring peak of norovirus around March was observed in the past 3 years. Conclusion Viral infections were predominant, and norovirus played a leading role. A seasonal distribution was observed and an emerging spring peak of norovirus was noted. Our findings highlight the necessity for conducting an active, comprehensive surveillance in adults, to monitor changing dynamics in the epidemiology and aetiology of infectious diarrhoea.