Dawei Cui

Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome

Summary Background Human infection with avian influenza A H7N9 virus emerged in eastern China in February, 2013, and has been associated with exposure to poultry. We report the clinical and microbiological features of patients infected with influenza A H7N9 virus and compare genomic features of the human virus with those of the virus in market poultry in Zhejiang, China. Methods Between March 7 and April 8, 2013, we included hospital inpatients if they had new-onset respiratory symptoms, unexplained radiographic infiltrate, and laboratory-confirmed H7N9 virus infection. We recorded histories and results of haematological, biochemical, radiological, and microbiological investigations. We took throat and sputum samples, used RT-PCR to detect M, H7, and N9 genes, and cultured samples in Madin-Darby canine kidney cells. We tested for co-infections and monitored serum concentrations of six cytokines and chemokines. We collected cloacal swabs from 86 birds from epidemiologically linked wet markets and inoculated embryonated chicken eggs with the samples. We identified and subtyped isolates by RT-PCR sequencing. RNA extraction, complementary DNA synthesis, and PCR sequencing were done for one human and one chicken isolate. We characterised and phylogenetically analysed the eight gene segments of the viruses in the patients and the chickens isolates, and constructed phylogenetic trees of H, N, PB2, and NS genes. Findings We identified four patients (mean age 56 years), all of whom had contact with poultry 3–8 days before disease onset. They presented with fever and rapidly progressive pneumonia that did not respond to antibiotics. Patients were leucopenic and lymphopenic, and had impaired liver or renal function, substantially increased serum cytokine or chemokine concentrations, and disseminated intravascular coagulation with disease progression. Two patients died. Sputum specimens were more likely to test positive for the H7N9 virus than were samples from throat swabs. The viral isolate from the patient was closely similar to that from an epidemiologically linked market chicken. All viral gene segments were of avian origin. The H7 of the isolated viruses was closest to that of the H7N3 virus from domestic ducks in Zhejiang, whereas the N9 was closest to that of the wild bird H7N9 virus in South Korea. We noted Gln226Leu and Gly186Val substitutions in human virus H7 (associated with increased affinity for α-2,6-linked sialic acid receptors) and the PB2 Asp701Asn mutation (associated with mammalian adaptation). Ser31Asn mutation, which is associated with adamantane resistance, was noted in viral M2. Interpretation Cross species poultry-to-person transmission of this new reassortant H7N9 virus is associated with severe pneumonia and multiorgan dysfunction in human beings. Monitoring of the viral evolution and further study of disease pathogenesis will improve disease management, epidemic control, and pandemic preparedness. Funding Larry Chi-Kin Yung, National Key Program for Infectious Diseases of China.

Avian-Origin Influenza A(H7N9) Infection in Influenza A(H7N9)–Affected Areas of China: A Serological Study

Serological surveillance conducted in areas of an outbreak of influenza A(H7N9) infection in China found no seropositivity for antibodies specific for avian-origin influenza A(H7N9) among 1129 individuals of the general population, whereas >6% of 396 poultry workers were positive (on the basis of a hemagglutination inhibition titer of ≥ 80) for this subtype, confirming that infected poultry is the principal source of human infections and that subclinical infections are possible. Fourteen days after symptom onset, elevated levels of antibodies to A(H7N9) were found in 65.8% of patients (25/38) who survived but in only 28.6% of those (2/7) who died, suggesting that the presence of antibodies may improve clinical outcome in infected patients.

Serotypes, genotypes and antimicrobial resistance patterns of human diarrhoeagenic Escherichia coli isolates circulating in southeastern China

Diarrhoeagenic Escherichia coli (DEC) infection is a major health problem in developing countries. The prevalence and characteristics of DEC have not been thoroughly investigated in China. Consecutive faecal specimens from outpatients with acute diarrhoea in nine sentinel hospitals in southeastern China were collected from July 2009 to June 2011. Bacterial and viral pathogens were detected by culture and RT-PCR, respectively. DEC isolates were further classified into five pathotypes using multiplex PCR. The O/H serotypes, sequence types (STs) and antimicrobial susceptibility profiles of the DEC isolates were determined. A total of 2466 faecal specimens were collected, from which 347 (14.1%) DEC isolates were isolated. DEC was the dominant bacterial pathogen detected. The DEC isolates included 217 EAEC, 62 ETEC, 52 EPEC, 14 STEC, one EIEC and one EAEC/ETEC. O45 (6.6%) was the predominant serotype. Genotypic analysis revealed that the major genotype was ST complex 10 (87, 25.6%). Isolates belonging to the serogroups or genotypes of O6, O25, O159, ST48, ST218, ST94 and ST1491 were highly susceptible to the majority of antimicrobials. In contrast, isolates belonging to O45, O15, O1, O169, ST38, ST226, ST69, ST31, ST93, ST394 and ST648 were highly resistant to the majority of antimicrobials. DEC accounted for the majority of bacterial pathogens causing acute diarrhoea in southeastern China, and it is therefore necessary to test for all DEC, not only the EHEC O157:H7. Some serogroups or genotypes of DEC were highly resistant to the majority of antimicrobials. DEC surveillance should be emphasized.

Simultaneous Detection of Influenza A, Influenza B, and Respiratory Syncytial Viruses and Subtyping of Influenza A H3N2 Virus and H1N1 (2009) Virus by Multiplex Real-Time PCR

ABSTRACT A multiplex real-time PCR assay was developed to simultaneously detect and discriminate influenza A virus subtypes, including novel H1N1 (2009) and seasonal H3N2 virus, influenza B virus, and respiratory syncytial virus (RSV) in a single test tube, with detection sensitivity and specificity of 99% and 100%, respectively, for the four pathogens.

Dynamic variations in the peripheral blood lymphocyte subgroups of patients with 2009 pandemic H1N1 swine-origin influenza A virus infection

BackgroundNovel Influenza A (H1N1) is an acute respiratory infectious disease. Animal experiments indicated that when H1N1 virus infected early hosts, it showed strong CD4+, CD8+, and CD4+CD25+ T cell reactions. The aim of this study was to investigate the dynamic fluctuations of the peripheral blood lymphocyte subgroups in patients infected with H1N1 swine-origin influenza A virus (S-OIV).MethodsThe frequency of T cells, B cells, natural killer (NK) cells, and regulatory T cells (Treg) in 36 severe H1N1 and 40 moderate H1N1 patients were detected at different periods by flow cytometry. In parallel, serum cytokines were detected by enzyme-linked immunosorbent assay and C-reactive protein (CRP) was analyzed through an image-type automatic biochemical analyzer. In addition, 20 healthy volunteers, who were not infected with 2009 H1N1 virus, were selected as controls.ResultsThe frequency of NK cells were decreased in all cases and CD19+ B cells were increased in severe cases than those of the controls. At 1-2d from onset, the frequency of CD4+ and CD4+CD25+ T cells in moderate cases was higher than in the severe cases. Serum cytokines, specifically IL-2, IL-4, IL-6, IL-10, and IFN-γ exhibited no significant change both in the moderate and the severe cases during the whole monitoring process. In the early stage of the disease, serum CRP levels in the severe and moderate groups were significantly higher than that in the control group.ConclusionsPatients showed different lymphocyte subgroup distributions between mild and severe cases, which might affect the incidence and development of 2009 H1N1.

Viral agents associated with acute diarrhea among outpatient children in southeastern China

Background: Acute diarrhea is a leading cause of childhood morbidity and mortality worldwide, but there have been few reports on the causative viruses associated with acute diarrhea among outpatient children in developing countries. This study was conducted to identify the viral agents in outpatient children with acute diarrhea in southeastern China. Methods: Eight hundred eleven outpatient children 5 years or younger with acute diarrhea were enrolled. Five enteric viruses were determined by enzyme-linked immunosorbent assay and multiplex reverse transcription-polymerase chain reaction for each stool specimen. Results: At least 1 virus was detected in 353 (43.5%) of the subjects. The proportions of rotavirus, norovirus, sapovirus, adenovirus and astrovirus were 25.5%, 18.1%, 4.4%, 2.7% and 1.2%, respectively. G3P[8] was the most prevalent rotavirus strain. Mixed infections were observed in 65 cases, among which the most prevalent coinfection was rotavirus with other viruses (58 cases, 89.2%). Rotavirus and norovirus infections showed marked and opposing seasonal patterns. Mixed infection was significantly more common in children older than 1 year (12.2%) than in those younger than 1 year (7.1%) (P = 0.026). Clinically, rotavirus infection presented with a longer duration (4.3 ± 6.7 days) and higher frequency (5.9 ± 2.0 times/d) of diarrhea than any other viral infection. Vomiting was more common for mixed infections than for single infections (P = 0.010). Conclusions: All the 5 common etiologies were detected in this study, with rotavirus and norovirus being the 2 leading agents. Mixed viral infections were common in outpatient children with acute diarrhea, and rotavirus seemed to play a major role in mixed infections.

Functional variants regulating LGALS1 (Galectin 1) expression affect human susceptibility to influenza A(H7N9)

The fatality of avian influenza A(H7N9) infection in humans was over 30%. To identify human genetic susceptibility to A(H7N9) infection, we performed a genome-wide association study (GWAS) involving 102 A(H7N9) patients and 106 heavily-exposed healthy poultry workers, a sample size critically restricted by the small number of human A(H7N9) cases. To tackle the stringent significance cutoff of GWAS, we utilized an artificial imputation program SnipSnip to improve the association signals. In single-SNP analysis, one of the top SNPs was rs13057866 of LGALS1. The artificial imputation (AI) identified three non-genotyped causal variants, which can be represented by three anchor/partner SNP pairs rs13057866/rs9622682 (AI P = 1.81 × 10−7), rs4820294/rs2899292 (2.13 × 10−7) and rs62236673/rs2899292 (4.25 × 10−7) respectively. Haplotype analysis of rs4820294 and rs2899292 could simulate the signal of a causal variant. The rs4820294/rs2899292 haplotype GG, in association with protection from A(H7N9) infection (OR = 0.26, P = 5.92 × 10−7) correlated to significantly higher levels of LGALS1 mRNA (P = 0.050) and protein expression (P = 0.025) in lymphoblast cell lines. Additionally, rs4820294 was mapped as an eQTL in human primary monocytes and lung tissues. In conclusion, functional variants of LGALS1 causing the expression variations are contributable to the differential susceptibility to influenza A(H7N9).

Rapid diagnostic tests for identifying avian influenza A(H7N9) virus in clinical samples.

To determine sensitivity of rapid diagnostic tests for detecting influenza A(H7N9) virus, we compared rapid tests with PCR results and tested different types of clinical samples. Usefulness of seasonal influenza rapid tests for A(H7N9) virus infections is limited because of their low sensitivity for detecting virus in upper respiratory tract specimens.

An isoleucine-zipper motif enhances costimulation of human soluble trimeric GITR ligand

Glucocorticoid-induced tumor-necrosis factor receptor (GITR) and its ligand, GITRL, play significant roles in regulating immune responses. It is clear that human soluble GITRL (hsGITRL) transduces signal activity through multiple oligomerization states. To develop human soluble trimeric GITRL protein as a potential therapeutic target, we explored the link of the isoleucine-zipper (ILZ) motif to the N-terminus of the human soluble GITRL with two leucine sequences. hsGITRL, with the ILZ motif (ILZ-hsGITRL), was firstly expressed in Escherichia coli, which exhibited a predominant trimer when identified by Sephadex G-100 filtration and non-reducing SDS–polyacrylamide gel electrophoresis (SDS-PAGE). The significantly higher biological activity of the ILZ-hsGITRL compared with hsGITRL was confirmed by CD4+ T proliferation, interferon-γ (IFN-γ) secretion and binding activity assay. To reveal and compare the underlying mechanisms, the level of extracellular signal-regulated kinase-1/2 (ERK1/2) phosphorylation was examined, indicating that ILZ-hsGITRL induced more persistent and stronger ERK1/2 activation than hsGITRL. In conclusion, the incorporation of an ILZ motif could markedly improve the costimulation of hsGITRL.

Clinical and epidemiologic characteristics of hospitalized patients with laboratory-confirmed respiratory syncytial virus infection in eastern china between 2009 and 2013: a retrospective study

Respiratory syncytial virus (RSV) is a leading cause of morbidity and mortality worldwide in children aged <5 years and older adults with acute lower respiratory infections (ALRIs). However, few studies regarding the epidemiology of hospitalizations for RSV infection have been performed previously in China. Here, we aimed to describe the clinical and epidemiologic characteristics of hospitalized patients with laboratory-confirmed RSV infection in eastern China. Active surveillance for hospitalized ALRI patients using a broad case definition based on symptoms was performed from 2009–2013 in 12 sentinel hospitals in eastern China. Clinical and epidemiologic data pertaining to hospitalized patients of all ages with laboratory-confirmed RSV infection by PCR assay were collected and analyzed in this study. From 2009 to 2013, 1046 hospitalized patients with laboratory-confirmed RSV infection were enrolled in this study, and 14.7% of patients had subtype A, 24.2% of patients had subtype B, 23.8% of patients with subtype not performed, and 37.3% of patients had RSV coinfections with other viruses. RSV and influenza coinfections (33.3%) were the most common coinfections noted in this study. Moreover, young children aged <5 years (89.1%, 932/1046), particularly young infants aged <1 year (43.3%, 453/1046), represented the highest proportion of patients with RSV infections. In contrast, older adults aged ≥60 years (1.1%, 12/1046) represented the lowest proportion of patients with RSV infections among enrolled patients. The peak RSV infection period occurred mainly during autumn and winter, and 57% and 66% of patients exhibited symptoms such as fever (body temperature ≥38°C) and cough separately. Additionally, only a small number of patients were treated with broad-spectrum antiviral drugs, and most of patients were treated with antimicrobial drugs that were not appropriate for RSV infection. RSV is a leading viral pathogen and a common cause of viral infection in young children aged <5 years with ALRIs in eastern China. Effective vaccines and antiviral agents targeting RSV are needed to mitigate its large public health impact.