George F. Gao

Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus

BACKGROUND Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus. METHODS We obtained and analyzed clinical, epidemiologic, and virologic data from these patients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase-polymerase-chain-reaction assays, viral culturing, and sequence analyses. RESULTS A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from all three patients and was identified as H7N9. Sequencing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 virus. A T160A mutation was identified at the 150-loop in the HA gene of all three viruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed diffuse opacities and consolidation. Complications included acute respiratory distress syndrome and multiorgan failure. All three patients died. CONCLUSIONS Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).

Rapid health transition in China, 1990-2010: findings from the Global Burden of Disease Study 2010.

Summary Background China has undergone rapid demographic and epidemiological changes in the past few decades, including striking declines in fertility and child mortality and increases in life expectancy at birth. Popular discontent with the health system has led to major reforms. To help inform these reforms, we did a comprehensive assessment of disease burden in China, how it changed between 1990 and 2010, and how Chinas health burden compares with other nations. Methods We used results of the Global Burden of Diseases, Injuries, and Risk Factors Study 2010 (GBD 2010) for 1990 and 2010 for China and 18 other countries in the G20 to assess rates and trends in mortality, causes of death, years of life lost (YLLs), years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE). We present results for 231 diseases and injuries and for 67 risk factors or clusters of risk factors relevant to China. We assessed relative performance of China against G20 countries (significantly better, worse, or indistinguishable from the G20 mean) with age-standardised rates and 95% uncertainty intervals. Findings The leading causes of death in China in 2010 were stroke (1·7 million deaths, 95% UI 1·5–1·8 million), ischaemic heart disease (948 700 deaths, 774 500–1 024 600), and chronic obstructive pulmonary disease (934 000 deaths, 846 600–1 032 300). Age-standardised YLLs in China were lower in 2010 than all emerging economies in the G20, and only slightly higher than noted in the USA. China had the lowest age-standardised YLD rate in the G20 in 2010. China also ranked tenth (95% UI eighth to tenth) for HALE and 12th (11th to 13th) for life expectancy. YLLs from neonatal causes, infectious diseases, and injuries in children declined substantially between 1990 and 2010. Mental and behavioural disorders, substance use disorders, and musculoskeletal disorders were responsible for almost half of all YLDs. The fraction of DALYs from YLDs rose from 28·1% (95% UI 24·2–32·5) in 1990 to 39·4% (34·9–43·8) in 2010. Leading causes of DALYs in 2010 were cardiovascular diseases (stroke and ischaemic heart disease), cancers (lung and liver cancer), low back pain, and depression. Dietary risk factors, high blood pressure, and tobacco exposure are the risk factors that constituted the largest number of attributable DALYs in China. Ambient air pollution ranked fourth (third to fifth; the second highest in the G20) and household air pollution ranked fifth (fourth to sixth; the third highest in the G20) in terms of the age-standardised DALY rate in 2010. Interpretation The rapid rise of non-communicable diseases driven by urbanisation, rising incomes, and ageing poses major challenges for Chinas health system, as does a shift to chronic disability. Reduction of population exposures from poor diet, high blood pressure, tobacco use, cholesterol, and fasting blood glucose are public policy priorities for China, as are the control of ambient and household air pollution. These changes will require an integrated government response to improve primary care and undertake required multisectoral action to tackle key risks. Analyses of disease burden provide a useful framework to guide policy responses to the changing disease spectrum in China. Funding Bill & Melinda Gates Foundation.

Emergence of fatal PRRSV variants: unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark.

Porcine reproductive and respiratory syndrome (PRRS) is a severe viral disease in pigs, causing great economic losses worldwide each year. The causative agent of the disease, PRRS virus (PRRSV), is a member of the family Arteriviridae. Here we report our investigation of the unparalleled large-scale outbreaks of an originally unknown, but so-called “high fever” disease in China in 2006 with the essence of PRRS, which spread to more than 10 provinces (autonomous cities or regions) and affected over 2,000,000 pigs with about 400,000 fatal cases. Different from the typical PRRS, numerous adult sows were also infected by the “high fever” disease. This atypical PRRS pandemic was initially identified as a hog cholera-like disease manifesting neurological symptoms (e.g., shivering), high fever (40–42°C), erythematous blanching rash, etc. Autopsies combined with immunological analyses clearly showed that multiple organs were infected by highly pathogenic PRRSVs with severe pathological changes observed. Whole-genome analysis of the isolated viruses revealed that these PRRSV isolates are grouped into Type II and are highly homologous to HB-1, a Chinese strain of PRRSV (96.5% nucleotide identity). More importantly, we observed a unique molecular hallmark in these viral isolates, namely a discontinuous deletion of 30 amino acids in nonstructural protein 2 (NSP2). Taken together, this is the first comprehensive report documenting the 2006 epidemic of atypical PRRS outbreak in China and identifying the 30 amino-acid deletion in NSP2, a novel determining factor for virulence which may be implicated in the high pathogenicity of PRRSV, and will stimulate further study by using the infectious cDNA clone technique.

Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses

BACKGROUND On March 30, 2013, a novel avian influenza A H7N9 virus that infects human beings was identified. This virus had been detected in six provinces and municipal cities in China as of April 18, 2013. We correlated genomic sequences from avian influenza viruses with ecological information and did phylogenetic and coalescent analyses to extrapolate the potential origins of the virus and possible routes of reassortment events. METHODS We downloaded H7N9 virus genome sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) database and public sequences used from the Influenza Virus Resource. We constructed phylogenetic trees and did 1000 bootstrap replicates for each tree. Two rounds of phylogenetic analyses were done. We used at least 100 closely related sequences for each gene to infer the overall topology, removed suspicious sequences from the trees, and focused on the closest clades to the novel H7N9 viruses. We compared our tree topologies with those from a bayesian evolutionary analysis by sampling trees (BEAST) analysis. We used the bayesian Markov chain Monte Carlo method to jointly estimate phylogenies, divergence times, and other evolutionary parameters for all eight gene fragments. We used sequence alignment and homology-modelling methods to study specific mutations regarding phenotypes, specifically addressing the human receptor binding properties. FINDINGS The novel avian influenza A H7N9 virus originated from multiple reassortment events. The HA gene might have originated from avian influenza viruses of duck origin, and the NA gene might have transferred from migratory birds infected with avian influenza viruses along the east Asian flyway. The six internal genes of this virus probably originated from two different groups of H9N2 avian influenza viruses, which were isolated from chickens. Detailed analyses also showed that ducks and chickens probably acted as the intermediate hosts leading to the emergence of this virulent H7N9 virus. Genotypic and potential phenotypic differences imply that the isolates causing this outbreak form two separate subclades. INTERPRETATION The novel avian influenza A H7N9 virus might have evolved from at least four origins. Diversity among isolates implies that the H7N9 virus has evolved into at least two different lineages. Unknown intermediate hosts involved might be implicated, extensive global surveillance is needed, and domestic-poultry-to-person transmission should be closely watched in the future. FUNDING China Ministry of Science and Technology Project 973, National Natural Science Foundation of China, China Health and Family Planning Commission, Chinese Academy of Sciences.

Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2.

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8αα or the heterodimer αβ stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class I/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7Å resolution of a complex between CD8αα and the human MHC molecule HLA-A2, which is associated with peptide. CD8αα binds one HLA-A2/peptide molecule, interfacing with the α2 and α3 domains of HLA-A2 and also contacting β2-microglobulin. A flexible loop of the α3 domain (residues 223–229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody–antigen interaction, precluding the binding of a second MHC molecule. The position of the α3 domain is different from that in uncomplexed HLA-A2 (refs 3, 4), being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in α3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is clearly consistent with an avidity-based contribution from CD8 to TCR–peptide–MHC interactions.

Open-Source Genomic Analysis of Shiga-Toxin-Producing E. coli O104:H4

An outbreak caused by Shiga-toxin–producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance.

The crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor

A newly identified severe acute respiratory syndrome coronavirus (SARS-CoV), is the etiological agent responsible for the outbreak of SARS. The SARS-CoV main protease, which is a 33.8-kDa protease (also called the 3C-like protease), plays a pivotal role in mediating viral replication and transcription functions through extensive proteolytic processing of two replicase polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). Here, we report the crystal structures of the SARS-CoV main protease at different pH values and in complex with a specific inhibitor. The protease structure has a fold that can be described as an augmented serine-protease, but with a Cys-His at the active site. This series of crystal structures, which is the first, to our knowledge, of any protein from the SARS virus, reveal substantial pH-dependent conformational changes, and an unexpected mode of inhibitor binding, providing a structural basis for rational drug design.

TCR Binding to Peptide-MHC Stabilizes a Flexible Recognition Interface

The binding of TCRs to their peptide-MHC ligands is characterized by a low affinity, slow kinetics, and a high degree of cross-reactivity. Here, we report the results of a kinetic and thermodynamic analysis of two TCRs binding to their peptide-MHC ligands, which reveal two striking features. First, significant activation energy barriers must be overcome during both association and dissociation, suggesting that conformational adjustments are required. Second, the low affinity of binding is a consequence of highly unfavorable entropic effects, indicative of a substantial reduction in disorder upon binding. This is evidence that the TCR and/or peptide-MHC have flexible binding surfaces that are stabilized upon binding. Such conformational flexibility, which may also be a feature of primary antibodies, is likely to contribute to cross-reactivity in antigen recognition.

Streptococcal Toxic Shock Syndrome Caused by Streptococcus suis Serotype 2

Background Streptococcus suis serotype 2 ( S. suis 2, SS2) is a major zoonotic pathogen that causes only sporadic cases of meningitis and sepsis in humans. Most if not all cases of Streptococcal toxic shock syndrome (STSS) that have been well-documented to date were associated with the non-SS2 group A streptococcus (GAS). However, a recent large-scale outbreak of SS2 in Sichuan Province, China, appeared to be caused by more invasive deep-tissue infection with STSS, characterized by acute high fever, vascular collapse, hypotension, shock, and multiple organ failure. Methods and Findings We investigated this outbreak of SS2 infections in both human and pigs, which took place from July to August, 2005, through clinical observation and laboratory experiments. Clinical and pathological characterization of the human patients revealed the hallmarks of typical STSS, which to date had only been associated with GAS infection. Retrospectively, we found that this outbreak was very similar to an earlier outbreak in Jiangsu Province, China, in 1998. We isolated and analyzed 37 bacterial strains from human specimens and eight from pig specimens of the recent outbreak, as well as three human isolates and two pig isolates from the 1998 outbreak we had kept in our laboratory. The bacterial isolates were examined using light microscopy observation, pig infection experiments, multiplex-PCR assay, as well as restriction fragment length polymorphisms (RFLP) and multiple sequence alignment analyses. Multiple lines of evidence confirmed that highly virulent strains of SS2 were the causative agents of both outbreaks. Conclusions We report, to our knowledge for the first time, two outbreaks of STSS caused by SS2, a non-GAS streptococcus. The 2005 outbreak was associated with 38 deaths out of 204 documented human cases; the 1998 outbreak with 14 deaths out of 25 reported human cases. Most of the fatal cases were characterized by STSS; some of them by meningitis or severe septicemia. The molecular mechanisms underlying these human STSS outbreaks in human beings remain unclear and an objective for further study.

Biological features of novel avian influenza A (H7N9) virus

Human infection associated with a novel reassortant avian influenza H7N9 virus has recently been identified in China. A total of 132 confirmed cases and 39 deaths have been reported. Most patients presented with severe pneumonia and acute respiratory distress syndrome. Although the first epidemic has subsided, the presence of a natural reservoir and the disease severity highlight the need to evaluate its risk on human public health and to understand the possible pathogenesis mechanism. Here we show that the emerging H7N9 avian influenza virus poses a potentially high risk to humans. We discover that the H7N9 virus can bind to both avian-type (α2,3-linked sialic acid) and human-type (α2,6-linked sialic acid) receptors. It can invade epithelial cells in the human lower respiratory tract and type II pneumonocytes in alveoli, and replicated efficiently in ex vivo lung and trachea explant culture and several mammalian cell lines. In acute serum samples of H7N9-infected patients, increased levels of the chemokines and cytokines IP-10, MIG, MIP-1β, MCP-1, IL-6, IL-8 and IFN-α were detected. We note that the human population is naive to the H7N9 virus, and current seasonal vaccination could not provide protection.