Potential Geographic Distribution of the Novel Avian-Origin Influenza A (H7N9) Virus

Background

In late March 2013, a new avian-origin influenza virus emerged in eastern China. This H7N9 subtype virus has since infected 240 people and killed 60, and has awakened global concern as a potential pandemic threat. Ecological niche modeling has seen increasing applications as a useful tool in mapping geographic potential and risk of disease transmission.

Methodology/Principals

We developed two datasets based on seasonal variation in Normalized Difference Vegetation Index (NDVI) from the MODIS sensor to characterize environmental dimensions of H7N9 virus. One-third of well-documented cases was used to test robustness of models calibrated based on the remaining two-thirds, and model significance was tested using partial ROC approaches. A final niche model was calibrated using all records available.

Conclusions/Significance

Central-eastern China appears to represent an area of high risk for H7N9 spread, but suitable areas were distributed more spottily in the north and only along the coast in the south; highly suitable areas also were identified in western Taiwan. Areas identified as presenting high risk for H7N9 spread tend to present consistent NDVI values through the year, whereas unsuitable areas show greater seasonal variation.     

Novel Avian-Origin Influenza A (H7N9) Virus Attachment to the Respiratory Tract of Five Animal Models

We determined the pattern of attachment of the avian-origin H7N9 influenza viruses A/Anhui/1/2013 and A/Shanghai/1/2013 to the respiratory tract in ferrets, macaques, mice, pigs, and guinea pigs and compared it to that in humans. The H7N9 attachment pattern in macaques, mice, and to a lesser extent pigs and guinea pigs resembled that in humans more closely than the attachment pattern in ferrets. This information contributes to our knowledge of the different animal models for influenza.     

Origin and Characteristics of Internal Genes Affect Infectivity of the Novel Avian-Origin Influenza A (H7N9) Virus

Background

Human infection with a novel avian-origin influenza A (H7N9) virus occurred continuously in China during the first half of 2013, with high infectivity and pathogenicity to humans. In this study, we investigated the origin of internal genes of the novel H7N9 virus and analyzed the relationship between internal genes and infectivity of the virus.

Methodology and Principal findings

We tested the environmental specimens using real-time RT-PCR assays and isolated five H9N2 viruses from specimens that were positive for both H7 and H9. Results of recombination and phylogeny analysis, performed based on the entire sequences of 221 influenza viruses, showed that one of the Zhejiang avian H9N2 isolates, A/environment/Zhejiang/16/2013, shared the highest identities on the internal genes with the novel H7N9 virus A/Anhui/1/2013, ranging from 98.98% to 100%. Zhejiang avian H9N2 isolates were all reassortant viruses, by acquiring NS gene from A/chicken/Dawang/1/2011-like viruses and other five internal genes from A/brambling/Beijing/16/2012-like viruses. Compared to A/Anhui/1/2013 (H7N9), the homology on the NS gene was 99.16% with A/chicken/Dawang/1/2011, whereas only 94.27-97.61% with A/bramnling/Beijing/16/2012-like viruses. Analysis on the relationship between internal genes and the infectivity of novel H7N9 viruses were performed by comparing amino acid sequences with the HPAI H5N1 viruses, the H9N2 and the earlier H7N9 avian influenza viruses. There were nine amino acids on the internal genes found to be possibly associated with the infectivity of the novel H7N9 viruses.

Conclusions

These findings indicate that the internal genes, sharing the highest similarities with A/environment/Zhejiang/16/2013-like (H9N2) viruses, may affect the infectivity of the novel H7N9 viruses.     

Characterization of Influenza A (H7N9) Viruses Isolated from Human Cases Imported into Taiwan

A novel avian influenza A (H7N9) virus causes severe human infections and was first identified in March 2013 in China. The H7N9 virus has exhibited two epidemiological peaks of infection, occurring in week 15 of 2013 and week 5 of 2014. Taiwan, which is geographically adjacent to China, faces a large risk of being affected by this virus. Through extensive surveillance, launched in April 2013, four laboratory-confirmed H7N9 cases imported from China have been identified in Taiwan. The H7N9 virus isolated from imported case 1 in May 2013 (during the first wave) was found to be closest genetically to a virus from wild birds and differed from the prototype virus, A/Anhui/1/2013, in the MP gene. The other three imported cases were detected in December 2013 and April 2014 (during the second wave). The viruses isolated from cases 2 and 4 were similar in the compositions of their 6 internal genes and distinct from A/Anhui/1/2013 in the PB2 and MP genes, whereas the virus isolated from case 3 exhibited a novel reassortment that has not been identified previously and was different from A/Anhui/1/2013 in the PB2, PA and MP genes. The four imported H7N9 viruses share similar antigenicity with A/Anhui/1/2013, and their HA and NA genes grouped together in their respective phylogenies. In contrast with the HA and NA genes, which exhibited a smaller degree of diversity, the internal genes were heterogeneous and provided potential distinctions between transmission sources in terms of both geography and hosts. It is important to strengthen surveillance of influenza and to share viral genetic data in real-time for reducing the threat of rapid and continuing evolution of H7N9 viruses.     

Novel Avian Influenza A (H7N9) Virus Induces Impaired Interferon Responses in Human Dendritic Cells

In March 2013 a new avian influenza A(H7N9) virus emerged in China and infected humans with a case fatality rate of over 30%. Like the highly pathogenic H5N1 virus, H7N9 virus is causing severe respiratory distress syndrome in most patients. Based on genetic analysis this avian influenza A virus shows to some extent adaptation to mammalian host. In the present study, we analyzed the activation of innate immune responses by this novel H7N9 influenza A virus and compared these responses to those induced by the avian H5N1 and seasonal H3N2 viruses in human monocyte-derived dendritic cells (moDCs). We observed that in H7N9 virus-infected cells, interferon (IFN) responses were weak although the virus replicated as well as the H5N1 and H3N2 viruses in moDCs. H7N9 virus-induced expression of pro-inflammatory cytokines remained at a significantly lower level as compared to H5N1 virus-induced “cytokine storm” seen in human moDCs. However, the H7N9 virus was extremely sensitive to the antiviral effects of IFN-α and IFN-β in pretreated cells. Our data indicates that different highly pathogenic avian viruses may show considerable differences in their ability to induce host antiviral responses in human primary cell models such as moDCs. The unexpected appearance of the novel H7N9 virus clearly emphasizes the importance of the global influenza surveillance system. It is, however, equally important to systematically characterize in normal human cells the replication capacity of the new viruses and their ability to induce and respond to natural antiviral substances such as IFNs.     

Entry Properties and Entry Inhibitors of a Human H7N9 Influenza Virus

The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC₅₀ of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.     

Clinical,Virological and Immunological Features from Patients Infected with Re-Emergent Avian-Origin Human H7N9 Influenza Disease of Varying Severity in Guangdong Province

BackgroundThe second wave of avian influenza H7N9 virus outbreak in humans spread to the Guangdong province of China by August of 2013 and this virus is now endemic in poultry in this region.MethodsFive patients with H7N9 virus infection admitted to our hospital during August 2013 to February 2014 were intensively investigated. Viral load in the respiratory tract was determined by quantitative polymerase chain reaction (Q-PCR) and cytokine levels were measured by bead-based flow cytometery.ResultsFour patients survived and one died. Viral load in different clinical specimens was correlated with cytokine levels in plasma and broncho-alveolar fluid (BALF), therapeutic modalities used and clinical outcome. Intravenous zanamivir appeared to be better than peramivir as salvage therapy in patients who failed to respond to oseltamivir. Higher and more prolonged viral load was found in the sputum or endotracheal aspirates compared to throat swabs. Upregulation of proinflammatory cytokines IP-10, MCP-1, MIG, MIP-1α/β, IL-1β and IL-8 was found in the plasma and BALF samples. The levels of cytokines in the plasma and viral load were correlated with disease severity. Reactivation of herpes simplex virus type 1(HSV-1) was found in three out of five patients (60%).ConclusionExpectorated sputum or endotracheal aspirate specimens are preferable to throat swabs for detecting and monitoring H7N9 virus. Severity of the disease was correlated to the viral load in the respiratory tract as well as the extents of cytokinemia. Reactivation of HSV-1 may contribute to clinical outcome.     

Prognosis of 18 H7N9 Avian Influenza Patients in Shanghai

Purpose

To provide prognosis of an 18 patient cohort who were confirmed to have H7N9 lung infection in Shanghai.

Methods

Patients'' history, clinical manifestation, laboratory test, treatment strategy and mortality were followed and recorded for data analysis.

Results

A total of 18 patients had been admitted into Shanghai Public Health Clinical Center from April 8^th to July 29, 2013. 22.2% of the patients were found to have live poultry contact history and 80% were aged male patients with multiple co-morbidities including diabetes, hypertension and/or chronic obstructive pulmonary disease (COPD). This group of patients was admitted to the clinical center around 10 days after disease onset. According to laboratory examinations, increased C reactive protein (CRP), Procalcitonin (PCT), Plasma thromboplastin antecedent (PTA) and virus positive time (days) were indicative of patients'' mortality. After multivariate analysis, only CRP level showed significant prediction of mortality (P = 0.013) while results of prothrombin time (PT) analysis almost reached statistical significance (P = 0.056).

Conclusions

H7N9 infection induced pneumonia of different severity ranging from mild to severe pneumonia or acute lung injury/acute respiratory distress syndrome to multiple organ failure. Certain laboratory parameters such as plasma CRP, PCT, PTA and virus positive days predicted mortality of H7N9 infection and plasma CRP is an independent predictor of mortality in these patients.     

Seasonal Patterns in Human A (H5N1) Virus Infection: Analysis of Global Cases

Background

Human cases of highly pathogenic avian influenza (HPAI) A (H5N1) have high mortality. Despite abundant data on seasonal patterns in influenza epidemics, it is unknown whether similar patterns exist for human HPAI H5N1 cases worldwide. Such knowledge could help decrease avian-to-human transmission through increased prevention and control activities during peak periods.

Methods

We performed a systematic search of published human HPAI H5N1 cases to date, collecting month, year, country, season, hemisphere, and climate data. We used negative binomial regression to predict changes in case incidence as a function of season. To investigate hemisphere as a potential moderator, we used AIC and the likelihood-ratio test to compare the season-only model to nested models including a main effect or interaction with hemisphere. Finally, we visually assessed replication of seasonal patterns across climate groups based on the Köppen-Geiger climate classification.

Findings

We identified 617 human cases (611 with complete seasonal data) occurring in 15 countries in Southeast Asia, Africa, and the Middle East. Case occurrence was much higher in winter (n = 285, p = 0.03) than summer (n = 64), and the winter peak occurred across diverse climate groups. There was no significant interaction between hemisphere and season.

Interpretation

Across diverse climates, HPAI H5N1 virus infection in humans increases significantly in winter. This is consistent with increased poultry outbreaks and HPAI H5N1 virus transmission during cold and dry conditions. Prioritizing prevention and control activities among poultry and focusing public health messaging to reduce poultry exposures during winter months may help to reduce zoonotic transmission of HPAI H5N1 virus in resource-limited settings.     

A Combination of Serological Assays to Detect Human Antibodies to the Avian Influenza A H7N9 Virus

Human infection with avian influenza A H7N9 virus was first identified in March 2013 and represents an ongoing threat to public health. There is a need to optimize serological methods for this new influenza virus. Here, we compared the sensitivity and specificity of the hemagglutinin inhibition (HI), microneutralization (MN), and Western blot (WB) assays for the detection of human antibodies against avian influenza A (H7N9) virus. HI with horse erythrocytes (hRBCs) and a modified MN assay possessed greater sensitivity than turkey erythrocytes and the standard MN assay, respectively. Using these assays, 80% of tested sera from confirmed H7N9 cases developed detectable antibody to H7N9 after 21 days. To balance sensitivity and specificity, we found serum titers of ≥20 (MN) or 160 (HI) samples were most effective in determining seropositive to H7N9 virus. Single serum with HI titers of 20–80 or MN titer of 10 could be validated by each other or WB assay. Unlike serum collected from adult or elderly populations, the antibody response in children with mild disease was low or undetectable. These combinations of assays will be useful in case diagnosis and serologic investigation of human cases.     

Human H7N9 Influenza A Viruses Replicate in Swine Respiratory Tissue Explants

Recently, novel H7N9 influenza viruses have caused an unprecedented outbreak in humans. Pigs are an important intermediate host for influenza; thus, we assessed the replication ability of three human H7N9 viruses (A/Anhui/1/2013, A/Shanghai/1/2013, A/Shanghai/2/2013) in swine tissue explants. All viruses tested replicated efficiently in explants from tracheas and bronchi, with limited replication in alveolar cells. Swine respiratory tissue explants can serve as an efficient model for screening replication potential of newly emerging H7N9 viruses.     

Evolution of Influenza A H7N9 Virus with an Emphasis on Gene Constellation

The H7 subtype avian influenza viruses, including H7N2, HTN3 and HTN7, have posed a public health threat worldwide. Except one H7N7 fatal case in the Netherlands in 2003, the other H7 human cases have resulted in self-limiting conjunc- tivitis or mild upper respiratory illness.