We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R²=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.
Animals ; Reproducibility of Results ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Chromatography, Gel ; Virion ; Lasers

The conserved hemagglutinin (HA) stem region of avian influenza virus (AIV) is an important target for designing broad-spectrum vaccines, therapeutic antibodies and diagnostic reagents. Previously, we obtained a monoclonal antibody (mAb) (5D3-1B5) which was reactive with the HA stem epitope (aa 428-452) of H7N9 subtype AIV. To systematically characterize the mAb, we determined the antibody titers, including the HA-binding IgG, hemagglutination-inhibition (HI) and virus neutralizing (VN) titers. In addition, the antigenic epitope recognized by the antibody as well as the sequence and structure of the antibody variable region (VR) were also determined. Moreover, we evaluated the cross-reactivity of the antibody with influenza virus strains of different subtypes. The results showed that the 5D3-1B5 antibody had undetectable HI and VN activities against H7N9 virus, whereas it exhibited strong reactivity with the HA protein. Using the peptide-based enzyme-linked immunosorbent assay and biopanning with a phage-displayed random peptide library, a motif with the core sequence (⁴³¹W-⁴³³Y-⁴³⁷L) in the C-helix domain in the HA stem was identified as the epitope recognized by 5D3-1B5. Moreover, the mAb failed to react with the mutant H7N9 virus which contains mutations in the epitope. The VR of the antibody was sequenced and the complementarity determining regions in the VR of the light and heavy chains were determined. Structural modeling and molecular docking analysis of the VR verified specific binding between the antibody and the C-helix domain of the HA stem. Notably, 5D3-1B5 showed a broad cross-reactivity with influenza virus strains of different subtypes belonging to groups 1 and 2. In conclusion, 5D3-1B5 antibody is a promising candidate in terms of the development of broad-spectrum virus diagnostic reagents and therapeutic antibodies. Our findings also provided new information for understanding the epitope characteristics of the HA protein of H7N9 subtype AIV.
Animals ; Antibodies, Monoclonal ; Antibodies, Viral ; Hemagglutinin Glycoproteins, Influenza Virus/genetics* ; Hemagglutinins ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Molecular Docking Simulation

BACKGROUND: The new emerging avian influenza A H7N9 virus, causing severe human infection with a mortality rate of around 41%. This study aims to provide a novel treatment option for the prevention and control of H7N9. METHODS: H7 hemagglutinin (HA)-specific B cells were isolated from peripheral blood plasma cells of the patients previously infected by H7N9 in Jiangsu Province, China. The human monoclonal antibodies (mAbs) were generated by amplification and cloning of these HA-specific B cells. First, all human mAbs were screened for binding activity by enzyme-linked immunosorbent assay. Then, those mAbs, exhibiting potent affinity to recognize H7 HAs were further evaluated by hemagglutination-inhibiting (HAI) and microneutralization in vitro assays. Finally, the lead mAb candidate was selected and tested against the lethal challenge of the H7N9 virus using murine models. RESULTS: The mAb 6-137 was able to recognize a panel of H7 HAs with high affinity but not HA of other subtypes, including H1N1 and H3N2. The mAb 6-137 can efficiently inhibit the HA activity in the inactivated H7N9 virus and neutralize 100 tissue culture infectious dose 50 (TCID50) of H7N9 virus (influenza A/Nanjing/1/2013) in vitro, with neutralizing activity as low as 78 ng/mL. In addition, the mAb 6-137 protected the mice against the lethal challenge of H7N9 prophylactically and therapeutically. CONCLUSION The mAb 6-137 could be an effective antibody as a prophylactic or therapeutic biological treatment for the H7N9 exposure or infection.
Animals ; Antibodies, Monoclonal/therapeutic use* ; Antibodies, Neutralizing/therapeutic use* ; Antibodies, Viral ; Hemagglutinins ; Humans ; Influenza A Virus, H1N1 Subtype ; Influenza A Virus, H3N2 Subtype ; Influenza A Virus, H7N9 Subtype ; Influenza Vaccines ; Influenza in Birds ; Influenza, Human/prevention & control* ; Mice

The avian influenza A (H7N9) virus is a zoonotic virus that is closely associated with live poultry markets. It has caused infections in humans in China since 2013. Five waves of the H7N9 influenza epidemic occurred in China between March 2013 and September 2017. H7N9 with low-pathogenicity dominated in the first four waves, whereas highly pathogenic H7N9 influenza emerged in poultry and spread to humans during the fifth wave, causing wide concern. Specialists and officials from China and other countries responded quickly, controlled the epidemic well thus far, and characterized the virus by using new technologies and surveillance tools that were made possible by their preparedness efforts. Here, we review the characteristics of the H7N9 viruses that were identified while controlling the spread of the disease. It was summarized and discussed from the perspectives of molecular epidemiology, clinical features, virulence and pathogenesis, receptor binding, T-cell responses, monoclonal antibody development, vaccine development, and disease burden. These data provide tools for minimizing the future threat of H7N9 and other emerging and re-emerging viruses, such as SARS-CoV-2.
Animals ; COVID-19 ; China/epidemiology* ; Humans ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds/epidemiology* ; Influenza, Human/prevention & control* ; Poultry ; SARS-CoV-2

BACKGROUND: Six epidemic waves of human infection with avian influenza A (H7N9) virus have emerged in China with high mortality. However, study on quantitative relationship between clinical indices in ill persons and H7N9 outcome (fatal and non-fatal) is still unclear. A retrospective cohort study was conducted to collect laboratory-confirmed cases with H7N9 viral infection from 2013 to 2015 in 23 hospitals across 13 cities in Guangdong Province, China. METHODS: Multivariable logistic regression model and classification tree model analyses were used to detect the threshold of selected clinical indices and risk factors for H7N9 death. The receiver operating characteristic curve (ROC) and analyses were used to compare survival and death distributions and differences between indices. A total of 143 cases with 90 survivors and 53 deaths were investigated. RESULTS: Average age (Odds Ratio (OR) = 1.036, 95% Confidence Interval (CI) = 1.016-1.057), interval days between dates of onset and confirmation (OR = 1.078, 95% CI = 1.004-1.157), interval days between onset and oseltamivir treatment (OR = 5.923, 95% CI = 1.877-18.687), body temperature (BT) (OR = 3.612, 95% CI = 1.914-6.815), white blood cell count (WBC) (OR = 1.212, 95% CI = 1.092-1.346) were significantly associated with H7N9 death after adjusting for confounders. The chance of death from H7N9 infection was 80.0% if BT was over 38.1 °C, and chance of death is 67.4% if WBC count was higher than 9.5 (10/L). Only 27.1% of patients who began oseltamivir treatment less than 9.5 days after disease onset died, compared to 68.8% of those who started treatment more than 15.5 days after onset. CONCLUSIONS The intervals between date of onset and confirmation of diagnosis, between date of onset to oseltamivir treatment, age, BT and WBC are found to be the best predictors of H7N9 mortality.
Adult ; Aged ; China ; epidemiology ; Confidence Intervals ; Female ; Hospitalization ; statistics & numerical data ; Humans ; Influenza A Virus, H7N9 Subtype ; pathogenicity ; Influenza, Human ; epidemiology ; mortality ; virology ; Logistic Models ; Male ; Middle Aged ; ROC Curve ; Retrospective Studies ; Risk Factors ; Young Adult

OBJECTIVETo assess the risk of local outbreaks of H7N9 avian influenza infection in Zhejiang province and to explore the semi-quantitative assessment method for public health risks in emergency.

METHODSRisk index system of human infection with H7N9 avian influenza caused by local transmission were reviewed. The weights of indexes were calculated by analytic hierarchy process, which was combined with the TOPSIS method to calculate the risk comprehensive index.

RESULTSFour primary indexes and 23 secondary indexes were identified for risk assessment in local outbreaks of H7N9 avian influenza infection. The weights ranked on the top five were:morbidity (0.0972), closure measures (0.0718), sterilization measures (0.0673), fatality rate (0.0651), and epidemic spread (0.0616). The comprehensive index of the risk of local outbreaks of H7N9 avian influenza ranged from high to low were Hangzhou (0.5910), Shaoxing (0.5711), Jiaxing (0.5199), Taizhou (0.5198), Huzhou (0.4662), Ningbo (0.3828), Wenzhou (0.3719), Jinhua (0.3392), Lishui (0.2727), Quzhou (0.2001) and Zhoushan (0.0508).

CONCLUSIONSA semi-quantitative method has been established in this study, which provides scientific basis for prevention and control of H7N9 avian influenza epidemic in Zhejiang province.

Animals ; Birds ; China ; Humans ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; transmission ; Influenza, Human ; transmission ; Risk Assessment

Since 2013, the Hospital-based Influenza Morbidity and Mortality (HIMM) surveillance system began a H7N9 influenza surveillance scheme for returning travelers in addition to pre-existing emergency room (ER)-based influenza-like illness (ILI) surveillance and severe acute respiratory infection (SARI) surveillance. Although limited to eastern China, avian A/H7N9 influenza virus is considered to have the highest pandemic potential among currently circulating influenza viruses. During the study period between October 1st, 2013 and April 30th, 2016, 11 cases presented with ILI within seven days of travel return. These patients visited China, Hong Kong, or neighboring Southeast Asian countries, but none of them visited a livestock market. Seasonal influenza virus (54.5%, 6 among 11) was the most common cause of ILI among returning travelers, and avian A/H7N9 influenza virus was not detected during the study period.
Asian Continental Ancestry Group ; China ; Emergency Service, Hospital ; Hong Kong ; Humans ; Influenza A Virus, H7N9 Subtype ; Influenza, Human ; Livestock ; Mortality ; Orthomyxoviridae ; Pandemics ; Seasons

Adolescent ; Humans ; Influenza A Virus, H7N9 Subtype ; immunology ; pathogenicity ; Lymphopenia ; immunology ; microbiology ; Male ; T-Lymphocytes ; metabolism

In 2013, two episodes of influenza emerged in China and caused worldwide concern. A new H7N9 avian influenza virus (AIV) first appeared in China on February 19, 2013. By August 31, 2013, the virus had spread to ten provinces and two metropolitan cities. Of 134 patients with H7N9 influenza, 45 died. From then on, epidemics emerged sporadically in China and resulted in several victims. On November 30, 2013, a 73-year-old woman presented with an influenza-like illness. She developed multiple organ failure and died 9 d after the onset of disease. A novel reassortant AIV, H10N8, was isolated from a tracheal aspirate specimen that was obtained from the patient 7 d after onset. This case was the first human case of influenza A subtype H10N8. On 4 February, 2014, another death due to H10N8 avian influenza was reported in Jiangxi Province, China.
Aged ; China ; epidemiology ; Female ; Humans ; Influenza A Virus, H10N8 Subtype ; classification ; Influenza A Virus, H7N9 Subtype ; classification ; Influenza A Virus, H9N2 Subtype ; classification ; Influenza, Human ; epidemiology ; virology ; Phylogeny ; Reassortant Viruses ; classification

Objective: To understand the epidemiological characteristics of human infection with avian influenza A (H7N9) virus in China, and provide evidence for the prevention and control of human infection with H7N9 virus. Methods: The published incidence data of human infection with H7N9 virus in China from March 2013 to April 2017 were collected. Excel 2007 software was used to perform the analysis. The characteristics of distribution of the disease, exposure history, cluster of the disease were described. Results: By the end of April 2017, a total of 1 416 cases of human infection with H7N9 virus were confirmed in China, including 559 deaths, the case fatality rate was 39.5%. In 2016, the case number was lowest (127 cases), with the highest fatality rate (57.5%). The first three provinces with high case numbers were Zhejiang, Guangdong and Jiangsu. The median age of the cases was 55 years and the male to female ratio was 2.3∶1. Up to 66% of cases had clear live poultry exposure history before illness onset, 31% of cases had unknown exposure history and only 3% of the cases had no live poultry exposure history. There were 35 household clusters (5 in 2013, 9 in 2014, 6 in 2015, 5 in 2016, 10 in 2017), which involved 72 cases, accounting for 5% of the total cases. Conclusions: The epidemic of human infection with H7N9 virus in China during 2013-2017 had obvious seasonality and spatial distribution. There was limited family clustering. Infection cases were mostly related to poultry contact.
Adult ; Age Distribution ; Aged ; Aged, 80 and over ; Animals ; China/epidemiology* ; Cluster Analysis ; Disease Outbreaks ; Epidemics ; Female ; Humans ; Incidence ; Influenza A Virus, H7N9 Subtype/isolation & purification* ; Influenza, Human/virology* ; Male ; Middle Aged ; Poultry ; Sex Distribution