Neutralizing antibody responses against contemporary and future influenza A(H3N2) viruses in paradoxical clades elicited by repeated and single vaccinations.

As one of the most effective measures to prevent seasonal influenza viruses, annual influenza vaccination is globally recommended. Nevertheless, evidence regarding the impact of repeated vaccination to contemporary and future influenza has been inconclusive. A total of 100 subjects singly or repeatedly immunized with influenza vaccines including 3C.2a1 or 3C.3a1 A(H3N2) during 2018-2019 and 2019-2020 influenza season were recruited. We investigated neutralization antibody by microneutralization assay using four antigenically distinct A(H3N2) viruses circulating from 2018 to 2023, and tracked the dynamics of B cell receptor (BCR) repertoire for consecutive vaccinations. We found that vaccination elicited cross-reactive antibody responses against future emerging strains. Broader neutralizing antibodies to A(H3N2) viruses and more diverse BCR repertoires were observed in the repeated vaccination. Meanwhile, a higher frequency of BCR sequences shared among the repeated-vaccinated individuals with consistently boosting antibody response was found than those with a reduced antibody response. Our findings suggest that repeated seasonal vaccination could broaden the breadth of antibody responses, which may improve vaccine protection against future emerging viruses.

Smart senior care cognition and health among Chinese elderly: A moderated mediation model featuring parent-child relationship and internet use.

In the context of digital transformation, smart senior care (SSC) cognition among elderly individuals has become an important contributor to their health. Using a sample of 345 older adults derived from the cross-sectional data of a questionnaire survey on the application of home-based SSC services and products among elderly individuals, this study explored how the parent‒child relationship mediated the linkage between SSC cognition and elderly health. To examine the moderating role of internet use, we applied the multigroup structural equation modeling (SEM) approach to test whether significant disparities exist between older adults who use the internet and those who do not on the pathways in the mediation model. After controlling for gender, age, hukou (household registration permit), ethnicity, income, marital status, and education level, we found that SSC cognition exerted significant positive effects on elderly health, in which the parent‒child relationship exerted a mediating effect. As for differences between the elderly with internet use and those without, on the three pathways connecting SSC cognition and health, SSC cognition and parent‒child relationship, and parent‒child relationship and health among elderly individuals, older adults who used the internet were more susceptible than those who did not. The findings are helpful for improving the policy-making on elderly health and may serve as a practical guide and theoretical reference for the promotion of active aging.

Comparison of percutaneous transluminal angioplasty and surgical revision after intraoperative dilatation with biliary tract probes for arteriovenous fistula stenosis at juxta-anastomosis.

BACKGROUND: Percutaneous transluminal angioplasty (PTA) is widely used for stenosis of vascular access (VA) for hemodialysis. We aimed to evaluate the effectiveness of both PTA and surgical revision after intraoperative dilatation with biliary tract probes for juxta-anastomotic stenosis in autogenous radiocephalic arteriovenous fistulas (RCAVFs). METHODS: We performed a retrospective analysis of PTA and surgical revision after intraoperative dilatation with biliary tract probes; these were the first interventions after RCAVF establishment in 112 patients with juxta-anastomotic stenosis. Anatomical (number of stenoses) and clinical variables (age and gender of the patient, time of hemodialysis, AVF age, presence of diabetes mellitus, and cause of end-stage renal disease) were reviewed. Technical success, clinical success, and post-intervention primary patency were evaluated. RESULTS: Our study enrolled 35 patients in the PTA group and 77 patients in the surgical revision group. Clinical and technical success rates of both groups were 100%. There were no complications, such as bleeding or hematomas. Using the Kaplan-Meier method, the post-intervention primary patency rates at 3, 6, 9, 12, 18, and 24 months in the PTA group were 100%, 94.28%, 77.1%, 60%, 54.29%, and 45.71%, respectively, and those in the surgical revision group were 100%, 94.81%, 92.2%, 90.91%, 81.82%, and 76.62%, respectively. The post-intervention primary patency rates at 9-24 months in the surgical revision group were significantly higher than those in the PTA group (χ2 = 19.04, p < 0.0001). CONCLUSION: The post-intervention long-term primary patency rate of surgical revision after intraoperative dilatation with biliary tract probes is higher than that of PTA for the first intervention of patients with juxta-anastomotic stenosis in RCAVFs. The surgical revision method is safe and effective, especially in hospitals that have not yet carried out PTA.

Application of a three-dimensional visualization model in intraoperative guidance of percutaneous nephrolithotomy.

OBJECTIVES: To establish a three-dimensional visualization model of percutaneous nephrolithotomy, apply it to guiding intraoperative puncture in a mixed reality environment, and evaluate its accuracy and clinical value. METHODS: Patients with percutaneous nephrolithotomy indications were prospectively divided into three-dimensional group and control group with a ratio of 1:2. For patients in three-dimensional group, positioning markers were pasted on the skin and enhanced computed tomography scanning was performed in the prone position. Holographic three-dimensional models were made and puncture routes were planned before operation. During the operation, the three-dimensional model was displayed through HoloLens glass and visually registered with the patient's body. Puncture of the target renal calyx was performed under three-dimensional-image guiding and ultrasonic monitoring. Patients in the control group underwent routine percutaneous nephrolithotomy in the prone position under the monitoring of B-ultrasound. Deviation distance of the kidney, puncture time, puncture attempts, channel coincidence rate, stone clearance rate, and postoperative complications were assessed. RESULTS: Twenty-one and 40 patients were enrolled in three-dimensional and control group, respectively. For three-dimensional group, the average deviation between virtual and real kidney was 3.1 ± 2.9 mm. All punctures were performed according to preoperative planning. Compared with the control group, the three-dimensional group had shorter puncture time (8.9 ± 3.3 vs 14.5 ± 6.1 min, P < 0.001), fewer puncture attempts (1.4 ± 0.6 vs 2.2 ± 1.5, P = 0.009), and might also have a better performance in stone clearance rate (90.5% vs 72.5%, P = 0.19) and postoperative complications (P = 0.074). CONCLUSIONS: The percutaneous nephrolithotomy three-dimensional model manifested acceptable accuracy and good value for guiding puncture in a mixed reality environment.

The predictive value of bioimpedance-derived fluid parameters for cardiovascular events in patients undergoing hemodialysis.

BACKGROUND: It is becoming increasingly evident that the accurate assessment of fluid status is critical to ensure optimal care in patients undergoing hemodialysis (HD). Various fluid parameters, including overhydration (OH) and overhydration/extracellular water (OH/ECW%), which can be obtained using a bioimpedance spectroscopy device have been used to indicate the hydration status in such patients. This study aimed to explore the effect of these fluid parameters on cardiovascular events and determine which parameter was a better predictor of cardiovascular events (CVEs). METHODS: A total of 227 patients who underwent HD at the Hangzhou Hospital of Traditional Chinese Medicine were enrolled in this prospective study between December 2017 and August 2018. Clinical data were collected, and the fluid status of patients was assessed using a body composition monitor. The patients were followed up until December 2020. The primary outcomes were CVEs. The association between fluid parameters and CVEs was analyzed using Cox proportional hazards models. The areas under the curve (AUCs) of receiver operating characteristic analysis and improvement in the global chi-squared value were used to compare the predictive values of fluid parameters for CVEs. RESULTS: During a median follow-up of 31 months, 66 CVEs were recorded. The patients with a higher absolute hydration index (OH) and a relative hydration index (OH/ECW%) exhibited an increased risk of developing CVEs. After adjusting for confounding factors, both OH [hazard ratio (HR) 1.279 per L, 95% confidence interval (CI) 1.047-1.562; p = 0.016] and OH/ECW% (HR 1.061 per %, 95% CI 1.017-1.108; p = 0.006) were independently associated with CVEs. The predictive ability of the absolute hydration index was superior to the relative hydration index based on AUC calculations for CVEs. Furthermore, a greater change in χ2 in predicting CVEs was noted for the absolute hydration index. CONCLUSIONS: Both absolute hydration index and relative hydration index were found to be independent predictors of CVEs in univariate and multivariate analyses. Furthermore, the absolute hydration index had a better additive predictive value than the relative hydration index in predicting CVEs.

The effect of single amino acid substitution at position 220 in the hemagglutinin glycoprotein on avian influenza H7N9 candidate vaccine virus.

Influenza vaccines represent the most effective preventive strategy to control influenza virus infections; however, adaptive mutations frequently occur in the hemagglutinin (HA) glycoprotein during the preparation of candidate vaccine virus and production of vaccine in embryonated eggs. In our previous study, we constructed candidate vaccine virus (HA-R) to match the highly pathogenic avian influenza H7N9 viruses A/Guangdong/17SF003/2016 as part of a pandemic preparedness program. However, mixed amino acids (R, G, and I) were presented at position 220 (H3 numbering) in HA during passage in embryonated eggs. The residue at position 220 is located close to the receptor-binding site and the biological characteristics of this site remain to be elucidated. Therefore, in this study, using reverse genetics, we constructed two viruses carrying the single substitution in position 220 of HA (HA-G and HA-I) and evaluated the biological effects of substitution (R with G/I) on receptor binding, neuraminidase (NA) activity, growth characteristics, genetic stability, and antigenicity. The results revealed both mutant viruses exhibited lower HA binding affinities to two receptor types (sialic acid in alpha2,3- and alpha2,6-linkage to galactose, P < 0.001) and significant better growth characteristics compared to HA-R in two cells. Moreover, under similar NA enzymatic activity, the two mutant viruses eluted more easily from agglutinated erythrocytes than HA-R. Collectively, these results implied the balance of HA and NA in mutant viruses was a stronger determinant of viral growth than the individual amino acid in the HA position 220 in HA-R without strong binding between HA and sialylated receptors. Importantly, both the substitutions conferred altered antigenicity to the mutant viruses. In conclusion, amino acid substitutions at position 220 can substantially influence viral biological properties.

A sandwich ELISA for detecting the hemagglutinin of avian influenza A (H10N8) virus.

Novel influenza A virus (H10N8) infected human with fatality in China during 2013-2014. It is important to detect such nonprevalent subtype influenza A virus in clinic and regular surveillance in the early stage for effective control and prevention from the potential pandemic. Unavailability of convenient rapid diagnosis for this subtype virus in resources-limited setting is an obstacle for timely recognizing human case. In the present study, a panel of mouse H10 specific monoclonal antibodies (mAbs) was generated, two of which were used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for detecting the hemagglutinin of avian influenza A (H10N8) virus. ELISA results showed high sensitivity with the lowest detection limit of 0.5HAU/50 µL for live virus, which laid a foundation for clinic use as a promising diagnostic methodology.

Establishment of sandwich ELISA for detecting the H7 subtype influenza A virus.

Avian H7N9 subtype influenza virus infects human with high case-fatality rate since it emerged in 2013. Although the vaccination has been rapidly used in poultry due to the emergence of highly pathogenic strain, this virus remains prevalent in this region. Thus, rapid diagnosis both in poultry and human clinic is critically important for the control and prevention of H7N9 infection. In this study, a batch of H7 subtype-specific monoclonal antibodies (mAbs) were developed and a pair of mAb, 2B6, and 5E9 were used to establish a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to quantify H7 protein and detect influenza A virus baring H7 subtype HA. The lowest detection limit for the recombinant H7 protein was 10 ng/mL and 0.5 HAU/50 µL of A/Guangdong/17SF003/2016(H7N9), 2 HAU/50 µL of A/Netherlands/219/2003(H7N7) and A/Anhui/1/2013(H7N9) for live virus, respectively. The ELISA could not only detect the prevailing H7N9 virus, but also antigenic drift H7 subtype viruses, showing excellent sensitivity and high specificity. Hence, it could serve as a valuable approach to diagnose H7 subtype virus which showed great potential to cause pandemic, as well as antigen quantification.

Highly pathogenic avian influenza H7N9 viruses with reduced susceptibility to neuraminidase inhibitors showed comparable replication capacity to their sensitive counterparts.

BACKGROUND: Human infection with avian influenza H7N9 virus was first reported in 2013. Since the fifth epidemic, a highly pathogenic avian influenza (HPAI) H7N9 virus has emerged and caused 33 human infections. Several potential NAI resistance sites have been found in human cases. However, the drug susceptibility and replication ability of HPAI H7N9 virus with such substitutions have not yet been studied. METHODS: Thirty-three HPAI H7N9 virus strains were isolated from human cases in China, and then sequences were analyzed to identify potential NAI resistance sites. Recombinant influenza viruses were generated to evaluate the effect of NA amino acid substitutions on NAI (oseltamivir or zanamivir) susceptibility and viral replication efficiency in MDCK cells. RESULTS: Four potential NAI resistance sites, R292 K, E119V, A246T or H274Y, were screened. All four substitutions conferred either reduced or highly reduced susceptibility to oseltamivir or zanamivir. 292 K not only highly reduced the susceptibility of HPAI H7N9 to oseltamivir but also induced an increase in the IC50 of zanamivir. 119 V or 274Y conferred reduced susceptibility of HPAI H7N9 to oseltamivir. Additionally, 246 T conferred reduced susceptibility to zanamivir. All tested NAI-resistant viruses were capable of replication in MDCK cells. The virus yields of rg006-NA292K were lower than those of rg006-NA292R at 24, 48, 72 and 96 h postinfection (P<0.05). Rg006-NA119V, rg006-NA246T or rg006-NA274Y showed comparable replication capacity to wild-type virus (except for rg006-NA274Y at 96 h, P<0.05). CONCLUSIONS: All 4 amino acid substitutions (R292 K, E119V, A246T or H274Y) in NA reduced the susceptibility of HPAI H7N9 to NAIs. The NAI-resistant mutations in HPAI H7N9, in most cases, did not reduce the replication ability of the virus in mammalian cells. Special attention needs to be paid to these mutations, and the development of new anti-H7N9 drugs is of great importance.

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-1ß, 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.

Ultrapotent Human Neutralizing Antibody Repertoires Against Middle East Respiratory Syndrome Coronavirus From a Recovered Patient.

Background: The Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infection with a high (~35%) mortality rate. Neutralizing antibodies targeting the spike of MERS-CoV have been shown to be a therapeutic option for treatment of lethal disease. Methods: We describe the germline diversity and neutralizing activity of 13 potent human monoclonal antibodies (mAbs) that target the MERS-CoV spike (S) protein. Biological functions were assessed by live MERS-CoV, pseudotype particle and its variants, and structural basis was also determined by crystallographic analysis. Results: Of the 13 mAbs displaying strong neutralizing activity against MERS-CoV, two with the immunoglobulin heavy-chain variable region (IGHV)1-69-derived heavy chain (named MERS-GD27 and MERS-GD33) showed the most potent neutralizing activity against pseudotyped and live MERS-CoV in vitro. Mutagenesis analysis suggested that MERS-GD27 and MERS-GD33 recognized distinct regions in S glycoproteins, and the combination of 2 mAbs demonstrated a synergistic effect in neutralization against pseudotyped MERS-CoV. The structural basis of MERS-GD27 neutralization and recognition revealed that its epitope almost completely overlapped with the receptor-binding site. Conclusions: Our data provide new insights into the specific antibody repertoires and the molecular determinants of neutralization during natural MERS-CoV infection in humans. This finding supports additional efforts to design and develop novel therapies to combat MERS-CoV infections in humans.

Population-Based Epidemiology of Sepsis in a Subdistrict of Beijing.

OBJECTIVE: Information about the epidemiology of sepsis in community residents in China remains scarce and incomplete. The purpose of this study was to describe the occurrence rate and outcome of sepsis in Yuetan Subdistrict of Beijing and to estimate the occurrence rate of sepsis in China. DESIGN: Retrospective cohort study. SETTING: All public hospitals serving residents in Yuetan Subdistrict, Beijing. PATIENTS: All patients (n = 1,716) meeting criteria for sepsis based on American College of Chest Physicians/Society of Critical Care Medicine consensus definition. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: We screened all adult residents in Yuetan Subdistrict who were hospitalized from July 1, 2012, to June 30, 2014, and reviewed medical records. Patients with sepsis were included in the analysis. We enrolled 1,716 patients with sepsis out of 21,191 hospitalized adults screened, among whom severe sepsis developed in 256 patients, and septic shock developed in 233 patients. The crude annual occurrence rates of sepsis, severe sepsis, and septic shock in Yuetan Subdistrict were 667, 103, and 91 cases per 100,000 population, corresponding to standardized occurrence rates of 461, 68, and 52 cases per 100,000 population per year, respectively. Both occurrence rate and mortality increased significantly with age, although males had higher age-adjusted occurrence rate and mortality. The occurrence rate of sepsis also exhibited seasonal variation, peaking in winter season. The overall hospital mortality rate of sepsis was 20.6%, yielding a standardized mortality rate of 79 cases per 100,000 population per year. CONCLUSIONS: Sepsis is a common and frequently fatal syndrome in Yuetan Subdistrict, Beijing. The occurrence rate and mortality of sepsis are significantly higher in males and elderly people.

Two Outbreak Sources of Influenza A (H7N9) Viruses Have Been Established in China.

UNLABELLED: Due to enzootic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. The Yangtze River Delta region, which is located in eastern China, is well recognized as the original source for H7N9 outbreaks. Based on the evolutionary analysis of H7N9 viruses from all three outbreak waves since 2013, we identified the Pearl River Delta region as an additional H7N9 outbreak source. H7N9 viruses are repeatedly introduced from these two sources to the other areas, and the persistent circulation of H7N9 viruses occurs in poultry, causing continuous outbreak waves. Poultry movements may contribute to the geographic expansion of the virus. In addition, the AnH1 genotype, which was predominant during wave 1, was replaced by JS537, JS18828, and AnH1887 genotypes during waves 2 and 3. The establishment of a new source and the continuous evolution of the virus hamper the elimination of H7N9 viruses, thus posing a long-term threat of H7N9 infection in humans. Therefore, both surveillance of H7N9 viruses in humans and poultry and supervision of poultry movements should be strengthened. IMPORTANCE: Since its occurrence in humans in eastern China in spring 2013, the avian H7N9 viruses have been demonstrating the continuing pandemic threat posed by the current influenza ecosystem in China. As the viruses are silently circulated in poultry, with potentially severe outcomes in humans, H7N9 virus activity in humans in China is very important to understand. In this study, we identified a newly emerged H7N9 outbreak source in the Pearl River Delta region. Both sources in the Yangtze River Delta region and the Pearl River Delta region have been established and found to be responsible for the H7N9 outbreaks in mainland China.

Biological characterisation of the emerged highly pathogenic avian influenza (HPAI) A(H7N9) viruses in humans, in mainland China, 2016 to 2017.

With no or low virulence in poultry, avian influenza A(H7N9) virus has caused severe infections in humans. In the current fifth epidemic wave, a highly pathogenic avian influenza (HPAI) H7N9 virus emerged. The insertion of four amino acids (KRTA) at the haemagglutinin (HA) cleavage site enabled trypsin-independent infectivity of this virus. Although maintaining dual receptor-binding preference, its HA antigenicity was distinct from low-pathogenic avian influenza A(H7N9). The neuraminidase substitution R292K conferred a multidrug resistance phenotype.

Health-related quality of life of among elders in rural China: the effect of widowhood.

PURPOSE: China has an enormous and rapidly growing population of widowed elders. Little is known about how losing a spouse affects elders' health-related quality of life (QOL), especially in the rural areas where most Chinese elders live. This article analyzes QOL data collected in 2014 among rural Chinese elders to address this question. METHODS: SF12 questionnaires and information about individual and household characteristics were collected from 3053 elders aged 60 and above in rural China. We compared the physical component summary (PCS) and mental component summary (MCS) scores between 1925 married elders and 1060 widowed elders in a bivariate model stratifying by gender and age group and in a general factorial ANOVA multivariate analysis that examined and controlled for other predictors of PCS and MCS scores including education, chronic disease, and family and household factors. RESULTS: Widowed male and female elders' physical health and mental health were in decline with age. Widowed men had lower PCS and MCS scores than married men. Widowed women also had lower PCS scores, but differences in MCS scores did not reach statistical significance. In multivariate analysis, widowhood was associated with lower PCS and MCS scores overall. Support from children was associated with better QOL and, based on interaction analysis, appeared to mitigate negative effects of widowhood. CONCLUSIONS: Widowed rural elders in China have lower physical and mental quality of life than their married counterparts. These elders rely on their children for care, and a supportive family is associated with better QOL.

Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study.

BACKGROUND: Human infections with different avian influenza viruses--eg, H5N1, H9N2, and H7N9--have raised concerns about pandemic potential worldwide. We report the first human infection with a novel reassortant avian influenza A H10N8 virus. METHODS: We obtained and analysed clinical, epidemiological, and virological data from a patient from Nanchang City, China. Tracheal aspirate specimens were tested for influenza virus and other possible pathogens by RT-PCR, viral culture, and sequence analyses. A maximum likelihood phylogenetic tree was constructed. FINDINGS: A woman aged 73 years presented with fever and was admitted to hospital on Nov 30, 2013. She developed multiple organ failure and died 9 days after illness onset. A novel reassortant avian influenza A H10N8 virus was isolated from the tracheal aspirate specimen obtained from the patient 7 days after onset of illness. Sequence analyses revealed that all the genes of the virus were of avian origin, with six internal genes from avian influenza A H9N2 viruses. The aminoacid motif GlnSerGly at residues 226-228 of the haemagglutinin protein indicated avian-like receptor binding preference. A mixture of glutamic acid and lysine at residue 627 in PB2 protein--which is associated with mammalian adaptation--was detected in the original tracheal aspirate samples. The virus was sensitive to neuraminidase inhibitors. Sputum and blood cultures and deep sequencing analysis indicated no co-infection with bacteria or fungi. Epidemiological investigation established that the patient had visited a live poultry market 4 days before illness onset. INTERPRETATION: The novel reassortant H10N8 virus obtained is distinct from previously reported H10N8 viruses. The virus caused human infection and could have been associated with the death of a patient. FUNDING: Emergency Research Project on human infection with avian influenza H7N9 virus, the National Basic Research Program of China, and the National Mega-projects for Infectious Diseases.

Limited effect of recombinant human mannose-binding lectin on the infection of novel influenza A (H7N9) virus in vitro.

Mannose-binding lectin (MBL), a pattern-recognition molecule in serum, recognizes specific hexose sugars rich in mannose and N-acetylglucosamine on bacterium, yeasts, viruses as well as apoptotic cells. It has been well-identified that MBL has antiviral effects via binding to seasonal influenza H1 and H3 subtype viruses. Influenza A (H7N9) virus, a novel reassortant virus to human population, possesses the surface hemagglutinin (HA) and neuraminidase (NA) genes from duck and wild-bird influenza viruses and internal genes from poultry H9N2 viruses. As of Dec 7th, 2014, a total of 467 human infections and 183 fatal cases have been identified. Here, recombinant human (rh) MBL was tested for its binding and effects on hemagglutination inhibition (HI) and NA activity inhibition (NAI) of avian H7N9, H9N2 and human H3N2 viruses. We discovered that rhMBL exhibited a strong binding to H7N9 virus as human H3N2 did at high virus titers. However, it performed a significantly weaker HI activity effect on H7N9 comparing to those of H3N2 and H9N2, even at a much higher concentration (3.67 ± 0.33 vs. 0.026 ± 0.001 and 0.083 ± 0.02 µg/mL, respectively). Similarly, minor NAI effect of rhMBL, even at up to 10 µg/mL, was found on H7N9 virus while it displayed significant effects on both H3N2 and H9N2 at a lowest concentration of 0.0807 ± 0.009 and 0.0625 µg/mL, respectively. The HI and NAI effects of rhMBL were calcium-dependent and mediated by lectin domain. Our findings suggest that MBL, the host innate molecule, has differential interference effects with human and avian influenza virus and limited antiviral effect against H7N9 virus.

Residues 41V and/or 210D in the NP protein enhance polymerase activities and potential replication of novel influenza (H7N9) viruses at low temperature.

BACKGROUND: The influenza A (H7N9) virus emerged in the spring of 2013 in China. It contained six internal genes from Y280-like H9N2 viruses, which have co-circulated with G1-like lineage viruses throughout poultry in China. Accompanied with continuous reassortment among H7N9 and H9N2 viruses in poultry, it is possible for H7N9 viruses to acquire internal genes of G1-lineage viruses. Thus, it is important to evaluate potential impact of G1-like viruses on the H7N9 viruses. FINDINGS: We used in vitro assays of polymerase activities and growth kinetics to evaluate the potential contribution of G1-like virus genes to the replication abilities of H7N9 viruses. Two mutations in the NP protein (41V and/or 210D) could enhance H7N9 RNP activities, especially at low temperature (33°C, which is similar to the temperature of human upper respiratory tract). Meanwhile, G1 viruses with V41I or D210E substitutions exhibited poor growth ability in the early infection stage at low temperature. The D210E substitution also reduced the replication ability of G1 virus at 12 and 24 hour post infection at 37°C. In both tested temperatures, V41I could compensate for the defective virus replication induced by the D210E mutation. CONCLUSIONS: Mutations 41V and/or 210D in the NP protein conferred improved RNP activity in H7N9 viruses and promoted the replication ability of H9N2 viruses, particularly at lower temperature. Substitutions at these two positions may promote the replication ability of H7N9 viruses in low temperature and thus might contribute to viral transmissibility. While these two residues have not yet been observed in H7N9 viruses, attention should be devoted to these two residues.

Structural and functional characterization of K339T substitution identified in the PB2 subunit cap-binding pocket of influenza A virus.

Influenza virus RNA-dependent RNA polymerase is a heterotrimer composed of PA, PB1, and PB2 subunits. RNA-dependent RNA polymerase is required for both transcription and replication of influenza viral RNA taking place in the nucleus of infected cells. A "cap-snatching" mechanism is used to generate a 5'-capped primer for transcription in which the cap-binding domain of PB2 (PB2cap) captures the 5' cap of the host pre-mRNA. Our statistical analysis of PB2 sequences showed that residue Lys(339) located in the cap-binding pocket of H5N1 PB2cap was gradually replaced by Thr(339) over the past decade. To understand the role of this amino acid polymorphism, we solved the crystal structures of PB2cap with or without a pre-mRNA cap analog, m(7)GTP, in the presence of Lys(339) or Thr(339). The structures showed that Lys(339) contributes to binding the γ-phosphate group of m(7)GTP, and the replacement of Lys(339) by Thr eliminates this interaction. Isothermal titration calorimetry analysis showed that Thr(339) attenuated the PB2cap cap binding activity in vitro compared with Lys(339). Further functional studies confirmed that Thr(339)-PB2-containing ribonucleoprotein complex has a reduced influenza polymerase activity and RNA synthesis activity, and a reconstituted H5N1 virus containing the Thr(339) substitution exhibited a lower virulence to mice but more active replication in Madin-Darby canine kidney cells. The K339T substitution in the cap-binding pocket of PB2 modulates the polymerase activity and virulence by regulating the cap binding activity. It is informative to track variations in the cap-binding pocket of PB2 in surveillance of the evolution and spread of influenza virus.