Influenza A virus replicates productively in primary human kidney cells and induces factors and mechanisms related to regulated cell death and renal pathology observed in virus-infected patients.

Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.

Seasonal influenza viruses decay more rapidly at intermediate humidity in droplets containing saliva compared to respiratory mucus.

Expulsions of virus-laden aerosols or droplets from the oral and nasal cavities of an infected host are an important source of onward respiratory virus transmission. However, the presence of infectious influenza virus in the oral cavity during infection has not been widely considered, and thus, little work has explored the environmental persistence of influenza virus in oral cavity expulsions. Using the ferret model, we detected infectious virus in the nasal and oral cavities, suggesting that the virus can be expelled into the environment from both anatomical sites. We also assessed the stability of two influenza A viruses (H1N1 and H3N2) in droplets of human saliva or respiratory mucus over a range of relative humidities. We observed that influenza virus infectivity decays rapidly in saliva droplets at intermediate relative humidity, while viruses in airway surface liquid droplets retain infectivity. Virus inactivation was not associated with bulk protein content, salt content, or droplet drying time. Instead, we found that saliva droplets exhibited distinct inactivation kinetics during the wet and dry phases at intermediate relative humidity, and droplet residue morphology may lead to the elevated first-order inactivation rate observed during the dry phase. Additionally, distinct differences in crystalline structure and nanobead localization were observed between saliva and airway surface liquid droplets. Together, our work demonstrates that different respiratory fluids exhibit unique virus persistence profiles and suggests that influenza viruses expelled from the oral cavity may contribute to virus transmission in low- and high-humidity environments.IMPORTANCEDetermining how long viruses persist in the environment is important for mitigating transmission risk. Expelled infectious droplets and aerosols are composed of respiratory fluids, including saliva and complex mucus mixtures, but how well influenza viruses survive in such fluids is largely unknown. Here, we find that infectious influenza virus is present in the oral cavity of infected ferrets, suggesting that saliva-containing expulsions can play a role in onward transmission. Additionally, influenza virus in droplets composed of saliva degrades more rapidly than virus within respiratory mucus. Droplet composition impacts the crystalline structure and virus localization in dried droplets. These results suggest that viruses from distinct sites in the respiratory tract could have variable persistence in the environment, which will impact viral transmission fitness.

Expression of TMPRSS2 is up-regulated by bacterial flagellin, LPS, and Pam3Cys in human airway cells.

Many viruses require proteolytic activation of their envelope proteins for infectivity, and relevant host proteases provide promising drug targets. The transmembrane serine protease 2 (TMPRSS2) has been identified as a major activating protease of influenza A virus (IAV) and various coronaviruses (CoV). Increased TMPRSS2 expression has been associated with a higher risk of severe influenza infection and enhanced susceptibility to SARS-CoV-2. Here, we found that Legionella pneumophila stimulates the increased expression of TMPRSS2-mRNA in Calu-3 human airway cells. We identified flagellin as the dominant structural component inducing TMPRSS2 expression. The flagellin-induced increase was not observed at this magnitude for other virus-activating host proteases. TMPRSS2-mRNA expression was also significantly increased by LPS, Pam3Cys, and Streptococcus pneumoniae, although less pronounced. Multicycle replication of H1N1pdm and H3N2 IAV but not SARS-CoV-2 and SARS-CoV was enhanced by flagellin treatment. Our data suggest that bacteria, particularly flagellated bacteria, up-regulate the expression of TMPRSS2 in human airway cells and, thereby, may support enhanced activation and replication of IAV upon co-infections. In addition, our data indicate a physiological role of TMPRSS2 in antimicrobial host response.

How avian influenza viruses spill over to mammals.

The H3N2 canine influenza virus - which originally came from birds - is evolving to become more transmissible between dogs.

Using machine learning to determine the time of exposure to infection by a respiratory pathogen.

Given an infected host, estimating the time that has elapsed since initial exposure to the pathogen is an important problem in public health. In this paper we use longitudinal gene expression data from human challenge studies of viral respiratory illnesses for building predictive models to estimate the time elapsed since onset of respiratory infection. We apply sparsity driven machine learning to this time-stamped gene expression data to model the time of exposure by a pathogen and subsequent infection accompanied by the onset of the host immune response. These predictive models exploit the fact that the host gene expression profile evolves in time and its characteristic temporal signature can be effectively modeled using a small number of features. Predicting the time of exposure to infection to be in first 48 h after exposure produces BSR in the range of 80-90% on sequestered test data. A variety of machine learning experiments provide evidence that models developed on one virus can be used to predict exposure time for other viruses, e.g., H1N1, H3N2, and HRV. The interferon [Formula: see text] signaling pathway appears to play a central role in keeping time from onset of infection. Successful prediction of the time of exposure to a pathogen has potential ramifications for patient treatment and contact tracing.

Wearable Sensor-Based Detection of Influenza in Presymptomatic and Asymptomatic Individuals.

BACKGROUND: The COVID-19 pandemic highlighted the need for early detection of viral infections in symptomatic and asymptomatic individuals to allow for timely clinical management and public health interventions. METHODS: Twenty healthy adults were challenged with an influenza A (H3N2) virus and prospectively monitored from 7 days before through 10 days after inoculation, using wearable electrocardiogram and physical activity sensors. This framework allowed for responses to be accurately referenced to the infection event. For each participant, we trained a semisupervised multivariable anomaly detection model on data acquired before inoculation and used it to classify the postinoculation dataset. RESULTS: Inoculation with this challenge virus was well-tolerated with an infection rate of 85%. With the model classification threshold set so that no alarms were recorded in the 170 healthy days recorded, the algorithm correctly identified 16 of 17 (94%) positive presymptomatic and asymptomatic individuals, on average 58 hours postinoculation and 23 hours before the symptom onset. CONCLUSIONS: The data processing and modeling methodology show promise for the early detection of respiratory illness. The detection algorithm is compatible with data collected from smartwatches using optical techniques but needs to be validated in large heterogeneous cohorts in normal living conditions. Clinical Trials Registration. NCT04204493.

Synthesis and structure-activity relationship of L-methionine-coupled 1,3,4-thiadiazole derivatives with activity against influenza virus.

In previous investigations, we identified a class of 1,3,4-thiadiazole derivatives with antiviral activity. N-{3-(Methylsulfanyl)-1-[5-(phenylamino)-1,3,4-thiadiazole-2-yl]propyl}benzamide emerged as a relevant lead compound for designing novel influenza A virus inhibitors. In the present study, we elaborated on this initial lead by performing chemical synthesis and antiviral evaluation of a series of structural analogues. During this research, thirteen novel 1,3,4-thiadiazole derivatives were synthesized by the cyclization of the corresponding thiosemicarbazides as synthetic precursors. The structures and the purities of the synthesized compounds were confirmed through chromatographic and spectral data. Four L-methionine-based 1,3,4-thiadiazole derivatives displayed activity against influenza A virus, the two best compounds being 24 carrying a 5-(4-chlorophenylamino)-1,3,4-thiadiazole moiety and 30 possessing a 5-(benzoylamino)-1,3,4-thiadiazole structure [antiviral EC50 against influenza A/H3N2 virus: 4.8 and 7.4 µM, respectively]. The 1,3,4-thiadiazole derivatives were inactive against influenza B virus and a wide panel of unrelated DNA and RNA viruses. Compound 24 represents a new class of selective influenza A virus inhibitors acting during the virus entry process, as evidenced by our findings in a time-of-addition assay. Molecular descriptors and in silico prediction of ADMET properties of the active compounds were calculated. According to in silico ADMET and drug similarity studies, active compounds have been estimated to be good candidates for oral administration with no apparent toxicity considerations.

Age-seroprevalence curves for the multi-strain structure of influenza A virus.

The relationship between age and seroprevalence can be used to estimate the annual attack rate of an infectious disease. For pathogens with multiple serologically distinct strains, there is a need to describe composite exposure to an antigenically variable group of pathogens. In this study, we assay 24,402 general-population serum samples, collected in Vietnam between 2009 to 2015, for antibodies to eleven human influenza A strains. We report that a principal components decomposition of antibody titer data gives the first principal component as an appropriate surrogate for seroprevalence; this results in annual attack rate estimates of 25.6% (95% CI: 24.1% - 27.1%) for subtype H3 and 16.0% (95% CI: 14.7% - 17.3%) for subtype H1. The remaining principal components separate the strains by serological similarity and associate birth cohorts with their particular influenza histories. Our work shows that dimensionality reduction can be used on human antibody profiles to construct an age-seroprevalence relationship for antigenically variable pathogens.

Pre-existing immunity and vaccine history determine hemagglutinin-specific CD4 T cell and IgG response following seasonal influenza vaccination.

Effectiveness of seasonal influenza vaccination varies between individuals and might be affected by vaccination history among other factors. Here we show, by monitoring frequencies of CD4 T cells specific to the conserved hemagglutinin epitope HA118-132 and titres of IgG against the corresponding recombinant hemagglutinin protein, that antigen-specific CD4 T cell and antibody responses are closely linked to pre-existing immunity and vaccine history. Upon immunization, a strong early reaction is observed in all vaccine naïve participants and also in vaccine experienced individuals who have not received the respective seasonal vaccine in the previous year. This response is characterized by HA118-132 specific CD4 T cells with a follicular helper T cell phenotype and by ascending titers of hemagglutinin-specific antibodies from baseline to day 28 following vaccination. This trend was observed in only a proportion of those participants who received the seasonal vaccine the year preceding the study. Regardless of history, levels of pre-existing antibodies and CD127 expression on CD4 T cells at baseline were the strongest predictors of robust early response. Thus, both pre-existing immunity and vaccine history contribute to the response to seasonal influenza vaccines.

Replication and transmission of an influenza A(H3N2) virus harboring the polymerase acidic I38T substitution, in guinea pigs.

The polymerase acidic (PA) I38T substitution is a dominant marker of resistance to baloxavir. We evaluated the impact of I38T on the fitness of a contemporary influenza A(H3N2) virus. Influenza A/Switzerland/9715293/2013 (H3N2) wild-type (WT) virus and its I38T mutant were rescued by reverse genetics. Replication kinetics were compared using ST6GalI-MDCK and A549 cells and infectivity/contact transmissibility were evaluated in guinea pigs. Nasal wash (NW) viral titres were determined by TCID50 ml-1 in ST6GalI-MDCK cells. Competition experiments were performed and the evolution of viral population was assessed by droplet digital RT-PCR. I38T did not alter in vitro replication. I38T induced comparable titres vs the WT in guinea pigs NWs and the two viruses transmitted equally by direct contact. However, a 50 %:50 % mixture inoculum evolved to mean WT/I38T ratios of 71 %:29 % and 66.4 %:33.6 % on days 4 and 6 p.i., respectively. Contemporary influenza A(H3N2)-I38T PA variants may conserve a significant level of viral fitness.

A natural variant in ANP32B impairs influenza virus replication in human cells.

Viruses require host factors to support their replication, and genetic variation in such factors can affect susceptibility to infectious disease. Influenza virus replication in human cells relies on ANP32 proteins, which are involved in assembly of replication-competent dimeric influenza virus polymerase (FluPol) complexes. Here, we investigate naturally occurring single nucleotide variants (SNV) in the human Anp32A and Anp32B genes. We note that variant rs182096718 in Anp32B is found at a higher frequency than other variants in either gene. This SNV results in a D130A substitution in ANP32B, which is less able to support FluPol activity than wild-type ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wild-type ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wild-type ANP32A and mutant ANP32B-D130A. We propose a model in which the D130A mutation impairs FluPol dimer formation, thus resulting in compromised replication. We suggest that both homozygous and heterozygous carriers of rs182096718 may have some genetic protection against influenza viruses.

Hemagglutinins of Avian Influenza Viruses Are Proteolytically Activated by TMPRSS2 in Human and Murine Airway Cells.

Cleavage of the influenza A virus (IAV) hemagglutinin (HA) by host proteases is indispensable for virus replication. Most IAVs possess a monobasic HA cleavage site cleaved by trypsin-like proteases. Previously, the transmembrane protease TMPRSS2 was shown to be essential for proteolytic activation of IAV HA subtypes H1, H2, H7, and H10 in mice. In contrast, additional proteases are involved in activation of certain H3 IAVs, indicating that HAs with monobasic cleavage sites can differ in their sensitivity to host proteases. Here, we investigated the role of TMPRSS2 in proteolytic activation of avian HA subtypes H1 to H11 and H14 to H16 in human and mouse airway cell cultures. Using reassortant viruses carrying representative HAs, we analyzed HA cleavage and multicycle replication in (i) lung cells of TMPRSS2-deficient mice and (ii) Calu-3 cells and primary human bronchial cells subjected to morpholino oligomer-mediated knockdown of TMPRSS2 activity. TMPRSS2 was found to be crucial for activation of H1 to H11, H14, and H15 in airway cells of human and mouse. Only H9 with an R-S-S-R cleavage site and H16 were proteolytically activated in the absence of TMPRSS2 activity, albeit with reduced efficiency. Moreover, a TMPRSS2-orthologous protease from duck supported activation of H1 to H11, H15, and H16 in MDCK cells. Together, our data demonstrate that in human and murine respiratory cells, TMPRSS2 is the major activating protease of almost all IAV HA subtypes with monobasic cleavage sites. Furthermore, our results suggest that TMPRSS2 supports activation of IAV with a monobasic cleavage site in ducks. IMPORTANCE Human infections with avian influenza A viruses upon exposure to infected birds are frequently reported and have received attention as a potential pandemic threat. Cleavage of the envelope glycoprotein hemagglutinin (HA) by host proteases is a prerequisite for membrane fusion and essential for virus infectivity. In this study, we identify the transmembrane protease TMPRSS2 as the major activating protease of avian influenza virus HAs of subtypes H1 to H11, H14 and H15 in human and murine airway cells. Our data demonstrate that inhibition of TMPRSS2 activity may provide a useful approach for the treatment of human infections with avian influenza viruses that should be considered for pandemic preparedness as well. Additionally, we show that a TMPRSS2-orthologous protease from duck can activate avian influenza virus HAs with a monobasic cleavage site and, thus, represents a potential virus-activating protease in waterfowl, the primary reservoir for influenza A viruses.

Induction of IFN-ß through TLR-3- and RIG-I-Mediated Signaling Pathways in Canine Respiratory Epithelial Cells Infected with H3N2 Canine Influenza Virus.

Canine influenza virus (CIV) induces acute respiratory disease in dogs. In this study, we aimed to determine the signaling pathways leading to the induction of IFN-ß in a canine respiratory epithelial cell line (KU-CBE) infected with the H3N2 subtype of CIV. Small interfering RNAs (siRNAs) specific to pattern recognition receptors (PRRs) and transcription factors were used to block the IFN-ß induction signals in H3N2 CIV-infected KU-CBE cells. Among the PRRs, only the TLR3 and RIG-I expression levels significantly (p < 0.001) increased in CIV-infected cells. Following transfection with siRNA specific to TLR3 (siTLR3) or RIG-I (siRIG-I), the mRNA expression levels of IFN-ß significantly (p < 0.001) decreased, and the protein expression of IFN-ß also decreased in infected cells. In addition, co-transfection with both siTLR3 and siRIG-I significantly reduced IRF3 (p < 0.001) and IFN-ß (p < 0.001) mRNA levels. Moreover, the protein concentration of IFN-ß was significantly (p < 0.01) lower in cells co-transfected with both siTLR3 and siRIG-I than in cells transfected with either siTLR3 or siRIGI alone. Also, the antiviral protein MX1 was only expressed in KU-CBE cells infected with CIV or treated with IFN-ß or IFN-α. Thus, we speculate that IFN-ß further induces MX1 expression, which might suppress CIV replication. Taken together, these data indicate that TLR3 and RIG-I synergistically induce IFN-ß expression via the activation of IRF3, and the produced IFN-ß further induces the production of MX1, which would suppress CIV replication in CIV-infected cells.

Utility of Three Nebulizers in Investigating the Infectivity of Airborne Viruses.

Laboratory-generated bioaerosols are widely used in aerobiology studies of viruses; however, few comparisons of alternative nebulizers exist. We compared aerosol production and virus survival for a Collison nebulizer, vibrating mesh nebulizer (VMN), and hydraulic spray atomizer (HSA). We also measured the dry size distribution of the aerosols produced and calculated the droplet sizes before evaporation and the dry size distribution from normal saline solution. Dry count median diameters of 0.11, 0.22, and 0.30 µm were found for normal saline from the Collison nebulizer, VMN, and HSA, respectively. The volume median diameters were 0.323, 1.70, and 1.30 µm, respectively. The effect of nebulization on the viability of two influenza A viruses (IAVs) (H1N1 and H3N2) and human rhinovirus 16 (HRV-16) was assessed by nebulization into an SKC BioSampler. The HSA had the least impact on surviving fractions (SFs) of H1N1 and H3N2 (89% ± 3% and 94% ± 2%, respectively), followed by the Collison nebulizer (83% ± 1% and 82% ± 2%, respectively). The VMN yielded SFs of 78% ± 2% and 76% ± 2%, respectively. Conversely, for HRV-16, the VMN produced higher SFs (87% ± 8%). Our findings indicate that there were no statistical differences between SFs of the viruses nebulized by these nebulizers. However, VMN produced higher aerosol concentrations within the airborne size range, making it more suitable where high aerosol mass production is required. IMPORTANCE Viral respiratory tract infections cause millions of lost days of work and physician visits globally, accounting for significant morbidity and mortality. Respiratory droplets and droplet nuclei from infected hosts are the potential carriers of such viruses within indoor environments. Laboratory-generated bioaerosols are applied in understanding the transmission and infection of viruses, modeling the physiological aspects of bioaerosol generation in a controlled environment. However, little comparative characterization exists for nebulizers used in infectious disease aerobiology, including Collison nebulizer, vibrating mesh nebulizer, and hydraulic spray atomizer. This study characterized the physical features of aerosols generated by laboratory nebulizers and their performance in producing aerosols at a size relevant to airborne transmission used in infectious disease aerobiology. We also determined the impact of nebulization mechanisms of these nebulizers on the viability of human respiratory viruses, including IAV H1N1, IAV H3N2, and HRV-16.

Intranasal vaccination with influenza HA/GO-PEI nanoparticles provides immune protection against homo- and heterologous strains.

Intranasal (i.n.) immunization is a promising vaccination route for infectious respiratory diseases such as influenza. Recombinant protein vaccines can overcome the safety concerns and long production phase of virus-based influenza vaccines. However, soluble protein vaccines are poorly immunogenic if administered by an i.n. route. Here, we report that polyethyleneimine-functionalized graphene oxide nanoparticles (GP nanoparticles) showed high antigen-loading capacities and superior immunoenhancing properties. Via a facile electrostatic adsorption approach, influenza hemagglutinin (HA) was incorporated into GP nanoparticles and maintained structural integrity and antigenicity. The resulting GP nanoparticles enhanced antigen internalization and promoted inflammatory cytokine production and JAWS II dendritic cell maturation. Compared with soluble HA, GP nanoparticle formulations induced significantly enhanced and cross-reactive immune responses at both systemic sites and mucosal surfaces in mice after i.n. immunization. In the absence of any additional adjuvant, the GP nanoparticle significantly boosted antigen-specific humoral and cellular immune responses, comparable to the acknowledged potent mucosal immunomodulator CpG. The robust immune responses conferred immune protection against challenges by homologous and heterologous viruses. Additionally, the solid self-adjuvant effect of GP nanoparticles may mask the role of CpG when coincorporated. In the absence of currently approved mucosal adjuvants, GP nanoparticles can be developed into potent i.n. influenza vaccines, providing broad protection. With versatility and flexibility, the GP nanoplatform can be easily adapted for constructing mucosal vaccines for different respiratory pathogens.

Time-dependent viral interference between influenza virus and coronavirus in the infection of differentiated porcine airway epithelial cells.

Coronaviruses and influenza viruses are circulating in humans and animals all over the world. Co-infection with these two viruses may aggravate clinical signs. However, the molecular mechanisms of co-infections by these two viruses are incompletely understood. In this study, we applied air-liquid interface (ALI) cultures of well-differentiated porcine tracheal epithelial cells (PTECs) to analyze the co-infection by a swine influenza virus (SIV, H3N2 subtype) and porcine respiratory coronavirus (PRCoV) at different time intervals. Our results revealed that in short-term intervals, prior infection by influenza virus caused complete inhibition of coronavirus infection, while in long-term intervals, some coronavirus replication was detectable. The influenza virus infection resulted in (i) an upregulation of porcine aminopeptidase N, the cellular receptor for PRCoV and (ii) in the induction of an innate immune response which was responsible for the inhibition of PRCoV replication. By contrast, prior infection by coronavirus only caused a slight inhibition of influenza virus replication. Taken together, the timing and the order of virus infection are important determinants in co-infections. This study is the first to show the impact of SIV and PRCoV co- and super-infection on the cellular level. Our results have implications also for human viruses, including potential co-infections by SARS-CoV-2 and seasonal influenza viruses.

Assessment of exposure to influenza A viruses in pigs between weaning and market age.

Influenza A viruses (IAVs) are common causes of respiratory infection in pigs. The objective of this study was to characterize the circulation of IAVs between weaning and market age on the basis of development of antibody response and molecular epidemiology of detected viruses. Two batches of weaned pigs were followed in the nursery and finisher barns with a sample of 81 and 75 pigs. Nasal swabs and blood samples were collected from individual pigs for virological and serological analyses. A H3N2 subtype virus, of cluster IV, was detected in Study 1, with a maximum of 97.9% identity to HA gene of viruses previously isolated in Ontario. In Study 2, a H1N1 subtype virus, of 2009 H1N1 pandemic lineage, was detected, with a maximum of 97.8% identity to HA gene of viruses previously isolated in Ontario. On the basis of HA gene, it was observed that pigs were being detected with the same virus over time. The existence of antibody titers for IAV other than the isolated one confirmed that more than one subtype can circulate in the same population. In Study 1, pigs with higher numbers of IAV detection had lower serological titers for the same virus that was confirmed to circulate in the nursery (P < 0.01). Thorough knowledge of all endemic viral strains is fundamental for development of infection and disease control, particularly in complex production systems. This may include consideration of sampling and testing strategies which could detect circulation of all IAV variants, even if they have low prevalence.

Cellular microRNAs influence replication of H3N2 canine influenza virus in infected cells.

MicroRNAs (miRNAs) are known to play important regulatory roles in host-virus interactions. Avian-origin H3N2 canine influenza virus (CIV) has emerged as the most prevalent subtype among dogs in Asia since 2007. To evaluate the roles of host miRNAs in H3N2 CIV infection, here, miRNA profiles obtained from primary canine bronchiolar epithelial cells (CBECs) and canine alveolar macrophages (CAMCs) were compared between infected and mock-infected cells with the H3N2 CIV JS/10. It was found that the expressions of cfa-miR-125b and cfa-miR-151, which have been reported to be associated with innate immunity and inflammatory response, were significantly decreased in CIV-infected canine primary cells. Bioinformatics prediction indicated that 5' seed regions of the two miRNAs are partially complementary to the mRNAs of nucleoprotein (NP) and non-structural protein 1 (NS1) of JS/10. As determined by virus titration, quantitative real-time PCR (qRT-PCR) and western blotting, overexpression of the two miRNAs inhibited CIV replication in cell culture, while their inhibition facilitated this replication, suggesting that the two miRNAs could act as negative regulators of CIV replication. Our findings support the notion that some cellular miRNAs can influence the outcome of virus infection, which helps to elucidate the resistance of host cells to viral infection and to clarify the pathogenesis of H3N2 CIV.

Cell-Culture Adaptation of H3N2 Influenza Virus Impacts Acid Stability and Reduces Airborne Transmission in Ferret Model.

Airborne transmission of seasonal and pandemic influenza viruses is the reason for their epidemiological success and public health burden in humans. Efficient airborne transmission of the H1N1 influenza virus relies on the receptor specificity and pH of fusion of the surface glycoprotein hemagglutinin (HA). In this study, we examined the role of HA pH of fusion on transmissibility of a cell-culture-adapted H3N2 virus. Mutations in the HA head at positions 78 and 212 of A/Perth/16/2009 (H3N2), which were selected after cell culture adaptation, decreased the acid stability of the virus from pH 5.5 (WT) to pH 5.8 (mutant). In addition, the mutant H3N2 virus replicated to higher titers in cell culture but had reduced airborne transmission in the ferret model. These data demonstrate that, like H1N1 HA, the pH of fusion for H3N2 HA is a determinant of efficient airborne transmission. Surprisingly, noncoding regions of the NA segment can impact the pH of fusion of mutant viruses. Taken together, our data confirm that HA acid stability is an important characteristic of epidemiologically successful human influenza viruses and is influenced by HA/NA balance.

Generation and properties of one strain of H3N2 influenza virus with enhanced replication.

H3N2 canine influenza virus (CIV) has been circulating in many countries since 2008. The epidemic spread of CIV could be a concern for public health because of the close contact between humans and companion animals. In this study, we used Madin-Darby canine kidney (MDCK) cells as a coinfection model of H3N2 CIV and the pandemic (2009) H1N1 influenza virus to investigate the possibility of genetic mutation or recombination. One of the resultant progeny viruses, designated as CP15, was identified with a significantly increased replication ability. For this viral strain all segments exhibit a homology close to 100 % with its parental strain A/Canine/Jiangsu/06/2010 (JS/10), except for two site mutations K156E and R201 K which occur in the receptor-binding sites of hemagglutinin (HA) and antigen binding sites of neuraminidase (NA), respectively. Virus growth in MDCK cells showed that CP15 had a higher virus titer (more than 10 times) than JS/10. Consistent with this, CP15 exhibited extensive tissue tropism and higher viral RNA loads in the spleen, kidney and lung of mice challenged with this virus compared to JS/10. However, body weight loss and lung injure score due to CP15 infection were greatly reduced. Importantly, anti-CP15 serum antibodies could confer a high neutralization activity against JS/10. These findings indicated that the CP15 strain of high replication ability represents a promising candidate to develop an efficient CIV vaccine.