Genomic features associated with sustained mammalian transmission of avian influenza A viruses.

In this work we simulated in amouse model anaturally occurring situation of humans, who overcame an infection with epidemic strains of influenza A, and were subsequently exposed to avian influenza Aviruses (IAV). The antibody response to avian IAV in mice previously infected with human IAV was analyzed. We used two avian IAV (A/Duck/Czechoslovakia/1956 (H4N6) and the attenuated virus rA/Viet Nam/1203-2004 (H5N1)) as well as two human IAV isolates (virus A/Mississippi/1/1985 (H3N2) of medium virulence and A/Puerto Rico/8/1934 (H1N1) of high virulence). Two repeated doses of IAV of H4 or of H5 virus elicited virus-specific neutralizing antibodies in mice. Exposure of animals previously infected with human IAV (of H3 or H1 subtype) to IAV of H4 subtype led to the production of antibodies neutralizing H4 virus in alevel comparable with the level of antibodies against the human IAV used for primary infection. In contrast, no measurable levels of virus-neutralizing (VN) antibodies specific to H5 virus were detected in mice infected with H5 virus following aprevious infection with human IAV. In both cases the secondary infection with avian IAV led to asignificant increase of the titer of VN antibodies specific to the corresponding human virus used for primary infection. Moreover, cross-reactive HA2-specific antibodies were also induced by sequential infection. By virtue of these results we suggest that the differences in the ability of avian IAV to induce specific antibodies inhibiting virus replication after previous infection of mice with human viruses can have an impact on the interspecies transmission and spread of avian IAV in the human population.

The H1N1 pandemic (H1N1pdm09) lineage of influenza A viruses (IAV) emerged in North America in 2009. It spread rapidly due to efficient transmission and the limited immunity in humans, replacing the previous human seasonal H1. Human-to-swine transmission of H1N1pdm09 IAV has since contributed to genetic diversity in pigs. While most were not sustained, approximately 160 spillovers persisted in pigs in the United States for at least 1 year and reassorted with other endemic swine IAVs in most cases. We sought to identify how transmission and reassortment with endemic IAV in swine impact virus traits and zoonotic risk in this study. We conducted a swine pathogenesis and transmission study using four swine H1N1pdm09 viruses derived from different human influenza seasons that had acquired different gene segment combinations after spillovers into swine. To assess antigenic evolution, we compared the selected swine H1N1pdm09 strains against each other and to five human seasonal H1 vaccine strains. Ongoing circulation and reassortment resulted in viruses with variable virulence, shedding, and transmission kinetics. The H1N1pdm09 viruses retained antigenic similarities with the human vaccine strain of the same season of incursion but showed increasing antigenic distances with human seasonal H1N1 vaccine strains from other seasons. Human seasonal H1N1 viruses are capable of replicating and transmitting in swine, and there is potential for these human-to-swine spillovers to reassort with endemic swine IAV. Controlling IAV at the human-swine interface has the benefit of reducing IAV burden in swine and subsequent zoonotic risk.

Influenza A viruses (IAVs) can overcome species barriers by adaptation of the receptor-binding site of the hemagglutinin (HA). To initiate infection, HAs bind to glycan receptors with terminal sialic acids, which are either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc); the latter is mainly found in horses and pigs but not in birds and humans. We investigated the influence of previously identified equine NeuGc-adapting mutations (S128T, I130V, A135E, T189A, and K193R) in avian H7 IAVs in vitro and in vivo. We observed that these mutations negatively affected viral replication in chicken cells but not in duck cells and positively affected replication in horse cells. In vivo, the mutations reduced virus virulence and mortality in chickens. Ducks excreted high viral loads longer than chickens, although they appeared clinically healthy. To elucidate why these viruses infected chickens and ducks despite the absence of NeuGc, we re-evaluated the receptor binding of H7 HAs using glycan microarray and flow cytometry studies. This re-evaluation demonstrated that mutated avian H7 HAs also bound to α2,3-linked NeuAc and sialyl-LewisX, which have an additional fucose moiety in their terminal epitope, explaining why infection of ducks and chickens was possible. Interestingly, the α2,3-linked NeuAc and sialyl-LewisX epitopes were only bound when presented on tri-antennary N-glycans, emphasizing the importance of investigating the fine receptor specificities of IAVs. In conclusion, the binding of NeuGc-adapted H7 IAV to tri-antennary N-glycans enables viral replication and shedding by chickens and ducks, potentially facilitating interspecies transmission of equine-adapted H7 IAVs.IMPORTANCEInfluenza A viruses (IAVs) cause millions of deaths and illnesses in birds and mammals each year. The viral surface protein hemagglutinin initiates infection by binding to host cell terminal sialic acids. Hemagglutinin adaptations affect the binding affinity to these sialic acids and the potential host species targeted. While avian and human IAVs tend to bind to N-acetylneuraminic acid (sialic acid), equine H7 viruses prefer binding to N-glycolylneuraminic acid (NeuGc). To better understand the function of NeuGc-specific adaptations in hemagglutinin and to elucidate interspecies transmission potential NeuGc-adapted viruses, we evaluated the effects of NeuGc-specific mutations in avian H7 viruses in chickens and ducks, important economic hosts and reservoir birds, respectively. We also examined the impact on viral replication and found a binding affinity to tri-antennary N-glycans containing different terminal epitopes. These findings are significant as they contribute to the understanding of the role of receptor binding in avian influenza infection.

Influenza A viruses (IAVs) in swine can cause sporadic infections and pandemic outbreaks among humans, but how avian IAV emerges in swine is still unclear. Unlike domestic swine, feral swine are free ranging and have many opportunities for IAV exposure through contacts with various habitats and animals, including migratory waterfowl, a natural reservoir for IAVs. During the period from 2010 to 2013, 8,239 serum samples were collected from feral swine across 35 U.S. states and tested against 45 contemporary antigenic variants of avian, swine, and human IAVs; of these, 406 (4.9%) samples were IAV antibody positive. Among 294 serum samples selected for antigenic characterization, 271 cross-reacted with ≥1 tested virus, whereas the other 23 did not cross-react with any tested virus. Of the 271 IAV-positive samples, 236 cross-reacted with swine IAVs, 1 with avian IAVs, and 16 with avian and swine IAVs, indicating that feral swine had been exposed to both swine and avian IAVs but predominantly to swine IAVs. Our findings suggest that feral swine could potentially be infected with both avian and swine IAVs, generating novel IAVs by hosting and reassorting IAVs from wild birds and domestic swine and facilitating adaptation of avian IAVs to other hosts, including humans, before their spillover. Continued surveillance to monitor the distribution and antigenic diversities of IAVs in feral swine is necessary to increase our understanding of the natural history of IAVs.IMPORTANCE There are more than 5 million feral swine distributed across at least 35 states in the United States. In contrast to domestic swine, feral swine are free ranging and have unique opportunities for contact with wildlife, livestock, and their habitats. Our serological results indicate that feral swine in the United States have been exposed to influenza A viruses (IAVs) consistent with those found in both domestic swine and wild birds, with the predominant infections consisting of swine-adapted IAVs. Our findings suggest that feral swine have been infected with IAVs at low levels and could serve as hosts for the generation of novel IAVs at the interface of feral swine, wild birds, domestic swine, and humans.

Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their in vitro genetic compatibility and in vivo pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes in vitro, and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs.IMPORTANCE IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian and mammalian IAVs and represent a human health concern due to their susceptibility to infection, large global population, and close physical contact with humans. Our results suggest that humans are likely to have limited preexisting immunity to CIV-H3N2 and that CIV-H3N2 × pdmH1N1 reassortants have moderate genetic compatibility and are transmissible by direct contact in ferrets. Our study contributes to the increasing evidence that surveillance of the canine population for IAVs is an important component of pandemic preparedness.

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.

High throughput proteomics and transcriptomics has provided a platform to further understand viral – host interaction. This provides a window into the host proteome and transcriptome with and without infection. This leads to identification of potential biomarkers, understanding IAV pathogenesis and also drawing a comparison of how hosts respond to viral infection. This thesis used two independent high throughput approaches to explore the proteome and transcriptome of samples from hosts (in vitro and in vivo) infected with influenza A virus (IAV) compared to samples from hosts (in vitro and in vivo) non-infected with IAV. The independent high throughput approaches used were; proteomics on a Q-Exactive platform and transcriptomics on a MinION sequencer. These approaches were used to further understand IAV infection in these different hosts and secondly to explore and search further for a potential biomarker for the diagnosis of IAV and potential drug targets. To our knowledge, this is the first study that has used high throughput approaches to analyses samples from different hosts. This allowed for comparison across hosts but also provides vast amounts of data that are reliable and consistent. A549 cells that were mock-infected and IAV- infected were subjected to the Q-Exactive platform and MinION sequencing. This provided insight into the in vitro host-viral interaction on a cellular level. For the first time, showed the potential of MinION sequencing as a method for understanding the viral-host interaction of IAV infected cells and identifying potential biomarkers. This highlighted transcripts such as NUP54, RBM42, HPGD, GCLC, ANPEP, AKAP13, RACGAP1, CREB1, MAN2B1 and PRKCI. These transcripts were identified by bioinformatic analysis as host factors that play a crucial role in replication of IAV in the host. The corresponding proteins to these transcripts were also identified by proteomics. To better understand IAV in hosts, in vivo, Non-human primates (NHP) were infected with IAV and the broncho-alveolar fluid (BALF) was collected and compared to BALF from naive NHP. After analysis on the Q-Exactive platform, the results obtained drew parallels on a cellular level to that observed in vitro models. Proteins such as; DDX58, EIF3A, HSP90AA1, MAPK1, MX1 and STAT1 involved in the “replication of IAV” were highlighted. In addition to the cellular changes, the NHP studies provided insight into an immune response similar to that observed in humans following IAV infection. This provided an added dimension in understanding IAV infection. Finally, nasopharyngeal aspirates (NAs) from humans IAV- infected and IAV non-infected from three different cohorts (Alder Hey Children’s hospital (AHCH), Liverpool, Great Ormond Street Hospital (GOSH), London and Institute Pasteur Dakar (IPD)) were analysed on the Q-Exactive platform. This provided a full circle loop to compare if the changes observed in vitro model – A549 cells and in vivo model-NHP were relatable back to humans. Proteins identified in vitro and in vivo studies were concordant with proteins identified in the human NAs. These proteins include; COPA, STAT1, TUBB and HSPB1. Additionally, three proteins were identified in human NAs across all three cohorts; BPIFA1/SPLUNC1, Lactotransferrin and Fibrinogen A, B and G. These proteins play crucial roles in elucidating IAV infection in the host. This study presents the first time these proteins have been highlighted using label-free Mass spectrometry in human NAs across three cohorts from different geographical locations. This thesis illustrates how proteomic analysis of IAV-infected samples compared to non-infected samples can be used to identify markers that may serve as potential diagnostic indicators for IAV infection.

H3 influenza A viruses (IAV) have been shown to frequently cross the species barrier which can be an important factor in sustained transmission and spread. Machine learning methods have been widely explored for host prediction of IAV using genomic data; however, this is often done using data from only one of the eight IAV segments or by using all available IAV data to predict broad categories of hosts. The objective of this study was to combine machine learning algorithms with H3 IAV sequence data from all eight segments to train predictive machine learning models for distinct host prediction and validate model performance. Models were trained on both k-mers and amino acid properties alongside machine learning algorithms that included random forest and XGBoost for each of the eight IAV genome segments. Models were then validated on a test dataset through analytics of model class predicted probabilities and subsequently used to investigate between-species transmission patterns within case studies including canine H3N8, swine H3N2 2010.2, and duck H3 sequences. Models demonstrated strong performance in host prediction across all eight segments on the test dataset, with overall accuracies and κ (kappa) values ranging from 0.995-0.997, 0.984-0.990, respectively. Misclassified test dataset sequences with high predicted probabilities (> 90%) were validated using available literature and were identified to be frequently associated with between-species transmission events. Between-species transmission patterns within case study model class predicted probabilities were also identified to be consistent with the literature in cases of both correct and incorrect classification. These models allow for rapid and accurate host prediction of H3 IAV datasets from any of the eight IAV segments and provide a solid framework that allows for identification of variants with higher than typical between-species transmission potential. However, results obtained on selected case studies suggest further improvements of the training and validation processes should be considered.

BackgroundH3 influenza A viruses (IAV) have been shown to frequently cross the species barrier which can be an important factor in sustained transmission and spread. Machine learning methods have been widely explored for host prediction of IAV using genomic data; however, this is often done using data from only one of the eight IAV segments or by using all available IAV data to predict broad categories of hosts.ObjectiveThe objective of this study was to combine machine learning algorithms with H3 IAV sequence data from all eight segments to train predictive machine learning models for distinct host prediction and validate model performance.MethodsModels were trained on both k-mers and amino acid properties alongside machine learning algorithms that included random forest and XGBoost for each of the eight IAV genome segments. Models were then validated on a test dataset through analytics of model class predicted probabilities and subsequently used to investigate between-species transmission patterns within case studies including canine H3N8, swine H3N2 2010.2, and duck H3 sequences.ResultsModels demonstrated strong performance in host prediction across all eight segments on the test dataset, with overall accuracies and κ (kappa) values ranging from 0.995–0.997, 0.984–0.990, respectively. Misclassified test dataset sequences with high predicted probabilities (> 90%) were validated using available literature and were identified to be frequently associated with between-species transmission events. Between-species transmission patterns within case study model class predicted probabilities were also identified to be consistent with the literature in cases of both correct and incorrect classification.ConclusionsThese models allow for rapid and accurate host prediction of H3 IAV datasets from any of the eight IAV segments and provide a solid framework that allows for identification of variants with higher than typical between-species transmission potential. However, results obtained on selected case studies suggest further improvements of the training and validation processes should be considered.

Avian Influenza is caused by Influenza A virus which is a member of Orthomyxoviridae family. Influenza A virus is enveloped single stranded RNA with eight-segmented, negative polarity and filament or oval form, 50 – 120 by 200 – 300 nm diameters. Influenza A viruses have been found to infect birds, human, pig, horse and sometimes in the other mammalian such as seal and whale. The viruses are divided into different subtypes based on the antigenic protein which covers the virus surface i.e. Haemaglutinin (HA) and Neuraminidase (NA). In addition, the nomenclature of subtype virus is based on HA and NA i.e HxNx, for example H5N1, H9N2 and the others. According to pathogenic, it could be divided into two distinct groups, they are Highly Pathogenic Avian Influenza (HPAI) and Low Pathogenic Avian Influenza (LPAI). The Avian Influenza viruses have been continuously occurred and spread out in some continents such us America, Europe, Africa and Asian countries. The outbreak of Avian Influenza caused high mortality on birds and it has been reported that in human case Avian Influenza subtype H5N1 virus has caused several deaths. To anticipate this condition, an effort to prevent the transmission of Avian Influenza is needed. These strategic attempts include biosecurity, depopulation, vaccination, control of virus movement, monitoring and evaluation. Laboratory diagnostic plays an important role for successful prevention, control and eradication programs of Avian Influenza. Recently, there are two diagnostic methods for Avian Influenza. They are conventional (virological diagnosis) and molecular methods. The conventional method is usually used for initial diagnostic of Avian Influenza. The conventional method takes more time and more costly, whereas the molecular method is more effective than conventional method. Based on the available diagnostic technique, basically diagnostic of Avian Influenza is done by serology test, isolation and identification as well as pathogenicity test. Key words: Avian Influenza, Characterisation, Identification, Laboratory Diagnostic

Objective To monitor the environmental contamination with avian influenza virus (AIV) in Henan Province. Methods Environmental samples were collected every month from seven monitoring sites in Henan from 2013 to 2017. Real-time RT-PCR method was performed to detect the nucleic acid of influenza A (Flu A), H5, H7 and H9 viruses in poultry environmental samples. Results A total of 2 538 environmental samples were collected and 202 (7.96%) of them were positive for Flu A nucleic acid, including 16 positive for H5 (0.63%), eight positive for H7 (0.32%) and 161 positive for H9 (6.34%). The detection rate of Flu A increased dramatically from 2013 to 2017 except for a small fluctuation in 2015. However, H7 subtype AIV was detected only in 2015 and 2017. The highest detection rate of AIV was in February, followed by that in January. Among different environments, the highest detection rate of Flu A was in live poultry market, which was 13.69%, followed by that in poultry slaughtering plant (2.58%) and poultry farm (0.58%). The detection rates of Flu A in swab samples of poultry plucker and cutting board, stool specimens and poultry drinking water were 28.57%, 13.76%, 5.70% and 5.26%, respectively. Conclusion Contamination of H5/H7/H9 AIV did exist in poultry environment in Henan and was getting worse. Increasingly diversified sources and sale channels were the main causes of serious contamination of AIV. In order to effectively prevent and control human infection with AIV, live poultry in areas where human infection with AIV was confirmed should be blocked and banned to be sold to others areas. Key words: Avian influenza; H5/H7/H9 subtypes; Surveillance

Viruses and hosts have coevolved for millions of years, leading to the development of complex host–pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule–mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in a turkey in Massachusetts in 1965. Since 1997, H6 viruses with different neuraminidase (NA) subtypes have been detected frequently in the live poultry markets of southern China. Although sequence information has been gathered over the last few years, the H6 viruses have not been fully biologically characterized. To investigate the potential risk posed by H6 viruses to humans, here we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China from 2008 to 2011. Among the 257 H6 strains tested, 87 viruses recognized the human type receptor. Genome sequence analysis of 38 representative H6 viruses revealed 30 different genotypes, indicating that these viruses are actively circulating and reassorting in nature. Thirty-seven of 38 viruses tested in mice replicated efficiently in the lungs and some caused mild disease; none, however, were lethal. We also tested the direct contact transmission of 10 H6 viruses in guinea pigs and found that 5 viruses did not transmit to the contact animals, 3 viruses transmitted to one of the three contact animals, and 2 viruses transmitted to all three contact animals. Our study demonstrates that the H6 avian influenza viruses pose a clear threat to human health and emphasizes the need for continued surveillance and evaluation of the H6 influenza viruses circulating in nature. Avian influenza viruses continue to present a challenge to human health. Research and pandemic preparedness have largely focused on the H5 and H7 subtype influenza viruses in recent years. Influenza viruses of the H6 subtype have been isolated from wild and domestic aquatic and terrestrial avian species throughout the world since their first detection in the United States in 1965. Since 1997, H6 viruses have been detected frequently in the live poultry markets of southern China; however, the biological characterization of these viruses is very limited. Here, we assessed the receptor-binding preference, replication, and transmissibility in mammals of a series of H6 viruses isolated from live poultry markets in southern China and found that 34% of the viruses are able to bind human type receptors and that some of them are able to transmit efficiently to contact animals. Our study demonstrates that the H6 viruses pose a clear threat to human health.

To study the epidemic tendency of emerging influenza A (H1N1) in mainland China, and to explore the different patterns of spread on the disease under the following contexts: (1) To stop the temperature screening program at the border areas of the country; (2) To stop measures of prevention and control on those identified cases and their close contacts; (3) To strengthen programs for the foreign immigrants on 'home quarantine'. Under relevant parameters and information on the transmission link from different reference data, the patterns of influenza spread were simulated by Monte Carlo method. The temperature screening on border could inhibit the transmission of influenza A (H1N1) to some extent, so that after 3 months the cumulative number of cases will be reduced by 21.5% (1718 cases) and transmission speed of influenza A (H1N1) in mainland China will be delayed by about 4 days. Furthermore, taking positive measures of prevention and control could efficiently slow down the epidemic, so that after 3 months the cumulative number of cases will be reduced by 93.4% (about 90 thousand cases) and it would be delayed by about 15 days if influenza A (H1N1) spreads to the whole country. In addition, if the immigrants were able to practise quarantine measures consciously by themselves at home the effect of prevention and control against influenza A (H1N1) would be more significant. If 30%, 60% and 90% of immigrants would take quarantine measures home consciously, after 3 months the cumulative number of cases will be reduced by about 15% (about 940 cases), 34% (about 2230 cases) and 64% (about 4180 cases), respectively. Also, influenza A (H1N1) spreads to the whole country will be delayed by about 4 days, 10 days and 25 days, respectively. It is difficult to curb fully the development of the epidemic by taking existing control measures, and influenza A (H1N1) may spread to almost all provinces after about 3 months. The effects of existing prevention and control measures were objectively assessed and the results showed the necessity and effectiveness of these measures against the transmission of influenza A (H1N1), in the mainland of China.

Influenza A virus (IAV) is present in most swine-producing countries causing production losses and concerns on public health. In Brazil, influenza is endemic in pig herds, and a great genetic diversity has been described in swine IAVs due to multiple introductions of pre-2009 human-seasonal IAVs followed by reassortment events with 2009 pandemic H1N1 (H1N1pdm) virus. Here, we compile 14 years of IAV monitoring data and describe the subtypes and major lineages of H1 and H3 viruses co-circulating in Brazilian pigs. Using multiplex RT-qPCR and sequencing, we identified H1N1pdm as the most frequently detected virus, accounting for 41.3% of the subtyped samples (165/399), followed by H1huN2 (108/399), H3N2 (77/399), and H1N1hu (9/399). The three dominant subtypes were detected co-circulating annually and consistently in seven of the nine states sampled, as well as among pigs at different production phases. Other reassortants were found sporadically and included H1pdmN2 (22/399) and H1huN1pdm (4/399). The high diversity observed indicates that IAVs from distinct lineages are widely disseminated across the country. These findings strongly suggest substantial movement of pigs between regions and states, which may have implications for vaccine design, disease control, and updating of diagnostic tests. Continuous efforts to monitor IAV are crucial to better understand their ecology and to generate relevant data for pandemic preparedness.