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Point-of-care diagnostic technologies are becoming more widely available for production species. Here, we describe the application of reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect the matrix (M) gene of influenza A virus in swine (IAV-S). M-specific LAMP primers were designed based on M gene sequences from IAV-S isolated in the USA between 2017 and 2020. The LAMP assay was incubated at 65 °C for 30 min, with the fluorescent signal read every 20 s. The assay’s limit of detection (LOD) was 20 M gene copies for direct LAMP of the matrix gene standard, and 100 M gene copies when using spiked extraction kits. The LOD was 1000 M genes when using cell culture samples. Detection in clinical samples showed a sensitivity of 94.3% and a specificity of 94.9%. These results show that the influenza M gene RT-LAMP assay can detect the presence of IAV in research laboratory conditions. With the appropriate fluorescent reader and heat block, the assay could be quickly validated as a low-cost, rapid, IAV-S screening tool for use on farms or in clinical diagnostic labs.

Turkish people are facing several problems because of the novel coronavirus (COVID-19), as the pandemic has brought about drastic changes to their daily routines. This study mainly investigates the impact of this pandemic on the daily routines of Turkish. It also unveils how COVID-19 affects the air environment. The adopted methods for data collection are based on open-ended questions and Facebook interviews as per recommended by QSR-International (2012). The sample of this study comprises of Turkish students as well as professional workers. The findings of the research show that there are eighteen different results of COVID-19 that have been identified according to the Turkish people’s daily routines. Results reveal that increasing unemployment, decrease in air contamination, high stress and depression, a slowdown in the economic growth, and the tourism industry are profoundly affected due to the COVID-19 in Turkey. Furthermore, on the one hand, the consequences of the pandemic are segregated into social problems and psychological issues in daily routines. On the other hand, they have shown a positive impact on the air environment. This study concludes that, amid the COVID-19 pandemic, the lives of the people in Turkey are subject to deterioration, while the air environment of Turkey is gradually improving. Electronic supplementary material The online version of this article (10.1007/s11869-020-00943-2) contains supplementary material, which is available to authorized users.

This study aimed to determine the frequency and distribution of infectious diseases diagnosed through necropsy examination and histopathological analysis in growing/finishing pigs along 12 years (2005-2016) in Southern Brazil. We evaluated 1906 anatomopathological exams of pigs at growing/finishing phases, of which the infectious diseases corresponded to 75.6% of the cases (1,441/1,906). Porcine circovirus type 2 (PCV2) infections were the most frequent, accounting for 51.3% of the cases (739/1,441) with a higher frequency from 2005 to 2007, characterizing an epidemic distribution, with a gradual decline after 2008. Infectious diseases affecting the respiratory system were the second major cause with 30.1% of the cases. Among these, necrotizing bronchiolitis caused by swine Influenza (15.1%, 218/1,441) and bacterial pneumonia (15%, 216/1,441) were the main conditions. Influenza was mostly diagnosed from 2010 to 2013, accounting for 43.1% (167/387) of the cases. After this period, both respiratory infectious diseases were endemic. Digestive system infectious diseases accounted for 10.5% of the diagnoses (151/1,441), with the following main conditions: Salmonella spp. enterocolitis (43.7%, 66/151), Lawsonia spp. proliferative enteropathy (41.7%, 63/151), and Brachyspira spp. colitis (14.6%, 22/151). The latter had a higher incidence from 2012 to 2014 with all cases detected in this period. Polyserositis and bacterial meningitis represented, respectively, 5.8% (84/1,441) and 2.3% (33/1,441) of the cases diagnosed, with a constant endemic character. RESUMO: O objetivo deste estudo consistiu em determinar a frequência e a distribuição das doenças infecciosas diagnosticadas através de exame de necropsia e análise histopatológica em suínos nas fases de crescimento/terminação ao longo de 12 anos (2005-2016) no sul do Brasil. Foram avaliados 1906 laudos anatomopatológicos de suínos nas fases de crescimento/terminação, dos quais as doenças infecciosas corresponderam a 75,6% (1441/1906) do total. As infecções por circovírus suíno tipo 2 (PCV2) foram as mais frequentes, contabilizando 51,3% (739/1441) dos casos, com uma alta frequência de 2005 a 2007 caracterizando uma distribuição epidêmica neste período, e um declínio gradual após o ano de 2008. A segunda principal causa incluiu as doenças infecciosas que afetam o sistema respiratório (30,1% dos casos). Dentre essas, destacaram-se a influenza suína (15,1%; 218/1441) e pneumonias bacterianas (15%; 216/1441). O diagnóstico de influenza apresentou uma frequência elevada de 2010 a 2013, totalizando 43,1% (167/387) dos casos. Após este período, ambas doenças infecciosas respiratórias exibiram caráter endêmico. As doenças infecciosas do sistema digestório totalizaram 10,5% (151/1441) dos diagnósticos, com as seguintes principais condições: enterocolite por Salmonella spp. (43,7%; 66/151), enteropatia proliferativa por Lawsonia spp. (41,7%; 63/151) e colite por Brachyspira spp. (14,6%; 22/151). A colite por Brachyspira spp. apresentou uma alta incidência de 2012 a 2014 com todos os casos detectados no período. As polisserosites e meningites bacterianas representaram 5,8% (84/1441) e 2,3% (33/1441) dos casos diagnosticados, respectivamente, com um caráter endêmico constante.

Zoonotic infections of humans with influenza A viruses (IAVs) from animal reservoirs can result in severe disease in individuals and, in rare cases, lead to pandemic outbreaks; this is exemplified by numerous cases of human infection with avian IAVs (AIVs) and the 2009 swine influenza pandemic. In fact, zoonotic transmissions are strongly facilitated by manmade reservoirs that were created through the intensification and industrialization of livestock farming. This can be witnessed by the repeated introduction of IAVs from natural reservoirs of aquatic wild bird metapopulations into swine and poultry, and the accompanied emergence of partially- or fully-adapted human pathogenic viruses. On the other side, human adapted IAV have been (and still are) introduced into livestock by reverse zoonotic transmission. This link to manmade reservoirs was also observed before the 20th century, when horses seemed to have been an important reservoir for IAVs but lost relevance when the populations declined due to increasing industrialization. Therefore, to reduce zoonotic events, it is important to control the spread of IAV within these animal reservoirs, for example with efficient vaccination strategies, but also to critically surveil the different manmade reservoirs to evaluate the emergence of new IAV strains with pandemic potential.

Influenza A virus of swine (IAV-S) is an economically important swine pathogen. The IAV-S hemagglutinin (HA) surface protein is the main target for vaccine development. In this study, we evaluated the feasibility of using the recombinant tri-segmented Pichinde virus (rPICV) as a viral vector to deliver HA antigen to protect pigs against IAV-S challenge. Four groups of weaned pigs (T01–T04) were included in the study. T01 was injected with PBS to serve as a non-vaccinated control. T02 was inoculated with rPICV expressing green fluorescence protein (rPICV-GFP). T03 was vaccinated with rPICV expressing the HA antigen of the IAV-S H3N2 strain (rPICV-H3). T04 was vaccinated with the recombinant HA protein antigen of the same H3N2 strain. Pigs were vaccinated twice at day 0 and day 21 and challenged at day 43 by intra-tracheal inoculation with the homologous H3N2 IAV-S strain. After vaccination, all pigs in T03 and T04 groups were seroconverted and exhibited high titers of plasma neutralizing antibodies. After challenge, high levels of IAV-S RNA were detected in the nasal swabs and bronchioalveolar lavage fluid of pigs in T01 and T02 but not in the T03 and T04 groups. Similarly, lung lesions were observed in T01 and T02, but not in the T03 and T04 groups. No significant difference in terms of protection was observed between the T03 and T04 group. Collectively, our results demonstrate that the rPICV-H3 vectored vaccine elicited protective immunity against IAV-S challenge. This study shows that rPICV is a promising viral vector for the development of vaccines against IAV-S.

Swine influenza A virus (swIAV), which plays a major role in the porcine respiratory disease complex (PRDC), is eliminated from the respiratory tract within 7–9 days after infection. Therefore, diagnosis is complicated in endemically infected swine herds presenting no obvious clinical signs. This study aimed to investigate the right time point for sampling to detect swIAV. A cross-sectional study was performed in 131 farms from 12 European countries. The sampling protocol included suckling piglets, weaners, and nursery pigs. In each age group, 10 nasal swabs were collected and further examined in pools of 5 for swIAV by Matrix rRT-PCR, followed by a multiplex RT-PCR to determine the influenza subtype. SwIAV was detected in 284 (37.9%) of the samples and on 103 (78.6%) farms. Despite the highest number of animals with clinical signs being found in the nursery, the weaners were significantly more often virus-positive compared to nursery pigs (p = 0.048). Overall, the swIAV detection rate did not significantly differ between diseased or non-diseased suckling and nursery piglets, respectively; however, diseased weaners had significantly more positive pools than the non-diseased animals. Interestingly, in 9 farms, different subtypes were detected in different age groups. Our findings indicate that to detect all circulating swIAV subtypes on a farm, different age groups should be sampled. Additionally, the sampling strategy should also aim to include non-diseased animals, especially in the suckling period.

Pigs have long been recognized as “mixing vessels” in which new viruses are formed by reassortment involving various influenza virus lineages (avian, animal, human). However, surveillance of swine influenza viruses only gained real significance after the 2009 pandemic. A fundamentally important point is the fact that there is still no regular surveillance of swine flu in Russia, and the role of swine viruses is underestimated since, as a rule, they do not cause serious disease in animals. Since the pig population in Russia is large, it is obvious that the lack of monitoring and insufficient study of swine influenza evolution constitutes a gap in animal influenza surveillance, not only for Russia, but globally. A 6 year joint effort enabled identification of SIV subtypes that circulate in the pig population of Russia’s European geographic region. The swine influenza viruses isolated were antigenically and genetically diverse. Some were similar to human influenza viruses of A(H1N1)pdm09 and A(H3N2) subtype, while others were reassortant A(H1pdm09N2) and A(H1avN2) and were antigenically distinct from human H1N1 and H1N1pdm09 strains. Analysis of swine serum samples collected throughout the seasons showed that the number of sera positive for influenza viruses has increased in recent years. This indicates that swine populations are highly susceptible to infection with human influenza viruses. It also stresses the need for regular SIV surveillance, monitoring of viral evolution, and strengthening of pandemic preparedness.

The Thai Journal of Veterinary Medicine, 51, 1, 55

BackgroundThe objective was to document whether and to what extent there was an association between socioeconomic status (SES) and disease outcomes in the last five influenza pandemics.Methods/Principle FindingsThe review included studies published in English, Danish, Norwegian and Swedish. Records were identified through systematic literature searches in six databases. Results are summarized narratively and using meta-analytic strategies. We found studies only for the 1918 and 2009 pandemics. Of 14 studies on the 2009 pandemic including data on both medical and social risk factors, after controlling for medical risk factors 8 demonstrated independent impact of SES. A random effect analysis of 46 estimates from 35 studies found a pooled mean odds ratio of 1.4 (95% CI: 1.2 – 1.7), comparing the lowest to the highest SES, but with substantial effect heterogeneity across studies –reflecting differences in outcome measures and definitions of case and control samples. Analyses by pandemic period (1918 or 2009) and by level of SES measure (individual or ecological) indicate no differences along these dimensions. Studies using healthy controls tend to find low SES associated with worse influenza outcome, and studies using infected controls find low SES associated with more severe outcomes. Studies comparing severe outcomes (ICU or death) to hospital admissions are few but indicate no clear association. Studies with more unusual comparisons (e.g., pandemic vs seasonal influenza, seasonal influenza vs other patient groups) report no or negative associations.Conclusions/SignificanceResults show that social risk factors help to explain pandemic outcomes in 1918 and in 2009 although the mechanisms and types of social vulnerabilities leading to disparities in outcomes may differ over time. Studies of the 2009 pandemic also showed that social vulnerability could not always be explained by medical risk factors. To prepare for future pandemics, we must consider social along with medical vulnerability.The protocol for this study has been registered in PROSPERO (ref. no 87922) and has been published (1).

The evolution of circulating viruses is shaped by their need to evade antibody response, which mainly targets the viral spike. Because of the high density of spikes on the viral surface, not all antigenic sites are targeted equally by antibodies. We offer here a geometry-based approach to predict and rank the probability of surface residues of SARS spike (S protein) and influenza H1N1 spike (hemagglutinin) to acquire antibody-escaping mutations utilizing in-silico models of viral structure. We used coarse-grained MD simulations to estimate the on-rate (targeting) of an antibody model to surface residues of the spike protein. Analyzing publicly available sequences, we found that spike surface sequence diversity of the pre-pandemic seasonal influenza H1N1 and the sarbecovirus subgenus highly correlates with our model prediction of antibody targeting. In particular, we identified an antibody-targeting gradient, which matches a mutability gradient along the main axis of the spike. This identifies the role of viral surface geometry in shaping the evolution of circulating viruses. For the 2009 H1N1 and SARS-CoV-2 pandemics, a mutability gradient along the main axis of the spike was not observed. Our model further allowed us to identify key residues of the SARS-CoV-2 spike at which antibody escape mutations have now occurred. Therefore, it can inform of the likely functional role of observed mutations and predict at which residues antibody-escaping mutation might arise., Author summary The immune system responds to viruses by making neutralizing antibodies to regions of the viral spike protein, which mutates to escape. To inform vaccine design and understand how the fitness landscape of the viral spike changes over time, it is necessary to identify and quantify the factors directing its evolution. Based on the 3D structure of the viral surface and spike as captured with Cryo-EM and crystallography, we aimed to create a coarse-grained model for the effect of antibodies in forcing surface residues of the spike to mutate. We found that for pre-pandemic influenza (hemagglutinin) and the corona sarbecovirus subgenus (S protein), the location of a residue on the spike protein, which modulates its accessibility to antibodies, highly correlates with its propensity to mutate. Hence, a mechanistic approach can be used to identify aspects of viral spike sequence diversity related to antibody escape.

This study evaluated the genetic and antigenic evolution of swine influenza A viruses (swIAV) of the two main enzootic H1 lineages, i.e., HA-1C (H1av) and -1B (H1hu), circulating in France between 2000 and 2018. SwIAV RNAs extracted from 1220 swine nasal swabs were hemagglutinin/neuraminidase (HA/NA) subtyped by RT-qPCRs, and 293 virus isolates were sequenced. In addition, 146 H1avNy and 105 H1huNy strains were submitted to hemagglutination inhibition tests. H1avN1 (66.5%) and H1huN2 (25.4%) subtypes were predominant. Most H1 strains belonged to HA-1C.2.1 or -1B.1.2.3 clades, but HA-1C.2, -1C.2.2, -1C.2.3, -1B.1.1, and -1B.1.2.1 clades were also detected sporadically. Within HA-1B.1.2.3 clade, a group of strains named &ldquo, &Delta, 146-147&rdquo, harbored several amino acid mutations and a double deletion in HA, that led to a marked antigenic drift. Phylogenetic analyses revealed that internal segments belonged mainly to the &ldquo, Eurasian avian-like lineage&rdquo, with two distinct genogroups for the M segment. In total, 17 distinct genotypes were identified within the study period. Reassortments of H1av/H1hu strains with H1N1pdm virus were rarely evidenced until 2018. Analysis of amino acid sequences predicted a variability in length of PB1-F2 and PA-X proteins and identified the appearance of several mutations in PB1, PB1-F2, PA, NP and NS1 proteins that could be linked to virulence, while markers for antiviral resistance were identified in N1 and N2. Altogether, diversity and evolution of swIAV recall the importance of disrupting the spreading of swIAV within and between pig herds, as well as IAV inter-species transmissions.

Accurate and reliable short-term forecasts of influenza-like illness (ILI) visit volumes at emergency departments can improve staffing and resource allocation decisions within hospitals. In this paper, we developed a stacked ensemble model that averages the predictions from various competing methodologies in the current frontier for ILI-related forecasts. We also constructed a back-of-the-envelope prediction interval for the stacked ensemble, which provides a conservative characterization of the uncertainty in the stacked ensemble predictions. We assessed the accuracy and reliability of our model with 1 to 4 weeks ahead forecast targets using real-time hospital-level data on weekly ILI visit volumes during the 2012-2018 flu seasons in the Alberta Children’s Hospital, located in Calgary, Alberta, Canada. Our results suggest the forecasting performance of the stacked ensemble meets or exceeds the performance of the individual models over all forecast targets.

The household secondary attack risk (SAR), often called the secondary attack rate or secondary infection risk, is the probability of infectious contact from an infectious household member A to a given household member B, where we define infectious contact to be a contact sufficient to infect B if he or she is susceptible. Estimation of the SAR is an important part of understanding and controlling the transmission of infectious diseases. In practice, it is most often estimated using binomial models such as logistic regression, which implicitly attribute all secondary infections in a household to the primary case. In the simplest case, the number of secondary infections in a household with m susceptibles and a single primary case is modeled as a binomial(m, p) random variable where p is the SAR. Although it has long been understood that transmission within households is not binomial, it is thought that multiple generations of transmission can be neglected safely when p is small. We use probability generating functions and simulations to show that this is a mistake. The proportion of susceptible household members infected can be substantially larger than the SAR even when p is small. As a result, binomial estimates of the SAR are biased upward and their confidence intervals have poor coverage probabilities even if adjusted for clustering. Accurate point and interval estimates of the SAR can be obtained using longitudinal chain binomial models or pairwise survival analysis, which account for multiple generations of transmission within households, the ongoing risk of infection from outside the household, and incomplete follow-up. We illustrate the practical implications of these results in an analysis of household surveillance data collected by the Los Angeles County Department of Public Health during the 2009 influenza A (H1N1) pandemic., Author summary The household secondary attack risk (SAR), often called the secondary attack rate or secondary infection risk, is the probability of infectious contact from an infectious household member A to a given household member B, where we define infectious contact to be a contact sufficient to infect B if he or she is susceptible. The most common statistical models used to estimate the SAR are binomial models such as logistic regression, which implicitly assume that all secondary infections in a household are infected by the primary case. Here, we use analytical calculations and simulations to show that estimation of the SAR must account for multiple generations of transmission within households. As an example, we show that binomial models and statistical models that account for multiple generations of within-household transmission reach different conclusions about the household SAR for 2009 influenza A (H1N1) in Los Angeles County, with the latter models fitting the data better. In an epidemic, accurate estimation of the SAR allows rigorous evaluation of the effectiveness of public health interventions such as social distancing, prophylaxis or treatment, and vaccination.

Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018., While working overnight at a swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, Nanopore sequenced 13 IAV genomes from samples we collected, and predicted in real time that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current prepandemic candidate vaccine viruses (CVVs). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 h after unpacking the mobile lab. Exhibition swine are a known source for zoonotic transmission of IAV to humans and pose a potential pandemic risk. Genomic analyses of IAV in swine are critical to understanding this risk, the types of viruses circulating in swine, and whether current vaccines developed for use in humans would be predicted to provide immune protection. Nanopore sequencing technology has enabled genome sequencing in the field at the source of viral outbreaks or at the bedside or pen-side of infected humans and animals. The acquired data, however, have not yet demonstrated real-time, actionable public health responses. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes, A(H1N1), A(H3N2), and A(H1N2). Analysis of the hemagglutinin (HA) sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 of these viruses and the most closely related CVV. As an exercise in pandemic preparedness, all sequences were emailed to CDC collaborators who initiated the development of a synthetically derived CVV. IMPORTANCE Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018.

11 p.-5 fig.-3 tab., Despite the role of pigs as a source of new Influenza A Virus viruses (IAV) potentiallycapable of initiating human pandemics, immune responses toswine influenza virus(SwIV) in pigs are not fully understood. Several SwIV epitopes presented by swine MHC(SLA) class I have been identified using different approaches either in outbred pigs orin Babraham large white inbred pigs, which are 85% identicalby genome wide SNPanalysis. On the other hand, some class II SLA epitopes were recently described inoutbred pigs. In this work, Babraham large white inbred pigswere selected to identifySLA II epitopes from SwIV H1N1. PBMCs were screened for recognition of overlappingpeptides covering the NP and M1 proteins from heterologous IAV H1N1 in IFNγELISPOT.A novel SLA class II restricted epitope was identified in NP from swine H1N1. Thisconserved novel epitope could be the base for further vaccine approaches against H1N1in pigs., The research leading to these results has received funding from the European Community’s Seventh Framework Program (FP7,2007-2013), the Research Infrastructures Action under grant FP7-228393 (a NADIR project); from the project AGL2010-22200-C02-01 from the Spanish Ministry of Science and Innovation; the UK’s BBSRC grant BBS/E/I/00002014.

Influenza is an acute respiratory seasonal disease that affects millions of people worldwide and causes thousands of deaths in Europe alone. Estimating in a fast and reliable way the impact of an illness on a given country is essential to plan and organize effective countermeasures, which is now possible by leveraging unconventional data sources like web searches and visits. In this study, we show the feasibility of exploiting machine learning models and information about Wikipedia’s page views of a selected group of articles to obtain accurate estimates of influenza-like illnesses incidence in four European countries: Italy, Germany, Belgium, and the Netherlands. We propose a novel language-agnostic method, based on two algorithms, Personalized PageRank and CycleRank, to automatically select the most relevant Wikipedia pages to be monitored without the need for expert supervision. We then show how our model can reach state-of-the-art results by comparing it with previous solutions.

Influenza D virus (IDV) has been identified in domestic cattle, swine, camelid, and small ruminant populations across North America, Europe, Asia, South America, and Africa. Our study investigated seroprevalence and transmissibility of IDV in feral swine. During 2012-2013, we evaluated feral swine populations in 4 US states; of 256 swine tested, 57 (19.1%) were IDV seropositive. Among 96 archived influenza A virus-seropositive feral swine samples collected from 16 US states during 2010-2013, 41 (42.7%) were IDV seropositive. Infection studies demonstrated that IDV-inoculated feral swine shed virus 3-5 days postinoculation and seroconverted at 21 days postinoculation; 50% of in-contact naive feral swine shed virus, seroconverted, or both. Immunohistochemical staining showed viral antigen within epithelial cells of the respiratory tract, including trachea, soft palate, and lungs. Our findings suggest that feral swine might serve an important role in the ecology of IDV.

Background: Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. Objectives: To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. Methods: By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500 μg and 800 μg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. Results: When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800 μg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500 μg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) − as well as crossreactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dosedependent manner. Conclusion: The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination. info:eu-repo/semantics/publishedVersion

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

Recent studies have questioned the effect of maternal derived antibodies (MDAs) to protect piglets against infection with influenza A virus (IAV). The lack of protection against IAV infections provided by MDAs has encouraged alternative vaccination strategies targeting young piglets in an attempt to stimulate an early antibody response. There is a lack of studies documenting the efficacy of piglet vaccination. In the present study, we monitored a group of vaccinated and non-vaccinated piglets in a Danish sow herd that initiated piglet vaccination with ¼ dose of an inactivated swine influenza vaccine at the time of castration (day 3–4). A total of 160 piglets from 11 sows were included and either vaccinated with 0.5 mL inactivated swine influenza vaccine or sham-vaccinated. From week 0 until week 6, all included piglets were clinically examined and nasal swapped once per week and weighed at weeks 0, 3 and 6. Blood samples were collected from sows at week 0 and from piglets at week 3. Vaccination of piglets had limited effect on clinical signs, body weight, antibody development and viral shedding, within the first 6 weeks of life. At least 50% of all pigs of each treatment group tested positive for IAV at week 2, and very early onset of IAV shedding was observed. In total, 18 pigs were IAV positive in nasal swabs for more than one consecutive sampling time indicating prolonged shedding and 14 pigs were IAV positive with negative samplings in between indicating re-infection with the same IAV strain.

Across decades of co-circulation in humans, influenza A subtypes H1N1 and H3N2 have caused seasonal epidemics characterized by different age distributions of cases and mortality. H3N2 causes the majority of severe, clinically attended cases in high-risk elderly cohorts, and the majority of overall deaths, whereas H1N1 causes fewer deaths overall, and cases shifted towards young and middle-aged adults. These contrasting age profiles may result from differences in childhood imprinting to H1N1 and H3N2 or from differences in evolutionary rate between subtypes. Here we analyze a large epidemiological surveillance dataset to test whether childhood immune imprinting shapes seasonal influenza epidemiology, and if so, whether it acts primarily via homosubtypic immune memory or via broader, heterosubtypic memory. We also test the impact of evolutionary differences between influenza subtypes on age distributions of cases. Likelihood-based model comparison shows that narrow, within-subtype imprinting shapes seasonal influenza risk alongside age-specific risk factors. The data do not support a strong effect of evolutionary rate, or of broadly protective imprinting that acts across subtypes. Our findings emphasize that childhood exposures can imprint a lifelong immunological bias toward particular influenza subtypes, and that these cohort-specific biases shape epidemic age distributions. As a consequence, newer and less “senior” antibody responses acquired later in life do not provide the same strength of protection as responses imprinted in childhood. Finally, we project that the relatively low mortality burden of H1N1 may increase in the coming decades, as cohorts that lack H1N1-specific imprinting eventually reach old age., Author summary Influenza viruses of subtype H1N1 and H3N2 both cause seasonal epidemics in humans, but with different age-specific impacts. H3N2 causes a greater proportion of cases in older adults than H1N1, and more deaths overall. People tend to gain the strongest immune memory of influenza viruses encountered in childhood, and so differences in H1N1 and H3N2’s age-specific impacts may reflect that individuals born in different eras of influenza circulation have been imprinted with different immunological risk profiles. Another idea is that H3N2 may be more able to infect immunologically experienced adults because it evolves slightly faster than H1N1 and can more quickly escape immune memory. We analyzed a large epidemiological data set and found that birth year-specific differences in childhood immune imprinting, not differences in evolutionary rate, explain differences in H1N1 and H3N2’s age-specific impacts. These results can help epidemiologists understand how epidemic risk from specific influenza subtypes is distributed across the population and predict how population risk may shift as differently imprinted birth years grow older. Further, these results provide immunological clues to which facets of immune memory become biased in childhood, and then later play a strong role in protection during seasonal influenza epidemics.

The hemagglutinin (HA), a glycoprotein on the surface of influenza A virus (IAV), initiates the virus life cycle by binding to terminal sialic acid (SA) residues on host cells. The HA gradually accumulates amino acid substitutions that allow IAV to escape immunity through a mechanism known as antigenic drift. We recently confirmed that a small set of amino acid residues are largely responsible for driving antigenic drift in swine-origin H3 IAV. All identified residues are located adjacent to the HA receptor binding site (RBS), suggesting that substitutions associated with antigenic drift may also influence receptor binding. Among those substitutions, residue 145 was shown to be a major determinant of antigenic evolution. To determine whether there are functional constraints to substitutions near the RBS and their impact on receptor binding and antigenic properties, we carried out site-directed mutagenesis experiments at the single-amino-acid level. We generated a panel of viruses carrying substitutions at residue 145 representing all 20 amino acids. Despite limited amino acid usage in nature, most substitutions at residue 145 were well tolerated without having a major impact on virus replication in vitro. All substitution mutants retained receptor binding specificity, but the substitutions frequently led to decreased receptor binding. Glycan microarray analysis showed that substitutions at residue 145 modulate binding to a broad range of glycans. Furthermore, antigenic characterization identified specific substitutions at residue 145 that altered antibody recognition. This work provides a better understanding of the functional effects of amino acid substitutions near the RBS and the interplay between receptor binding and antigenic drift. IMPORTANCE The complex and continuous antigenic evolution of IAVs remains a major hurdle for vaccine selection and effective vaccination. On the hemagglutinin (HA) of the H3N2 IAVs, the amino acid substitution N 145 K causes significant antigenic changes. We show that amino acid 145 displays remarkable amino acid plasticity in vitro, tolerating multiple amino acid substitutions, many of which have not yet been observed in nature. Mutant viruses carrying substitutions at residue 145 showed no major impairment in virus replication in the presence of lower receptor binding avidity. However, their antigenic characterization confirmed the impact of the 145 K substitution in antibody immunodominance. We provide a better understanding of the functional effects of amino acid substitutions implicated in antigenic drift and its consequences for receptor binding and antigenicity. The mutation analyses presented in this report represent a significant data set to aid and test the ability of computational approaches to predict binding of glycans and in antigenic cartography analyses.

BACKGROUND: Pigs are natural hosts for influenza A viruses, and the infection is widely prevalent in swine herds throughout the world. Current commercial influenza vaccines for pigs induce a narrow immune response and are not very effective against antigenically diverse viruses. To control influenza in pigs, the development of more effective swine influenza vaccines inducing broader cross-protective immune responses is needed. Previously, we have shown that a polyvalent influenza DNA vaccine using vectors containing antibiotic resistance genes induced a broadly protective immune response in pigs and ferrets using intradermal injection followed by electroporation. However, this vaccination approach is not practical in large swine herds, and DNA vaccine vectors containing antibiotic resistance genes are undesirable.OBJECTIVES: To investigate the immunogenicity of an optimized version of our preceding polyvalent DNA vaccine, characterized by a next-generation expression vector without antibiotic resistance markers and delivered by a convenient needle-free intradermal application approach.METHODS: The humoral and cellular immune responses induced by three different doses of the optimized DNA vaccine were evaluated in groups of five to six pigs. The DNA vaccine consisted of six selected influenza genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase.RESULTS: Needle-free vaccination of growing pigs with the optimized DNA vaccine resulted in specific, dose-dependent immunity down to the lowest dose (200μg DNA/vaccination). Both the antibody-mediated and the recall lymphocyte immune responses demonstrated high reactivity against vaccine-specific strains and cross-reactivity to vaccine-heterologous strains.CONCLUSION: The results suggest that polyvalent DNA influenza vaccination may provide a strong tool for broad protection against swine influenza strains threatening animal as well as public health. In addition, the needle-free administration technique used for this DNA vaccine will provide an easy and practical approach for the large-scale vaccination of swine.

During influenza epidemics, influenza-like illnesses (ILIs) viruses cocirculate with influenza strains. If positive, rapid influenza diagnostic tests (RIDTs) identify influenza A/B, but false-negative RIDTs require retesting by viral polymerase chain reaction (PCR). Patient volume limits testing during influenza epidemics, and non-specific laboratory findings have been used for presumptive diagnosis pending definitive viral testing. In adults, the most useful laboratory abnormalities in influenza include relative lymphopenia, monocytosis, and thrombocytopenia. Lymphocyte:monocyte (L:M) ratios may be even more useful. L:M ratios 2 with human metapneumovirus (hMPV), rhinoviruses/enteroviruses (R/E), and respiratory syncytial virus (RSV), but not human parainfluenza virus type 3 (HPIV-3), which had L:M ratios 3 days, whereas with HPIV-3, L:M ratios 3 days of hospitalization.

Influenza, which is an acute respiratory disease caused by the influenza virus, represents a worldwide public health and economic problem owing to the significant morbidity and mortality caused by its seasonal epidemics and pandemics. Sensitive and convenient methodologies for the detection of influenza viruses are important for clinical care and infection control as well as epidemiological investigations. Here, we developed a multiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) with quencher/fluorescence oligonucleotides connected by a 5′ backward loop (LF or LB) primer for the detection of two subtypes of influenza viruses: Influenza A (A/H1 and A/H3) and influenza B. The detection limits of the multiplex RT-LAMP assay were 103 copies and 102 copies of RNA for influenza A and influenza B, respectively. The sensitivities of the multiplex influenza A/B/IC RT-LAMP assay were 94.62% and 97.50% for influenza A and influenza B clinical samples, respectively. The specificities of the multiplex influenza A/B/IC RT-LAMP assay were 100% for influenza A, influenza B, and healthy clinical samples. In addition, the multiplex influenza A/B/IC RT-LAMP assay had no cross-reactivity with other respiratory viruses.

Recurrent outbreaks of the influenza virus continue to pose a serious health threat all over the world. The role of mass media becomes increasingly important in modeling infectious disease transmission dynamics since it can provide public health information that influences risk perception and health behaviors. Motivated by the recent 2009 H1N1 influenza pandemic outbreak in South Korea, a mathematical model has been developed. In this work, a previous influenza transmission model is modified by incorporating two distinct media effect terms in the transmission rate function; (1) a theory-based media effect term is defined as a function of the number of infected people and its rage of change and (2) a data-based media effect term employs the real-world media coverage data during the same period of the 2009 influenza outbreak. The transmission rate and the media parameters are estimated through the least-squares fitting of the influenza model with two media effect terms to the 2009 H1N1 cumulative number of confirmed cases. The impacts of media effect terms are investigated in terms of incidence and cumulative incidence. Our results highlight that the theory-based and data-based media effect terms have almost the same influence on the influenza dynamics under the parameters obtained in this study. Numerical simulations suggest that the media can have a positive influence on influenza dynamics; more media coverage leads to a reduced peak size and final epidemic size of influenza.

Purpose of Review Zoonotic influenza viruses are those influenza viruses that cross the animal-human barrier and can cause disease in humans, manifesting from minor respiratory illnesses to multiorgan dysfunction. The increasing incidence of infections caused by these viruses worldwide has necessitated focused attention to improve both diagnostic as well as treatment modalities. In this second part of a two-part review, we discuss the clinical features, diagnostic modalities, and treatment of zoonotic influenza, and provide an overview of prevention strategies. Recent Findings Illnesses caused by novel reassortant avian influenza viruses continue to be detected and described; most recently, a human case of avian influenza A(H7N4) has been described from China. We continue to witness increasing rates of A(H7N9) infections, with the latest (fifth) wave, from late 2016 to 2017, being the largest to date. The case fatality rate for A(H7N9) and A(H5N1) infections among humans is much higher than that of seasonal influenza infections. Since the emergence of the A(H1N1) 2009 pandemic, and subsequently A(H7N9), testing and surveillance for novel influenzas have become more effective. Various newer treatment options, including peramivir, favipiravir (T-705), and DAS181, and human or murine monoclonal antibodies have been evaluated in vitro and in animal models. Summary Armed with robust diagnostic modalities, antiviral medications, vaccines, and advanced surveillance systems, we are today better prepared to face a new influenza pandemic and to limit the burden of zoonotic influenza than ever before. Sustained efforts and robust research are necessary to efficiently deal with the highly mutagenic zoonotic influenza viruses.

Introduction Pakistan is extremely susceptible to an influenza outbreak, as it shares borders with the most affected countries, namely China and India. The medical and dental students come into direct contact with the affected population and should be aware of the risk factors and signs and symptoms pertaining to swine influenza virus (SIV). Hence, this survey was conducted to assess the knowledge, perceptions and self-care practices of the medical and dental students with regards to this pandemic. Methods A descriptive, cross-sectional study was conducted to evaluate the swine flu-related knowledge, attitudes and practices of the medical and dental students at various institutions in Karachi, Pakistan. We approached 613 students that were available on the dates of this survey, keeping a medical to dental student ratio of 75:25. All students from first to final year comprised of the study population, and no internists or medical personnel were included. The questionnaire was divided into three sections, namely knowledge, attitudes and, practices. All questions were based on a multiple choice format. The data were entered and interpreted using the IBM Statistical Package for the Social Sciences 23.0 (IBM Corp., Armonk, New York). Results The majority of the students were aware that the swine flu is a transmittable disease (n=485, 80.8%). Most students identified the signs and symptoms correctly; however, diarrhea (15.5%) and vomiting (32.2%) were the least correct answers (n=93, n=193 respectively). Most of the preventative measures were reported accurately by the participants. Despite this, only 15.5% students (n=93) reported the use of a facemask when suffering from fever, cough and a runny nose. Conclusion There is a dire need for the routine integration of the awareness and management programs in the medical and dental schools. There exists a gap between the policy and practice, and it is high time we bridge the divide. The students should also be vaccinated annually for influenza A.

Background An association between AS03 adjuvanted pandemic influenza vaccine and the occurrence of Bell’s palsy was found in a population based cohort study in Stockholm, Sweden. To evaluate this association in a different population, we conducted a self-controlled case series in a primary health care database, THIN, in the United Kingdom. The aim of this study was to determine whether there was an increased risk of Bell’s palsy following vaccination with any influenza vaccine containing A/California/7/2009 (H1N1)-like viral strains. Secondly, we investigated whether risks were different following pandemic influenza A(H1N1)pdm09 vaccines and seasonal influenza vaccines containing the influenza A(H1N1)pdm09 strain. Methods The study population comprised all incident Bell’s palsy cases between 1 June 2009 and 30 June 2013 identified in THIN. We determined the relative incidence (RI) of Bell’s palsy during the 6 weeks following vaccination with either pandemic or seasonal influenza vaccine. All analyses were adjusted for seasonality and confounding variables. Results We found an incidence rate of Bell’s palsy of 38.7 per 100,000 person years. Both acute respiratory infection (ARI) consultations and pregnancy were found to be confounders. When adjusted for seasonality, ARI consultations and pregnancies, the RI during the 42 days after vaccination with an influenza vaccine was 0.85 (95% CI: 0.72–1.01). The RI was similar during the 42 days following seasonal vaccine (0.96, 95%CI: 0.82–1.13) or pandemic vaccine (0.73, 95%CI: 0.47–1.12). Conclusion We found no evidence for an increased incidence of Bell’s palsy following seasonal influenza vaccination overall, nor for monovalent pandemic influenza vaccine in 2009.

From 2010 to 2013 in Japan, we isolated 11 swine influenza viruses (SIVs) from pigs showing respiratory symptoms. Sequence and phylogenetic analyses showed that 6 H1N1 viruses originated from the pandemic (H1N1) 2009 (pdm 09) virus and the other 5 viruses were reassortants between SIVs and pdm 09 viruses, representing 4 genotypes. Two H1N2 viruses contained H1 and N2 genes originated from Japanese H1N2 SIV together with internal genes of pdm 09 viruses. Additionally, 1 H1N2 virus contained a further NP gene originating from Japanese H1N2 SIV. One H1N1 virus contained only the H1 gene originating from Japanese H1 SIV in a pdm 09 virus background. One H3N2 virus contained H3 and N2 genes originating from Japanese H3N2 SIV together with internal genes of pdm 09 virus. The results indicate that pdm 09 viruses are distributed widely in the Japanese swine population and that several reassortments with Japanese SIVs have occurred.

H1N1 is the earliest emerging subtype of influenza A viruses with available genomic sequences, has caused several pandemics and seasonal epidemics, resulting in millions of deaths and enormous economic losses. Timely determination of new antigenic variants is crucial for the vaccine selection and flu prevention. In this study, we chronologically divided the H1N1 strains into several periods in terms of the epidemics and pandemics. Computational models have been constructed to predict antigenic variants based on epidemic and pandemic periods. By sequence analysis, we demonstrated the diverse mutation patterns of HA1 protein on different periods and that an individual model built upon each period can not represent the variations of H1N1 virus. A stacking model was established for the prediction of antigenic variants, combining all the variation patterns across periods, which would help assess a new influenza strain's antigenicity. Three different feature extraction methods, i.e. residue-based, regional band-based and epitope region-based, were applied on the stacking model to verify its feasibility and robustness. The results showed the capability of determining antigenic variants prediction with accuracy as high as 0.908 which performed better than any of the single models. The prediction performance using the stacking model indicates clear distinctions of mutation patterns and antigenicity between epidemic and pandemic strains. It would also facilitate rapid determination of antigenic variants and influenza surveillance. Ministry of Education (MOE) Published version This study is supported by AcRF Tier 2 grant MOE2014-T2-2-023, Ministry of Education, Singapore.

The outbreaks of pandemic H1N1 influenza A virus (pdm-like H1N1 2009), detected for the first time in farrow-to-finish farms in Poland, were described. The nasal swabs and lung tissue collected from diseased/dead animals were tested using molecular techniques (RRT-PCR, MRT-PCR, RT-PCR, SSG-PCR, sequencing) and virus isolation. The amplification of the genetic material extracted from the tested samples confirmed the presence of the M1 gene sequence of type A influenza virus. Using MRT-PCRs no products characteristic for HA and NA of any swine influenza virus subtypes were obtained. Using SSGPCR, products specific for pandemic HA and NA gene fragments were detected. Six new pdm-like H1N1 2009 strains were isolated and characterised. Phylogenetic analysis of the HA and NA genes revealed that they belong to one lineage with the pandemic strain A/California/04/2009 and other human strains, including human strains isolated in Poland in 2011.

Introduction: The most frequent complication of A (H1N1) influenza and the leading cause of death was pneumonia with a primary viral or mixed viral and bacterial etiology. 182 patients had died because of a pandemic influenza in Poland by 31st July 2010.Material and Methods: A retrospective study of 6 fatal cases of pandemic influenza, aged 23-41, including 3 women, hospitalised between November 2009 and February 2011 in different Polish medical centres.Results: We present the clinical course of 6 late diagnosed cases of A (H1N1) influenza. All patients presented typical flu-like symptoms in the beginning. 4/6 patients had severe disease risk factors: pregnancy, arthritis, Wegener granulomatosis and obesity. All patients were seen by doctors, no one had received antiviral therapy, 4/5 were treated with antibiotics before they were hospitalized. One patient had nosocomial infection. Patients were admitted to the hospital on the 3rd to 8th day of the disease. They received oseltamivir treatment on the 4th to 9th day. All patients developed pneumonia complicated by acute respiratory distress syndrome. Death appeared between the 4th and 27th day after the onset of symptoms. Autopsies were performed in 5 cases and revealed haemorrhagic pneumonia in 2 patients.Conclusion: Delayed diagnosis and antiviral treatment initiation has a significant impact on mortality in A (H1N1) influenza. During the influenza epidemic, patients presenting typical symptoms should always be suspected of having influenza. Antiviral treatment has to be initiated immediately, especially ifthere are risk factors of severe disease.

To study influenza viruses in pigs in Sri Lanka, we examined samples from pigs at slaughterhouses. Influenza (H3N2) and A(H1N1)pdm09 viruses were prevalent during 2004-2005 and 2009-2012, respectively. Genetic and epidemiologic analyses of human and swine influenza viruses indicated 2 events of A(H1N1)pdm09 virus spillover from humans to pigs.

Infection with highly pathogenic avian influenza (HPAI) in birds and mammals is associated with severe pathology and increased mortality. We hypothesize that in contrast to low pathogenicity avian influenza (LPAI) infection, HPAI infection of chicken dendritic cells (DC) induces a cytokine deregulation which may contribute to their highly pathogenic nature. Infection of DC with LPAI H7N1 and H5N2 resulted in viral RNA and NP expression without increase in time, in contrast to HPAI H7N1 and H5N2 mRNA expression. No increase in IFN mRNA was detected after infection with LPAI, but after LPAI H5N2, and not LPAI H7N1, infection the level of bioactive IFNa/ß significantly increased. After HPAI H7N1 and H5N2 infection, significant increases in IL-8, IFN-a, IFN-¿ mRNA expression and in TLR1, 3, and 21 mRNA were observed. This enhanced activation of DC after HPAI infection may trigger deregulation of the immune response as seen during HPAI infection in chickens.

The paper describes an outbreak of swine influenza, which was the first H1N2 outbreak confirmed in pig farm in Poland. The outbreak occurred in October 2011 in the farrows to finish farm, first in the fattening units, and subsequently in the reproduction and weaning sectors. Samples of the lungs taken from dead gilts were tested by the use of real time and multiplex PCR, sequencing, and virus isolation methods. The amplification of the genetic material extracted from the lungs confirmed the presence of the M1 gene sequence of type A influenza virus. Using multiplex PCR, the bands of 241 bp, typical for HA1 human-like type, and of 791 bp, typical for NA2, were demonstrated. The new isolates of swine influenza virus, named A/swine/Poland/15817/2011, were obtained in embryonated SPF chicken eggs and MDCK cells. Phylogenetic analysis of the HA and NA genes of the isolate revealed that the virus is of human swine linkage closely related to European human-like viruses. On the basis of such results, it can be stated that A/Swine/Poland/15817/2011 is a new swine influenza virus, representing the H1N2 subtype strain, which emerged in Poland.

Introduction The aim of this review was to record systematically and assess the published literature relating to the occupational risk of influenza A (H1N1) infection among healthcare personnel during the 2009 pandemic. Methods The literature search was performed in June 2015. An update was carried out in May 2016. It was applied to the electronic databases EMBASE, MEDLINE, PsycINFO, PubMed, CINAHL and Google Scholar. The quality assessment was conducted with a tool using eight criteria. A meta-analysis was carried out to compute pooled effect estimates for influenza A (H1N1) infection. Results A total of 26 studies were included in the review, 15 studies met the criteria for the meta-analysis. After a sensitivity analysis the pooled analysis showed a significantly increased odds for influenza A (H1N1) infection for healthcare personnel compared to controls/comparisons (OR = 2.08, 95% CI = 1.73 to 2.51). The pooled prevalence rate for healthcare personnel alone was 6.3%. Conclusions This review corroborates the assumption that healthcare personnel were particularly at risk of influenza A (H1N1) infection during the 2009 pandemic. Healthcare facilities should intensify their focus on strategies to prevent infections among healthcare personnel, especially during the first period of pandemics.

A potentially fatal complication of influenza infection is the development of pneumonia, caused either directly by the influenza virus, or by secondary bacterial infection. Pneumonia related to the 2009 influenza A pandemic was found to be underestimated by commonly used pneumonia severity scores in many cases, and to be rapidly progressive, leading to respiratory failure. Confirmation of etiology by laboratory testing is warranted in such cases. Rapid antigen and immunofluorescence testing are useful screening tests, but have limited sensitivity. Confirmation of pandemic H1N1 influenza A infection can only be made by real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) or viral culture. The most effective preventive measure is annual influenza vaccination in selected individuals. Decisions to administer antiviral medications for influenza treatment or chemoprophylaxis should be based upon clinical and epidemiological factors, and should not be delayed by confirmatory laboratory testing results. Neuraminidase inhibitors (NI) are the agents of choice.

Influenza A H1N1 (2009) was declared by the World Health Organisation (WHO) as the first influenza pandemic of the 21st century. Rapid detection of influenza A and differentiation of influenza A H1N1 (2009) and seasonal influenza A is beneficial. In addition the rapid detection of antiviral resistant strains of influenza A H1N1 (2009) would be useful for clinicians to allow for change to an effective treatment at a much earlier stage if resistance is found. It was the aim of this study to develop a real-time RT-PCR that can detect all influenza A viruses and type simultaneously for influenza A H1N1 (2009) and oseltamivir resistant (H275Y) influenza A H1N1 (2009). This multiplex assay will allow laboratories to screen respiratory samples for all types of influenza A, influenza A H1N1 (2009) virus and oseltamivir resistant (H275Y) influenza A H1N1 (2009) virus in a rapid and cost effective format, ensuring that typing methods for seasonal and avian viruses are used on a smaller subset of samples. Since most virology laboratories already offer a molecular service for influenza A this assay could easily be implemented into most areas at little cost therefore increasing local access to resistance testing.

Objective To analyze the component law of Chinese patent medicines for anti-influenza and develop new prescriptions for anti-influenza by unsupervised data mining methods. Methods Chinese patent medicine recipes for anti-influenza were collected and recorded in the database, and then the correlation coefficient between herbs, core combinations of herbs and new prescriptions were analyzed by using modified mutual information, complex system entropy cluster and unsupervised hierarchical clustering, respectively. Results Based on analysis of 126 Chinese patent medicine recipes, the frequency of each herb occurrence in these recipes, 54 frequently-used herb pairs, 34 core combinations were determined, and 4 new recipes for influenza were developed. Conclusion Unsupervised data mining methods are able to mine the component law quickly and develop new prescriptions.

This study aimed to investigate prevalence and associated factors of influenza vaccination (IV) among children aged 12-23 months. Our cross-sectional survey interviewed 489 parents of children aged 12-23 months anonymously at twelve maternal and child health centers in Hong Kong. Results showed that only 11.5% of the children had ever received IV (64.3% being subsidized). Adjusted for the child's age, significant factors of the children's IV included parental knowledge about governmental policy/recommendation (Adjusted odds ratio [AOR] = 2.64, 95%CI = 1.09,6.40), knowledge about annual IV requirement (AOR = 2.30, 95%CI = 1.21,4.38), perceived safety-related barrier (AOR≥0.14, 95%CI = 0.06,0.33), cue to action (AOR = 7.79, 95%CI = 3.45,17.58), and subjective norm (AOR = 4.59, 95%CI = 2.34,9.00). IV prevalence of children aged 12-23 months remained low despite a subsidization scheme. The higher IV prevalence of older children reported by other studies suggested that parents postponed action. Promotion campaigns should shift emphases from cost reduction and mass media approaches to dissemination of knowledge about IV policy and safety, enhancement of health professionals' advice, and creation of supportive subjective norm.

In the spring of 2009, a novel (H1N1) influenza A virus began to spread among humans worldwide. Although the 2009 H1N1 is related genetically to swine influenza viruses, human infection has not been connected to pig exposure. Because the virus is now circulating widely in the human population, swine herds are at increased risk of becoming infected. In order to investigate potential outbreaks of the 2009 pandemic virus in pigs, a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) for the detection of the (H1N1) 2009 RNA in clinical specimens was developed. To evaluate the applicability of the test as a diagnostic tool in the screening of field specimens from swine, 64 field isolates of North American swine, 5 equine and 48 avian influenza viruses collected during diagnostic investigations were analyzed retrospectively as well as samples collected during an experimental in vivo infection with two novel H1N1 isolates, A/California/04/2009 (H1N1)v virus and A/Mexico/4108/2009 (H1N1)v. The sensitivity of the qRT-PCR was shown to be higher with respect to standard techniques such as virus isolation and the reproducibility was satisfactory. The present unique and highly sensitive assay is able to detect as little as 1 x 10(1) copies of RNA per microl of template and it represents a rapid and useful approach for the screening and quantitation of (H1N1) 2009 RNA in porcine specimens.

In April 2009 a novel influenza virus A H1N1 causing high morbidity and mortality spread first on the plateau of Mexico City subsequently followed by distribution to Texas and California in USA and to Spain; finally within three weeks this influenza virus was worldwide distributed. Its haemagglutinin showed epitope structures that were not recognized by the immune system of children and young adults as a memory response. The pandemic influenza virus consisted of quasispecies with a mixture of genome parts from human, avian, and American and Eurasian swine origin. This new virus caused all symptoms typically for influenza as abrupt onset, fever >39 °C, sore throat with all signs of inflammation, myalgia and arthralgia. In summer 2009 all infections in Europe were mild, while until winter there was a higher lethality in Brazil, Mexico and Argentine; but still in winter 2009 in Europe the pathogenic action of the circulating virus was mild. European isolates showed a neuraminidase inhibitor resistance of around 10%. Prevention of spread of the novel influenza virus H1N1 2009 is mainly by hygienic measurements as: avoid contact to infected, distance from infected, hand disinfection, protection of eye and nose mucous membranes, avoid aerosol formation during blowing the nose and sneezing. It is recommended to use single way tissue handkerchiefs and to depose them in a covered waste reservoir. For further prevention of influenza virus transmission three different vaccines are available in Europe since October 2009. This virus or at least some genomic parts will persist in the human population for the next 2 years.

Introduction Fluid accumulation is associated with adverse outcomes such as acute kidney injury (AKI) in critically ill patients. This study aimed to describe the factors associated with AKI in individuals with influenza A H1N1 severe pneumonia, and explore the relation of fluid accumulation with AKI and mortality. Material and methods We reviewed medical records of individuals with influenza A H1N1 severe pneumonia and no history of chronic kidney disease, attending a national referral center for respiratory diseases between November 2014 and May 2015. Demographic information, risk factors for AKI, physiologic and laboratory data, outcomes and information on fluid intake and output were recorded. Categorical variables were compared using the chi-square test. Quantitative variables were compared using the Mann-Whitney test. Factors associated with AKI and mortality were identified by binary logistic regression. Linear models of fluid accumulation rates for individuals and groups were estimated using segmented linear regression. Results Of 60 patients studied, 43 developed AKI (71.6%). Male gender was protective for AKI (p = 0.019). AKI was associated with nephrotoxic drugs (p = 0.016); PEEP>10 cm H2O on admission (p = 0.031); mortality (p = 0.037); and fluid accumulation ≥10% (fluid overload) at day 7 of hospitalization (p = 0.00026). Mortality was associated with older age (p = 0.009); nephrotoxic drugs (p = 0.034); and higher Pneumonia Severity Index score (112 vs. 76, p = 0.008) on admission. The Deceased-AKI group had a higher rate of fluid accumulation (expressed as ml/kg/body weight) than the Survivors-No AKI group during the study period of 7 days (Survivors-No AKI = 13.31 vs. Deceased-AKI = 22.76, p = 0.019). During the highest phase of fluid accumulation, the Survivors-No AKI group had a slower rate of fluid accumulation than the Survivors-AKI group (14.91 vs. 28.49, p = 0.001). Conclusions A high rate of fluid accumulation was associated with AKI and mortality. We support the approach of resuscitation in acute illness, with an early transition to neutral and then negative fluid balances.

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family consists of six mammalian members, and is shown to participate in signal transduction of a large number of receptor families including TNF receptor family (TNFR) and Toll-like receptors-interleukin-1 receptors (TLR-IL-1R) family. Upon receptor activation, TRAFs are directly or indirectly recruited to the intracellular domains of these receptors. They subsequently engage other signaling proteins to activate inhibitor of kappaB kinase (IKK) complex, TRAF family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1) and inducible I kappaB kinase (IKK-i) (also known as IKKepsilon), ultimately leading to activation of transcription factors such as NF-kappaB and interferon-regulatory factor (IRF) to induce immune and inflammatory responses.

From swine to humans : the A (H1N1) 2009 influenza pandemic. On April 25th 2009, Mexico notified the WHO of an outbreak of an influenza-like disease. The pathogen responsible was rapidly identified as an influenza virus of swine origin, the A(H1N1) 2009 influenza virus. The virus quickly spread worldwide, leading the WHO to announce a phase 6 pandemic alert on June 11th 2009. The majority of cases were mild, but 2 to 10 % of cases required hospitalization for respiratory distress, especially in children under 5, pregnant women, and patients with cardiovascular and respiratory diseases, diabetes or immunosuppression. By April 11th 2010, 17,798 deaths were officially notified to the WHO, a very low number compared to the usual death toll of seasonal influenza. This low death rate is due to the presence of immune resistance in the population over 60, a most likely consequence of sequence homologies between the 1918 and 2009 H1N1 pandemic viruses. Several anti-A (H1N1) 2009 vaccines were prepared in 2009, with and without adjuvants, and all were proven safe and immunogenic in Phase I/ II studies carried out in volunteers. However, the vaccination campaign met little enthusiasm, for reasons which will be reported. Swine appear once more as an important potential reservoir for influenza virus strains pathogenic for man, an observation which pleads for enhanced virological surveillance in this species., Le 25 avril 2009, le Mexique notifiait l’OMS de l’apparition d’un foyer de maladie de type grippal. Le virus fut identifié peu de temps après comme un virus influenza d’origine porcine, le virus influenza A (H1N1) 2009. Celui-ci diffusa ensuite très rapidement dans le monde entier, conduisant l’OMS à déclarer dès le 11 juin 2009 le niveau 6 de pandémie. Bien que la majorité des cas cliniques aient été bénins, deux à 10% ont nécessité une hospitalisation pour détresse respiratoire, notamment chez les enfants de moins de 5 ans, les femmes enceintes et les personnes souffrant de troubles cardiovasculaires ou pulmonaires, de diabète, ou d’immunodépression. Le nombre de décès dus à la grippe A (H1N1) 2009 officiellement notifiées à l’OMS au 11 avril 2010 ne s’élevait qu’à 17 798, un chiffre étonnamment bas par rapport aux chiffres habituels de mortalité de la grippe saisonnière. Cette faible mortalité reflète l’existence d’une résistance immunitaire chez les personnes âgées de plus de 60 ans, apparemment due à des homologies de séquences entre le virus pandémique de 2009 et celui de 1918. De nombreux vaccins anti-A (H1N1) 2009 ont été préparés courant 2009, avec ou sans adjuvant et tous ont fait la preuve de leur innocuité et de leur immunogénicité dans des études de Phase I/ II chez des volontaires. La campagne de vaccination, lancée en octobre 2009, ne devait cependant connaître qu’un succès mitigé, pour des raisons qui seront exposées. Le porc apparaît une fois de plus comme un réservoir potentiel important de souches de virus grippal pathogènes pour l’homme, ce qui implique de renforcer la surveillance virologique chez cette espèce., Girard Marc. Du porc à l'homme: la pandémie de grippe A (H1N1) 2009. In: Bulletin de l'Académie Vétérinaire de France tome 163 n°2, 2010. pp. 109-117.

The 1918 pandemic influenza virus has demonstrated significant pathogenicity in animal models and is the progenitor of ‘classical’ swine and modern seasonal human H1N1 lineages. Here we characterize the pathogenicity of an early ‘classical’ swine H1N1 influenza A virus isolated in 1931 compared to the pathogenicity of the 1918 pandemic virus and a seasonal H1N1 virus in mice and ferrets. A/Swine/Iowa/31 (Sw31) and the 1918 influenza viruses were uniformly lethal in mice at low doses and produced severe lung pathology. In ferrets, Sw31 and 1918 influenza viruses caused severe clinical disease and lung pathology with necrotizing bronchiolitis and alveolitis. The modern H1N1 virus caused little disease in either animal model. These findings revealed that in these models the virulence factors of the 1918 influenza virus are likely preserved in the Sw31 virus and suggest that early swine viruses may be a good surrogate model to study 1918 virulence and pathogenesis.

Background Swine influenza virus (SIV) is the cause of an acute respiratory disease that affects swine worldwide. In Brazil, SIV has been identified in pigs since 1978. After the emergence of pandemic H1N1 in 2009 (H1N1pdm09), few studies reported the presence of influenza virus in Brazilian herds. Objectives The objective of this study was to evaluate the serological profile for influenza virus in farrow-to-finish pig farms in Minas Gerais state, Brazil. Methods Thirty farms with no SIV vaccination history were selected from the four larger pig production areas in Minas Gerais state (Zona da Mata, Triângulo Mineiro/Alto Paranaiba, South/Southwest and the Belo Horizonte metropolitan area). At each farm, blood samples were randomly collected from 20 animals in each production cycle category: breeding animals (sows and gilts), farrowing crate (2–3 weeks), nursery (4–7 weeks), grower pigs (8–14 weeks), and finishing pigs (15–16 weeks), with 100 samples per farm and a total of 3000 animals in this study. The samples were tested for hemagglutination inhibition activity against H1N1 pandemic strain (A/swine/Brazil/11/2009) and H3N2 SIV (A/swine/Iowa/8548-2/98) reference strain. Results The percentages of seropositive animals for H1N1pdm09 and H3N2 were 26·23% and 1·57%, respectively, and the percentages of seropositive herds for both viruses were 96·6% and 13·2%, respectively. Conclusions The serological profiles differed for both viruses and among the studied areas, suggesting a high variety of virus circulation around the state, as well as the presence of seronegative animals susceptible to influenza infection and, consequently, new respiratory disease outbreaks.

A new subtype H3N1 may have arisen from reassortment of a H3N2 turkey isolate, a human H1N1 isolate, and currently circulating swine viruses., Influenza A virus infects various animal species and transmits among different hosts, especially between humans and swine. Swine may serve as a mixing vessel to create new reassortants that could infect humans. Thus, monitoring and characterizing influenza viruses in swine are important in preventing interspecies transmission. We report the emergence and characterization of a novel H3N1 subtype of swine influenza virus (SIV) in the United States. Phylogenetic analysis showed that the H3N1 SIVs may have acquired the hemagglutinin gene from an H3N2 turkey isolate, the neuraminidase gene from a human H1N1 isolate, and the remaining genes from currently circulating SIVs. The H3N1 SIVs were antigenically related to the turkey virus. Lung lesions and nasal shedding occurred in swine infected with the H3N1 SIVs, suggesting the potential to transmit among swine and to humans. Further surveillance will help determine whether this novel subtype will continue to circulate in swine populations.

The 1918 Influenza pandemic was one of the most virulent strains of influenza in history. This strain quickly dispatched previously held theories on influenza. World War One introduced new environmental stresses and speed of dissemination logistics never experienced by humans. In light of new phylogenic evidence the cause of this influenza outbreak is now being considered to have linkage to the avian influenza. Animals act as reservoirs for this influenza virus and research indicates the influenza virus often originates in the intestines of aquatic wildfowl. The virus is shed into the environment, which in turns infects domestic poultry, which in turn infects mammalian hosts. These animals, usually pigs, act as a transformer or converters; creating a strain that can more readily infect humans. Therefore swine can be infected with both avian and human influenza A viruses and serve as a source for infection for a number of species as the incidents of direct infection from birds to humans have been rare. Increased human habitation near poultry and swine raising facilities pose greater influenza outbreak risk. It was this combination of environmental factors that may have contributed to the greatest pandemic of recent times, and, moreover, similar conditions exist throughout Southeast Asia today.