Two novel human-like H3N2 influenza A virus strains, A/swine/Oklahoma/65980/2017 (H3N2) and A/swine/Oklahoma/65260/2017 (H3N2), were isolated from porcine samples submitted to the Iowa State University Veterinary Diagnostic Laboratory in the United States.

How influenza A viruses host-jump from animal reservoir species to humans, which can initiate global pandemics, is a central question in pathogen evolution. The zoonotic and spatial origins of the influenza virus associated with the "Spanish flu" pandemic of 1918 have been debated for decades. Outbreaks of respiratory disease in US swine occurred concurrently with disease in humans, raising the possibility that the 1918 virus originated in pigs. Swine also were proposed as "mixing vessel" intermediary hosts between birds and humans during the 1957 Asian and 1968 Hong Kong pandemics. Swine have presented an attractive explanation for how avian viruses overcome the substantial evolutionary barriers presented by different cellular environments in humans and birds. However, key assumptions underpinning the swine mixing-vessel model of pandemic emergence have been challenged in light of new evidence. Increased surveillance in swine has revealed that human-to-swine transmission actually occurs far more frequently than the reverse, and there is no empirical evidence that swine played a role in the emergence of human influenza in 1918, 1957, or 1968. Swine-to-human transmission occurs periodically and can trigger pandemics, as in 2009. But swine are not necessary to mediate the establishment of avian viruses in humans, which invites new perspectives on the evolutionary processes underlying pandemic emergence.

Genetic reassortment between influenza A viruses (IAVs) facilitate emergence of pandemic strains, and swine are proposed as a "mixing vessel" for generating reassortants of avian and mammalian IAVs that could be of risk to mammals, including humans. However, how a transmissible reassortant emerges in swine are not well understood. Genomic analyses of 571 isolates recovered from nasal wash samples and respiratory tract tissues of a group of co-housed pigs (influenza-seronegative, avian H1N1 IAV-infected, and swine H3N2 IAV-infected pigs) identified 30 distinct genotypes of reassortants. Viruses recovered from lower respiratory tract tissues had the largest genomic diversity, and those recovered from turbinates and nasal wash fluids had the least. Reassortants from lower respiratory tracts had the largest variations in growth kinetics in respiratory tract epithelial cells, and the cold temperature in swine nasal cells seemed to select the type of reassortant viruses shed by the pigs. One reassortant in nasal wash samples was consistently identified in upper, middle, and lower respiratory tract tissues, and it was confirmed to be transmitted efficiently between pigs. Study findings suggest that, during mixed infections of avian and swine IAVs, genetic reassortments are likely to occur in the lower respiratory track, and tissue tropism is an important factor selecting for a transmissible reassortant.

As a leading global city with a high population density, Singapore is at risk for the introduction of novel biological threats. This risk has been recently reinforced by human epidemics in Singapore of SARS coronavirus, 2009 pandemic H1N1 influenza A virus, and enterovirus 71. Other major threats to Singapore include MERS-coronavirus and various avian and swine influenza viruses. The ability to quickly identify and robustly track such threats to initiate an early emergency response remains a significant challenge. In an effort to enhance respiratory virus surveillance in Singapore, our team conducted a pilot study employing a noninvasive bioaerosol sampling method to detect respiratory viruses in Singapore's Mass Rapid Transit (MRT) network. Over a period of 52 weeks, 89 aerosol samples were collected during peak MRT ridership hours. Nine (10%) tested positive for adenovirus, four (4.5%) tested positive for respiratory syncytial virus type A, and one (1%) tested positive for influenza A virus using real-time RT-PCR/PCR. To our knowledge, this is the first time molecular evidence for any infectious respiratory agent has been collected from Singapore's MRT. Our pilot study data support the possibility of employing bioaerosol samplers in crowded public spaces to noninvasively monitor for respiratory viruses circulating in communities.

Split inactivated influenza vaccines remain one of the primary preventative strategies against severe influenza disease in the population. However, current vaccines are only effective against a limited number of matched strains. The need for broadly protective vaccines is acute due to the high mutational rate of influenza viruses and multiple strain variants in circulation at any one time. The neuraminidase (NA) glycoprotein expressed on the influenza virion surface has recently regained recognition as a valuable vaccine candidate. We sought to broaden the protection provided by NA within the N1 subtype by computationally engineering consensus NA sequences. Three NA antigens (NA5200, NA7900, NA9100) were designed based on sequence clusters encompassing three major groupings of NA sequence space; (i) H1N1 2009 pandemic and Swine H1N1, (ii) historical seasonal H1N1 and (iii) H1N1 viruses ranging from 1933 till current times. Recombinant NA proteins were produced as a vaccine and used in a mouse challenge model. The design of the protein dictated the protection provided against the challenge strains. NA5200 protected against H1N1 pdm09, a Swine isolate from 1998 and NIBRG-14 (H5N1). NA7900 protected against all seasonal H1N1 viruses tested, and NA9100 showed the broadest range of protection covering all N1 viruses tested. By passive transfer studies and serological assays, the protection provided by the cluster-based consensus (CBC) designs correlated to antibodies capable of mediating NA inhibition. Importantly, sera raised to the consensus NAs displayed a broader pattern of reactivity and protection than naturally occurring NAs, potentially supporting a predictive approach to antigen design.

Backyard pig populations are not monitored for influenza A virus (IAV) in Brazil and there are limited data about seroprevalence and risk factors in these populations. Our goal was to assess possible factors associated with IAV seroprevalence in backyard pig populations using an indirect ELISA protocol based on a recombinant nucleoprotein. Following the IAV screening using NP-ELISA, subtype-specific serology based on hemagglutination inhibition (HI) assay of the ELISA-positive pigs was conducted. The survey comprised a total of 1,667 sera samples collected in 2012 and 2014 in 479 holdings and the estimated seroprevalence was 5.3% (3.84%-7.33%) and 2.3% (1.34%-3.71%) in the respective years. In both years, H1N1pdm09 was the most prevalent subtype. The multivariable analysis showed main factors such as "age," "sex," "number of suckling pigs" and "neighbours raising pigs" that presented the greatest effect on IAV seroprevalence in these pig populations. These factors may be associated with the low biosecurity measures and management of backyard holdings. In addition, the low IAV seroprevalences found in these backyard pig populations could be related to a low number of animals in each pig holding and low animal movement/replacement that do not favour IAV transmission dynamics. This low frequency of H1N1pdm09 seropositive pigs could also be due to sporadic human-to-pig transmission of what is now a human seasonal influenza A virus; however, these factors should be explored in future studies. Herein, these results highlight the importance of IAV continued surveillance in backyard pig holdings, since it is poorly known which IAVs are circulating in these populations and the risk they could pose to public health and virus transmission to commercial farms.

The codon usage pattern can reveal the adaptive changes that allow virus survival and fitness adaptation to their particular host, as well as the external environment. Although still considered a novel influenza virus, there is an increasing number of influenza D viruses (IDV) reported. Considering the vital role of the hemagglutinin-esterase fusion (HEF) gene in in receptor binding, receptor degradation, and membrane fusion, we investigated the codon usage pattern of the IDV HEF gene to better understand its adaptive changes during evolution. Based on the HEF gene, three groups including, D/OK, D/660, and D/Japan were identified. We found a low codon usage bias, which allowed IDV to replicate in the corresponding hosts by reducing competition during evolution, that was mainly driven by natural selection and mutation pressure, with a profound role of natural selection. Furthermore, the interaction between the codon adaption index (CAI) and the relative codon deoptimization index (RCDI) revealed the adaption of IDV to multiple hosts, especially cattle which is currently considered its reservoir. Additionally, similarity index (SiD) analysis revealed that the swine exerted a stronger evolutionary pressure on IDV than cattle, though cattle is considered the primary reservoir. In addition, the conserved PB1 gene showed a similar pattern of codon usage compared to HEF. Therefore, we hypothesized that IDV has a preference to maintain infection in multiple hosts. The study aids the understanding of the evolutionary changes of IDV, which could assist this novel virus prevention and control.

Breed-to-wean pig farms play an important role in spreading influenza A virus (IAV) because suckling piglets maintain, diversify and transmit IAV at weaning to other farms. Understanding the nature and extent of which farm factors drive IAV infection in piglets is a prerequisite to reduce the burden of influenza in swine. We evaluated the association between IAV infection in piglets at weaning and farm factors including farm features, herd management practices and gilt- and piglet-specific management procedures performed at the farm. Voluntarily enrolled breed-to-wean farms (n = 83) agreed to share IAV diagnostic testing and farm data from July 2011 through March 2017 including data obtained via the administration of a survey. There were 23% IAV RT-PCR positive samples of the 12,814 samples submitted for IAV testing within 2989 diagnostic submissions with 30% positive submissions. Among all the factors evaluated (n = 24), and considering the season-adjusted multivariable analysis, only sow IAV vaccination and gilt IAV status at entry significantly reduced (p-value<0.05) IAV infections in piglets at weaning. Results from this study indicate that veterinarians and producers could manage these identified factors to reduce the burden of influenza in piglets prior to wean and perhaps, reduce the spread of IAV to other farms and people.

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