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Influenza A viruses are a One Health threat because they can spill over between host populations, including among humans, swine, and birds. Surveillance of swine influenza virus in Hanoi, Vietnam, during 2013-2019 revealed gene pool enrichment from imported swine from Asia and North America and showed long-term maintenance, persistence, and reassortment of virus lineages. Genome sequencing showed continuous enrichment of H1 and H3 diversity through repeat introduction of human virus variants and swine influenza viruses endemic in other countries. In particular, the North American H1-δ1a strain, which has a triple-reassortant backbone that potentially results in increased human adaptation, emerged as a virus that could pose a zoonotic threat. Co-circulation of H1-δ1a viruses with other swine influenza virus genotypes raises concerns for both human and animal health.

Background Recently, Influenza A virus (IAV) has been shown to activate several programmed cell death pathways that play essential roles in host defense. Indeed, cell death caused by viral infection may be mediated by a mixed pattern of cell death instead of a certain single mode. Ferroptosis is a novel form of regulated cell death (RCD) that is mainly mediated by iron-dependent lipid peroxidation. Based on the proteomic data, we wondered whether IAV causes ferroptosis in host cells. Method In this study, a quantitative proteomics approach based on an iTRAQ combined with LC–MS/MS was used to profile proteins expressed in A549 cells infected with H1N1 swine influenza virus (SIV). Meanwhile, we measured the intracellular iron content, reactive oxygen species (ROS) release and lipid peroxidation in response to SIV infection. Finally, a drug experiment was conducted to investigate the effects of ferroptosis on modulating SIV survival. Results The bioinformatics analysis revealed several proteins closely relevant to iron homeostasis and transport, and the ferroptosis signaling pathway are highly enriched in response to SIV infection. In our experiment, aberrant expression of iron-binding proteins disrupted labile iron uptake and storage after SIV infection. Meanwhile, SIV infection inhibited system the Xc − /GPX4 axis resulting in GSH depletion and the accumulation of lipid peroxidation products. Notably, cell death caused by SIV as a result of iron-dependent lipid peroxidation can be partially rescued by ferroptosis inhibitor. Additionally, blockade of the ferroptotic pathway by ferrostatin-1 (Fer-1) treatment decreased viral titers and inflammatory response. Conclusions This study revealed a new mode of cell death induced by IAV infection, and our findings might improve the understanding of the underlying mechanism involved in the interaction of virus and host cells.

Rapid and early detection of infectious diseases in pigs is important, especially for the implementation of control measures in suspected cases of African swine fever (ASF), as an effective and safe vaccine is not yet available in most of the affected countries. Additionally, analysis for swine influenza is of significance due to its high morbidity rate (up to 100%) despite a lower mortality rate compared to ASF. The wide distribution of swine influenza A virus (SwIAV) across various countries, the emergence of constantly new recombinant strains, and the danger of human infection underscore the need for rapid and accurate diagnosis. Several diagnostic approaches and commercial methods should be applied depending on the scenario, type of sample and the objective of the studies being implemented. At the early diagnosis of an outbreak, virus genome detection using a variety of PCR assays proves to be the most sensitive and specific technique. As the disease evolves, serology gains diagnostic value, as specific antibodies appear later in the course of the disease (after 7–10 days post-infection (DPI) for ASF and between 10–21 DPI for SwIAV). The ongoing development of commercial kits with enhanced sensitivity and specificity is evident. This review aims to analyse recent advances and current commercial kits utilised for the diagnosis of ASF and SwIAV.

•We evaluated swine influenza A transmission virus in piglets with different MDA statuses after a single-dose vaccination.•The transmission rate of the challenge strain was found 3.6 time greater in MDA-positive than in MDA-negative pigs.•Estimated parameters were unsed to feed a mechanistic model of SwIAV transmission in a farrow-to-finish pig farm.•An extended SwIAV within-farm persistence was observed when both sows and piglets were batch-to-batch vaccinated.•Alternative vaccination schemes need to be tested, accounting for population dynamics and hosts’ immune statuses. Swine influenza A virus (swIAV) is a major pathogen affecting pigs with a huge economic impact and potentially zoonotic. Epidemiological studies in endemically infected farms permitted to identify critical factors favoring on-farm persistence, among which maternally-derived antibodies (MDAs). Vaccination is commonly practiced in breeding herds and might be used for immunization of growing pigs at weaning. Althoughinterference between MDAs and vaccination was reported in young piglets, its impact on swIAV transmission was not yet quantified. To this aim, this study reports on a transmission experiment in piglets with or without MDAs, vaccinated with a single dose injection at four weeks of age, and challenged 17 days post-vaccination. To transpose small-scale experiments to real-life situation, estimated parameters were used in a simulation tool to assess their influence at the herd level. Based on a thorough follow-up of the infection chain during the experiment, the transmission of the swIAV challenge strain was highly dependent on the MDA status of the pigs when vaccinated. MDA-positive vaccinated animals showed a direct transmission rate 3.6-fold higher than the one obtained in vaccinated animals without MDAs, estimated to 1.2. Vaccination nevertheless reduced significantly the contribution of airborne transmission when compared with previous estimates obtained in unvaccinated animals. The integration of parameter estimates in a large-scale simulation model, representing a typical farrow-to-finish pig herd, evidenced an extended persistence of viral spread when vaccination of sows and single dose vaccination of piglets was hypothesized. When extinction was quasi-systematic at year 5 post-introduction in the absence of sow vaccination but with single dose early vaccination of piglets, the extinction probability fell down to 33% when batch-to-batch vaccination was implemented both in breeding herd and weaned piglets. These results shed light on a potential adverse effect of single dose vaccination in MDA-positive piglets, which might lead to longer persistence of the SwIAV at the herd level.

In Brazil, at least four lineages of influenza A virus circulate pig population: 2009 H1N1 flu pandemic (pH1N1), human-seasonal origin H3N2, H1N1 and H1N2 (huH1 lineages) viruses. Studies related to the occurrence of swine influenza A virus (SIAV) in Brazilian herds have been detecting an increase of occurrence of huH1 lineages. This study aimed to construct recombinant vaccines against the huH1N1 virus and test the immunogens in a murine model. The virus was constructed by reverse genetics using plasmids encoding the HA and NA sequences from a wild huH1N1 virus isolated from an infected pig. Amplified virus was inactivated, and oil-in-water (OW) and gel polymer (GP) adjuvants were used to formulate the vaccines. C57Bl6 mice received two doses with 3 weeks interval by the intramuscular route. Animals were randomly divided into 8 groups (G1-G8): G1 received OW vaccine and G2 PBS plus OW adjuvant; G3 received GP vaccine and G4 PBS plus GP adjuvant; G5 received the live virus by the intranasal route while G6 only PBS; G7 and G8 did not receive any treatment. Serum samples were collected before vaccination and after the first and second dose. Except for G8, three weeks post boost animals were challenged with a wild huH1N1 virus and observed for weight changes. After infection, bronchoalveolar lavage fluid (BALF) and lungs were collected from animals of each group for viral titers and immunohistochemistry (IHC) analysis, respectively. After booster, vaccinated groups seroconverted and the vaccines induced protection upon challenge. Reverse Genetics technique can be used to produce new and quickly updated swine influenza vaccines which is promising to control the virus in Brazilian herds. Future studies may focus on using the technology to produce multivalent recombinant vaccines against distinct strains of SIAVs circulating in Brazilian pig herds. •At least four lineages of swine influenza virus circulate in Brazilian pig herds.•The circulation of different strains poses challenges to control the disease in the country.•Searching for different vaccination methods with efficient adjuvants is crucial for swine influenza control.•The reverse genetics' technique constitutes an important alternative platform for obtaining safe and effective vaccines.

In recent years, the four main swine influenza A virus (IAV-S) subtypes circulating in swine in the EU have been H1avN1, H1huN2, H1N1pdm09, and H3N2. The latter emerged in 1984 from a reassortment event between a human seasonal H3N2 and H1avN1, and is currently detected at low prevalence in swine in Italy. Here, we describe nine H3N2 IAV-S isolates belonging to three novel genotypes, first detected in Italy in 2021, likely resulting from reassortment events between swine and human IAVs. The first genotype was characterized by a hemagglutinin (H3 HA) of human seasonal origin, a neuraminidase (N2 NA) derived from H1huN2 strains circulating in Italian swine, and an avian-like internal gene cassette (IGC). The second genotype differed in its IGC constellation: PB2, PB1, PA and NP segments were of pandemic origin (pdm09), while NS and M segments derived from the Eurasian avian-like lineage. The third genotype combined a human-derived H3, a Gent/84-derived N2, and a pdm09-origin IGC, except for an avian-like NS. This study aimed to characterize the genetic features of these novel H3huN2 and assess their epidemiological relevance, with implications for surveillance and control, improving preparedness and mitigating the risks posed by zoonotic influenza viruses.

We analyzed >4,000 pig samples from slaughterhouses in Cambodia and found higher influenza A seroprevalence (40.0%) and prevalence (1.5%) among pigs from commercial farms than smallholder farms (seroprevalence 8.9%; prevalence 0.6%). Multivariable analyses revealed evidence of transmission after leaving farms. Findings have implications for influenza risk and surveillance in emerging livestock systems.

Influenza A(H1N1)pdm09 (pH1N1) virus has become established in swine in the United Kingdom and currently co-circulates with previously enzootic swine influenza A virus (IAV) strains, including avian-like H1N1 and human-like H1N2 viruses. During 2010, a swine influenza A reassortant virus, H1N2r, which caused mild clinical disease in pigs in the United Kingdom, was isolated. This reassortant virus has a novel gene constellation, incorporating the internal gene cassette of pH1N1-origin viruses and hemagglutinin and neuraminidase genes of swine IAV H1N2 origin. We investigated the pathogenesis and infection dynamics of the H1N2r isolate in pigs (the natural host) and in ferrets, which represent a human model of infection. Clinical and virologic parameters were mild in both species and both intraspecies and interspecies transmission was observed when initiated from either infected pigs or infected ferrets. This novel reassortant virus has zoonotic and reverse zoonotic potential, but no apparent increased virulence or transmissibility, in comparison to pH1N1 viruses.

Pigs are important intermediate hosts for generating novel influenza viruses. The Eurasian avian-like H1N1 (EAH1N1) swine influenza viruses (SIVs) have circulated in pigs since 1979, and human cases associated with EAH1N1 SIVs have been reported in several countries. However, the biologic properties of EAH1N1 SIVs are largely unknown. Here, we performed extensive influenza surveillance in pigs in China and isolated 228 influenza viruses from 36,417 pigs. We found that 139 of the 228 strains from pigs in 10 provinces in China belong to the EAH1N1 lineage. These viruses formed five genotypes, with two distinct antigenic groups, represented by A/swine/Guangxi/18/2011 and A/swine/Guangdong/104/2013, both of which are antigenically and genetically distinct from the current human H1N1 viruses. Importantly, the EAH1N1 SIVs preferentially bound to human-type receptors, and 9 of the 10 tested viruses transmitted in ferrets by respiratory droplet. We found that 3.6% of children (≤10 y old), 0% of adults, and 13.4% of elderly adults (≥60 y old) had neutralization antibodies (titers ≥40 in children and ≥80 in adults) against the EAH1N1 A/swine/Guangxi/18/2011 virus, but none of them had such neutralization antibodies against the EAH1N1 A/swine/Guangdong/104/2013 virus. Our study shows the potential of EAH1N1 SIVs to transmit efficiently in humans and suggests that immediate action is needed to prevent the efficient transmission of EAH1N1 SIVs to humans.

•Chimeric bat influenza viruses as vaccines show no reassortment with classical influenza viruses.•Novel vaccines reduce virus replication and pathology of pigs infected with a heterologous virus.•Novel vaccines reduce virus nasal shedding in immunized and challenged pigs.•Novel vaccines induce robust mucosal and T-cell immune responses in pigs without VAERD.•Bat influenza vectored vaccines are effective and safe without VAERD and reassortment. Swine influenza is an important disease for the swine industry. Currently used whole inactivated virus (WIV) vaccines can induce vaccine-associated enhanced respiratory disease (VAERD) in pigs when the vaccine strains mismatch with the infected viruses. Live attenuated influenza virus vaccine (LAIV) is effective to protect pigs against homologous and heterologous swine influenza virus infections without inducing VAERD but has safety concerns due to potential reassortment with circulating viruses. Herein, we used a chimeric bat influenza Bat09:mH3mN2 virus, which contains both surface HA and NA gene open reading frames of the A/swine/Texas/4199–2/1998 (H3N2) and six internal genes from the novel bat H17N10 virus, to develop modified live-attenuated viruses (MLVs) as vaccine candidates which cannot reassort with canonical influenza A viruses by co-infection. Two attenuated MLV vaccine candidates including the virus that expresses a truncated NS1 (Bat09:mH3mN2-NS1-128, MLV1) or expresses both a truncated NS1 and the swine IL-18 (Bat09:mH3mN2-NS1-128-IL-18, MLV2) were generated and evaluated in pigs against a heterologous H3N2 virus using the WIV vaccine as a control. Compared to the WIV vaccine, both MLV vaccines were able to reduce lesions and virus replication in lungs and limit nasal virus shedding without VAERD, also induced significantly higher levels of mucosal IgA response in lungs and significantly increased numbers of antigen-specific IFN-γ secreting cells against the challenge virus. However, no significant difference was observed in efficacy between the MLV1 and MLV2. These results indicate that bat influenza vectored MLV vaccines can be used as a safe live vaccine to prevent swine influenza.