Genomic diversity and evolutionary dynamics of Influenza A viruses in Colombian swine: implications for one health surveillance and control.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

Detection and Characterization of Influenza A Virus Endemic Circulation in Suckling and Nursery Pigs Originating from Vaccinated Farms in the Same Production System.

Inactivated influenza A virus (IAV) vaccines help reduce clinical disease in suckling piglets, although endemic infections still exist. The objective of this study was to evaluate the detection of IAV in suckling and nursery piglets from IAV-vaccinated sows from farms with endemic IAV infections. Eight nasal swab collections were obtained from 135 two-week-old suckling piglets from four farms every other week from March to September 2013. Oral fluid samples were collected from the same group of nursery piglets. IAV RNA was detected in 1.64% and 31.01% of individual nasal swabs and oral fluids, respectively. H1N2 was detected most often, with sporadic detection of H1N1 and H3N2. Whole-genome sequences of IAV isolated from suckling piglets revealed an H1 hemagglutinin (HA) from the 1B.2.2.2 clade and N2 neuraminidase (NA) from the 2002A clade. The internal gene constellation of the endemic H1N2 was TTTTPT with a pandemic lineage matrix. The HA gene had 97.59% and 97.52% nucleotide and amino acid identities, respectively, to the H1 1B.2.2.2 used in the farm-specific vaccine. A similar H1 1B.2.2.2 was detected in the downstream nursery. These data demonstrate the low frequency of IAV detection in suckling piglets and downstream nurseries from farms with endemic infections in spite of using farm-specific IAV vaccines in sows.

Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine.

BACKGROUND: Influenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility. METHODS: This study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated. RESULTS: Only intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens. CONCLUSIONS: All findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

Introductions of Human-Origin Seasonal H3N2, H1N2 and Pre-2009 H1N1 Influenza Viruses to Swine in Brazil.

In South America, the evolutionary history of influenza A virus (IAV) in swine has been obscured by historically low levels of surveillance, and this has hampered the assessment of the zoonotic risk of emerging viruses. The extensive genetic diversity of IAV in swine observed globally has been attributed mainly to bidirectional transmission between humans and pigs. We conducted surveillance in swine in Brazil during 2011-2020 and characterized 107 H1N1, H1N2, and H3N2 IAVs. Phylogenetic analysis based on HA and NA segments revealed that human seasonal IAVs were introduced at least eight times into swine in Brazil since the mid-late 1980s. Our analyses revealed three genetic clades of H1 within the 1B lineage originated from three distinct spillover events, and an H3 lineage that has diversified into three genetic clades. The N2 segment from human seasonal H1N2 and H3N2 viruses was introduced into swine six times and a single introduction of an N1 segment from the human H1N1 virus was identified. Additional analysis revealed further reassortment with H1N1pdm09 viruses. All these introductions resulted in IAVs that apparently circulate only in Brazilian herds. These results reinforce the significant contributions of human IAVs to the genetic diversity of IAV in swine and reiterate the importance of surveillance of IAV in pigs.

Evaluation of two multiplex RT-PCR assays for detection and subtype differentiation of Brazilian swine influenza viruses.

Influenza A virus (IAV) subtypes H1N1, H1N2, and H3N2 are endemic in swine herds in most pork producing countries; however, the viruses circulating in different geographic regions are antigenically and genetically distinct. In this sense, the availability of a rapid diagnostic assay to detect locally adapted IAVs and discriminate the virus subtype in clinical samples from swine is extremely important for monitoring and control of the disease. This study describes the development and validation of a multiplex RT-PCR assay for detection and subtyping of IAV from pigs. The analytical and diagnostic specificity of the assays was 100% (94.3-100.0, CI 95%), and the limit of detection was 10-3 TCID50/mL. A total of 100 samples (IAV isolates and clinical specimens) were tested, and the virus subtype was determined for 80 samples (80%; 71.1-86.7, CI 95%). From these, 50% were H1N1, 22.5% were H1N2, and 7.5% were H3N2. Partial subtyping was determined for 8.75% samples (H1pdmNx and HxN2). Additionally, mixed infections with two virus subtypes (H1N2 + H3N2 and H1N1pdm + H1pdmN2; 2.5%) and reassortant viruses (H1pdmN2, 6.25%; and H1N1hu, 2.5%) were detected by the assay. A rapid detection of the most prevalent IAV subtypes and lineages in swine is provided by the assays developed here, improving the IAV diagnosis in Brazilian laboratories, and contributing to the IAV monitoring.

Infection of novel reassortant H1N2 and H3N2 swine influenza A viruses in the guinea pig model.

Novel H1N2 and H3N2 swine influenza A viruses (IAVs) were identified in commercial farms in Chile. These viruses contained H1, H3 and N2 sequences, genetically divergent from IAVs described worldwide, associated with pandemic internal genes. Guinea pigs were used as human surrogate to evaluate the infection dynamics of these reassortant viruses, compared with a pandemic H1N1 virus. All viruses replicated and were shed in the upper respiratory tract without prior adaptation although H1N2 viruses showed the highest shedding titers. This could have public health importance, emphasizing the need to carry out further studies to evaluate the zoonotic potential of these viruses.

Complete genome sequence of a novel influenza A H1N2 virus circulating in swine from Central Bajio region, Mexico.

The aim of this study was to perform the complete genome sequence of a swine influenza A H1N2 virus strain isolated from a pig in Guanajuato, México (A/swine/Mexico/GtoDMZC01/2014) and to report its seroprevalence in 86 counties at the Central Bajio zone. To understand the evolutionary dynamics of the isolate, we undertook a phylogenetic analysis of the eight gene segments. These data revealed that the isolated virus is a reassortant H1N2 subtype, as its genes are derived from human (HA, NP, PA) and swine (M, NA, PB1, PB2 and NS) influenza viruses. Pig serum samples were analysed by the hemagglutination inhibition test, using wild H1N2 and H3N2 strains (A/swine/México/Mex51/2010 [H3N2]) as antigen sources. Positive samples to the H1N2 subtype were processed using the field-isolated H1N1 subtype (A/swine/México/Ver37/2010 [H1N1]). Seroprevalence to the H1N2 subtype was 26.74% in the sampled counties, being Jalisco the state with highest seroprevalence to this subtype (35.30%). The results herein reported demonstrate that this new, previously unregistered influenza virus subtype in México that shows internal genes from other swine viral subtypes isolated in the past 5 years, along with human virus-originated genes, is widely distributed in this area of the country.

Swine Influenza Virus (H1N2) Characterization and Transmission in Ferrets, Chile.

Phylogenetic analysis of the influenza hemagglutinin gene (HA) has suggested that commercial pigs in Chile harbor unique human seasonal H1-like influenza viruses, but further information, including characterization of these viruses, was unavailable. We isolated influenza virus (H1N2) from a swine in a backyard production farm in Central Chile and demonstrated that the HA gene was identical to that in a previous report. Its HA and neuraminidase genes were most similar to human H1 and N2 viruses from the early 1990s and internal segments were similar to influenza A(H1N1)pdm09 virus. The virus replicated efficiently in vitro and in vivo and transmitted in ferrets by respiratory droplet. Antigenically, it was distinct from other swine viruses. Hemagglutination inhibition analysis suggested that antibody titers to the swine Chilean H1N2 virus were decreased in persons born after 1990. Further studies are needed to characterize the potential risk to humans, as well as the ecology of influenza in swine in South America.

Two years of surveillance of influenza a virus infection in a swine herd. Results of virological, serological and pathological studies.

Swine farms provide a dynamic environment for the evolution of influenza A viruses (IAVs). The present report shows the results of a surveillance effort of IAV infection in one commercial swine farm in Argentina. Two cross-sectional serological and virological studies (n=480) were carried out in 2011 and 2012. Virus shedding was detected in nasal samples from pigs from ages 7, 21 and 42-days old. More than 90% of sows and gilts but less than 40% of 21-days old piglets had antibodies against IAV. In addition, IAV was detected in 8/17 nasal swabs and 10/15 lung samples taken from necropsied pigs. A subset of these samples was further processed for virus isolation resulting in 6 viruses of the H1N2 subtype (δ2 cluster). Pathological studies revealed an association between suppurative bronchopneumonia and necrotizing bronchiolitis with IAV positive samples. Statistical analyses showed that the degree of lesions in bronchi, bronchiole, and alveoli was higher in lungs positive to IAV. The results of this study depict the relevance of continuing long-term active surveillance of IAV in swine populations to establish IAV evolution relevant to swine and humans.

Whole-Genome Characterization of a Novel Human Influenza A(H1N2) Virus Variant, Brazil.

We report the characterization of a novel reassortant influenza A(H1N2) virus not previously reported in humans. Recovered from a a pig farm worker in southeast Brazil who had influenza-like illness, this virus is a triple reassortant containing gene segments from subtypes H1N2 (hemagglutinin), H3N2 (neuraminidase), and pandemic H1N1 (remaining genes).

Swine Influenza Virus and Association with the Porcine Respiratory Disease Complex in Pig Farms in Southern Brazil.

Despite the putative endemic status of swine influenza A virus (swIAV) infections, data on the occurrence of swine influenza outbreaks are scarce in Brazil. The aim of this study was to detect and subtype swIAVs from six outbreaks of porcine respiratory disease complex (PRDC) in southern Brazil. Nasal swabs were collected from 66 piglets with signs of respiratory disease in six herds. Lung tissue samples were collected from six necropsied animals. Virus detection was performed by PCR screening and confirmed by virus isolation and hemagglutination (HA). Influenza A subtyping was performed by a real-time reverse transcriptase PCR (rRT-PCR) to detect the A(H1N1)pdm09; other swIAV subtypes were determined by multiplex RT-PCR. In lung tissues, the major bacterial and viral pathogens associated with PRDC (Pasteurella multocida, Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Haemophilus parasuis and PCV2) were investigated. In some affected pigs, clinico-pathological evaluations were conducted. Influenza A was detected by screening PCR in 46 of 66 swab samples and from five of six lungs. Virus was recovered from pigs of all six herds. Subtype A(H1N1)pdm09 was detected in four of six herds and H1N2 in the other two herds. In lung tissues, further agents involved in PRDC were detected in all cases; Pasteurella multocida was identified in five of six samples and Mycoplasma hyopneumoniae in three of six. Actinobacillus pleuropneumoniae (1/6), Haemophilus parasuis (1/6) and PCV2 (1/6) were also detected. These findings indicate that subtypes A(H1N1)pdm09 and H1N2 were present in pigs in southern Brazil and were associated with PRDC outbreaks.

A human-like H1N2 influenza virus detected during an outbreak of acute respiratory disease in swine in Brazil.

Passive monitoring for detection of influenza A viruses (IAVs) in pigs has been carried out in Brazil since 2009, detecting mostly the A(H1N1)pdm09 influenza virus. Since then, outbreaks of acute respiratory disease suggestive of influenza A virus infection have been observed frequently in Brazilian pig herds. During a 2010-2011 influenza monitoring, a novel H1N2 influenza virus was detected in nursery pigs showing respiratory signs. The pathologic changes were cranioventral acute necrotizing bronchiolitis to subacute proliferative and purulent bronchointerstitial pneumonia. Lung tissue samples were positive for both influenza A virus and A(H1N1)pdm09 influenza virus based on RT-qPCR of the matrix gene. Two IAVs were isolated in SPF chicken eggs. HI analysis of both swine H1N2 influenza viruses showed reactivity to the H1δ cluster. DNA sequencing was performed for all eight viral gene segments of two virus isolates. According to the phylogenetic analysis, the HA and NA genes clustered with influenza viruses of the human lineage (H1-δ cluster, N2), whereas the six internal gene segments clustered with the A(H1N1)pdm09 group. This is the first report of a reassortant human-like H1N2 influenza virus derived from pandemic H1N1 virus causing an outbreak of respiratory disease in pigs in Brazil. The emergence of a reassortant IAV demands the close monitoring of pigs through the full-genome sequencing of virus isolates in order to enhance genetic information about IAVs circulating in pigs.

Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus.

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.

Effective seroconversion and safety following the pandemic influenza vaccination (anti-H1N1) in patients with juvenile idiopathic arthritis.

OBJECTIVES: To assess the vaccine response in juvenile idiopathic arthritis (JIA) as an extension of previous observation of immunogenicity and safety of a non-adjuvanted influenza A H1N1/2009 vaccine in a large population of juvenile rheumatic diseases. Moreover, to assess the possible influence of demographic data, disease subtypes, disease activity, and treatment on immunogenicity and the potential deleterious effect of the vaccine in the disease itself, particularly in the number of arthritis and inflammatory markers. METHODS: A total of 95 patients with JIA and 91 healthy controls were evaluated before and 21 days after vaccination, and serology for anti-H1N1 was performed by haemagglutination inhibition assay (HIA). Patient and physician visual analogue scales (VAS), Childhood Health Assessment Questionnaire (CHAQ), number of active joints, acute phase reactants, and treatments were evaluated before and after vaccination. Adverse events were also reported. RESULTS: JIA patients and controls were comparable regarding mean current age (14.9 ± 3.2 vs. 14.6 ± 3.7 years, p = 0.182). After vaccination, the seroconversion rate was significantly lower in JIA patients compared to controls (83.2% vs. 95.6%, p = 0.008), particularly in the polyarticular subtype (80% vs. 95.6%, p = 0.0098). Of note, JIA subtypes, number of active joints, acute phase reactants, CHAQ, patient and physician VAS, and use of disease-modifying anti-rheumatic drugs (DMARDs)/immunosuppressive drugs were similar between seroconverted and non-seroconverted patients (p > 0.05). Regarding vaccine safety, no deterioration was observed in the number of active joints and acute phase reactants during the study period. CONCLUSION: Influenza A H1N1/2009 vaccination in JIA induces a lower but effective protective antibody response probably independent of disease parameters and treatment with an adequate disease safety profile.