A polyvalent virosomal influenza vaccine induces broad cellular and humoral immunity in pigs.

BACKGROUND: Influenza A virus (IAV) is endemic in pigs globally and co-circulation of genetically and antigenically diverse virus lineages of subtypes H1N1, H1N2 and H3N2 is a challenge for the development of effective vaccines. Virosomes are virus-like particles that mimic virus infection and have proven to be a successful vaccine platform against several animal and human viruses. METHODS: This study evaluated the immunogenicity of a virosome-based influenza vaccine containing the surface glycoproteins of H1N1 pandemic, H1N2 and H3N2 in pigs. RESULTS: A robust humoral and cellular immune response was induced against the three IAV subtypes in pigs after two vaccine doses. The influenza virosome vaccine elicited hemagglutinin-specific antibodies and virus-neutralizing activity. Furthermore, it induced a significant maturation of macrophages, and proliferation of B lymphocytes, effector and central memory CD4+ and CD8+ T cells, and CD8+ T lymphocytes producing interferon-γ. Also, the vaccine demonstrated potential to confer long-lasting immunity until the market age of pigs and proved to be safe and non-cytotoxic to pigs. CONCLUSIONS: This virosome platform allows flexibility to adjust the vaccine content to reflect the diversity of circulating IAVs in swine in Brazil. The vaccination of pigs may reduce the impact of the disease on swine production and the risk of swine-to-human transmission.

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.

Genetic diversity and evolution of the emerging picornavirus Senecavirus A.

Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32 % genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.

Porcine parvovirus VP1/VP2 on a time series epitope mapping: exploring the effects of high hydrostatic pressure on the immune recognition of antigens.

Porcine parvovirus (PPV) is a DNA virus that causes reproductive failure in gilts and sows, resulting in embryonic and fetal losses worldwide. Epitope mapping of PPV is important for developing new vaccines. In this study, we used spot synthesis analysis for epitope mapping of the capsid proteins of PPV (NADL-2 strain) and correlated the findings with predictive data from immunoinformatics. The virus was exposed to three conditions prior to inoculation in pigs: native (untreated), high hydrostatic pressure (350 MPa for 1 h) at room temperature and high hydrostatic pressure (350 MPa for 1 h) at - 18 °C, and was compared with a commercial vaccine produced using inactivated PPV. The screening of serum samples detected 44 positive spots corresponding to 20 antigenic sites. Each type of inoculated antigen elicited a distinct epitope set. In silico prediction located linear and discontinuous epitopes in B cells that coincided with several epitopes detected in spot synthesis of sera from pigs that received different preparations of inoculum. The conditions tested elicited antibodies against the VP1/VP2 antigen that differed in relation to the response time and the profile of structurally available regions that were recognized.

Low occurrence of pathogenic Yersinia enterocolitica in pig tonsils at slaughter in Southern Brazil.

Yersinia enterocolitica is a foodborne pathogen and pigs are the main reservoir of it in their tonsils. As Brazil is a large producer and exporter of pork meat and information regarding this pathogen is still quite scarce, this study aimed at evaluating the direct detection of Y. enterocolitica followed by pathogenic Y. enterocolitica (PYE) determination in tonsils of slaughtered pigs. For this purpose, 400 pig tonsils were collected from 15 farms in four federally certified slaughterhouses in Southern Brazil. Initially, samples were screened using conventional PCR targeting of the 16sRNA gene, followed by multiplex PCR (mPCR) in order to detect three virulence genes (ail, yadA, and virF) and quantitative real-time PCR (qPCR) for the detection of the ail gene. One hundred and one (25.2%) of the samples tested positive for the 16sRNA gene. However, a PYE was detected in one out of the 101 Y. enterocolitica positive samples. The three virulence genes were determined by mPCR and confirmed by partial DNA sequencing. Thus, a significant occurrence of Y. enterocolitica was observed in pig tonsils from federally inspected slaughterhouses in Brazil, although the presence of pathogenic strains was quite low.

Dynamics of vanishing of maternally derived antibodies of Ungulate protoparvovirus 1 suggests an optimal age for gilts vaccination.

The prevention of Ungulate protoparvovirus 1 (UPV1) infection and consequently the reproductive losses is based on vaccination of all pigs intended for breeding. As maternally derived antibodies (MDA) can interfere with the development of immunity following vaccination, it is important to know the duration of anti-UPV1 MDA to determine the optimal age for the best vaccination efficacy. To elucidate the association between dam and piglet antibody levels against UPV1 and to estimate the decrease rate of MDA, sera and colostrum of 127 gilts (before the first vaccination against UPV1; 15 days after the second vaccine dose; at farrowing; and during the second pregnancy) and sera of 276 piglets (prior to initial colostrum intake; at 7, 21, 57, 87, and 128 days-old) were tested by ELISA. Most gilts (85.8%) had anti-UPV1 antibodies before vaccination and after vaccination all were positive. At 7 days old, the majority of the piglets had anti-UPV1 antibodies, but around 57 days old, only 35.3% were positive and at 87 days old, all were negative. The estimated average half-life of MDA was 29.8 (28.8-30.9) days. A strong correlation was determined between piglet serum at 7 days old with gilt serum at farrowing time (r = 0.77, n = 248, P < 0.001) and with piglet serum at 7 days old with colostrum (r = 0.73, n = 248, P < 0.001). The MDA decreased earlier and the antibody half-life was a little longer than previously reported. Based on these findings, UPV1 vaccination can be performed earlier than usual.

Genetic characterization of porcine circovirus type 2 in captive wild boars in southern Brazil.

Porcine circovirus type 2 (PCV2) has been identified in pig population in Brazil since 2000, but scarce studies involving wild boars with PCV2 infection are reported in the country. This study aimed to perform the genetic characterization of PCV2 detected in clinically healthy captive wild boars from farms located in Southern Brazil. Bronchial and mesenteric lymph nodes from 129 clinically healthy captive wild boars were tested by nested PCR for PCV2 detection. Six out of 38 positive samples (29.5%) were submitted to a quantitative real time PCR (qPCR) and genetic sequencing. Viral load up to 1.19 × 109 viral DNA copies/uL was detected in lymph nodes samples by qPCR. According to the ORF2 gene sequence analysis, all PCV2 samples were classified into PCV2b genotype. Comparisons based on a 702 nt region of the ORF2 of all six isolates revealed a high degree of similarity between these isolates. The ORF2 sequences characterized here share 97.1-100% of nucleotide identity and 95.7-100% of amino acid identity with other PCV2b isolated in Brazil from wild boars and feral pigs. This study reports the first detection and genetic characterization of PCV2b in captive wild boars in Brazil and provides important information on PCV2 infection in this domesticated species.

Detection of the Emerging Picornavirus Senecavirus A in Pigs, Mice, and Houseflies.

Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.

Influenza A Viruses of Human Origin in Swine, Brazil.

The evolutionary origins of the influenza A(H1N1)pdm09 virus that caused the first outbreak of the 2009 pandemic in Mexico remain unclear, highlighting the lack of swine surveillance in Latin American countries. Although Brazil has one of the largest swine populations in the world, influenza was not thought to be endemic in Brazil's swine until the major outbreaks of influenza A(H1N1)pdm09 in 2009. Through phylogenetic analysis of whole-genome sequences of influenza viruses of the H1N1, H1N2, and H3N2 subtypes collected in swine in Brazil during 2009-2012, we identified multiple previously uncharacterized influenza viruses of human seasonal H1N2 and H3N2 virus origin that have circulated undetected in swine for more than a decade. Viral diversity has further increased in Brazil through reassortment between co-circulating viruses, including A(H1N1)pdm09. The circulation of multiple divergent hemagglutinin lineages challenges the design of effective cross-protective vaccines and highlights the need for additional surveillance.

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.

Case-control study evaluating the sow's risk factors associated with stillbirth piglets in Midwestern in Brazil.

Reproductive failure in swine herds is often difficult to diagnose and is important to swine production. The present study aims to identify the potential risk factors (infectious/noninfectious) for stillborn piglets in two commercial swine farms situated in midwestern region of Brazil. The potential risk factors were included in a multivariable logistic model, and the dependent variable was defined as the presence of at least one stillborn piglet in a given litter (yes or no). In the best fit model, two variables from the multivariable analysis, total litter size (p = 0.01), and average birth weight (p = 0.03) were significantly associated with the presence of stillborn piglets at the farms examined in this study. Porcine circovirus type 2 (PCV2) was detected in 29.1 % of the litters. Neither parvovirus (PPV) nor leptospirosis infections were identified in this study, suggesting that they have a minor impact on reproductive disease.

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.

Human-to-swine introductions and onward transmission of 2009 H1N1 pandemic influenza viruses in Brazil.

Introduction: Once established in the human population, the 2009 H1N1 pandemic virus (H1N1pdm09) was repeatedly introduced into swine populations globally with subsequent onward transmission among pigs. Methods: To identify and characterize human-to-swine H1N1pdm09 introductions in Brazil, we conducted a large-scale phylogenetic analysis of 4,141 H1pdm09 hemagglutinin (HA) and 3,227 N1pdm09 neuraminidase (NA) gene sequences isolated globally from humans and swine between 2009 and 2022. Results: Phylodynamic analysis revealed that during the period between 2009 and 2011, there was a rapid transmission of the H1N1pdm09 virus from humans to swine in Brazil. Multiple introductions of the virus were observed, but most of them resulted in self-limited infections in swine, with limited onward transmission. Only a few sustained transmission clusters were identified during this period. After 2012, there was a reduction in the number of human-to-swine H1N1pdm09 transmissions in Brazil. Discussion: The virus underwent continuous antigenic drift, and a balance was established between swine-to-swine transmission and extinction, with minimal sustained onward transmission from humans to swine. These results emphasize the dynamic interplay between human-to-swine transmission, antigenic drift, and the establishment of swine-to-swine transmission in shaping the evolution and persistence of H1N1pdm09 in swine populations.

A retrospective study of porcine reproductive and respiratory syndrome virus infection in Brazilian pigs from 2008 to 2020.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease characterized by reproductive impairment or failure in breeding animals, and a respiratory disease in pigs of any age. Brazil is the fourth largest pork producer and exporter globally, and PRRS virus (PRRSV) infection has never been reported in the country. This study aimed to investigate the status of porcine biological samples from commercial swine herds, quarantined imported boars, wild boars and feral pigs to update PRRS information in Brazil. A total of 14,382 samples were collected from 2008 to 2020, including sera (n = 12,841), plasma (n = 1,000) and oral fluids (n = 541), comprehending 137 herds and free-living pigs in eight Brazilian states. One out of 1,000 (0.1%) plasma and 15 out of 12,841 (0.11%) serum samples tested positive for PRRSV antibodies through ELISA. Upon ELISA retesting, only the plasma sample, from one 8-day-old piglet remained positive. All sixteen previously PRRSV antibody-positive samples were tested through RT-PCR and found to be negative. The presence of false-positive or singleton reactors are quite expected. Thus, the use of different/alternative diagnostic tests is indicated for an efficient PRRSV detection. Taken together, our findings demonstrated no conclusive evidence of PRRSV infection in the tested pigs, highlighting the importance to reinforce the surveillance program to prevent the introduction and eventual dissemination of PRRSV in Brazil.

High rate of viral evolution in the capsid protein of porcine parvovirus.

In recent years, it has been shown that some parvoviruses exhibit high substitution rates, close to those of RNA viruses. In order to monitor and determine new mutations in porcine parvovirus (PPV), recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analysed. These samples, together with sequences retrieved from GenBank, were included in three datasets, consisting of the complete NS1 and VP1 genes and a partial VP1 gene. For each dataset, the nucleotide substitution rate and the molecular clock were determined. Analysis of the PPV field isolates revealed that a recently described amino acid substitution, S436T, appeared to be common in the VP2 protein in the Austrian, Brazilian and German virus populations. Furthermore, new amino acid substitutions were identified, located mainly in the viral capsid loops. By inferring the evolutionary dynamics of the PPV sequences, nucleotide substitution rates of approximately 10(-5) substitutions per site per year for the non-structural protein gene and 10(-4) substitutions per site per year for the capsid protein gene (for both viral protein datasets) were found. The latter rate is similar to those commonly found in RNA viruses. An association of the phylogenetic tree with the molecular clock analysis revealed that the mutations on which the divergence for both capsid proteins was based occurred in the past 30 years. Based on these findings, it was concluded that PPV variants are continuously evolving and that vaccines, which are based mainly on strains isolated about 30 years ago, should perhaps be updated.

Porcine Circovirus 3a Field Strains in Free-Living Wild Boars in Paraná State, Brazil.

Porcine circovirus 3 (PCV-3) was identified in domestic pigs worldwide. Although PCV-3 has also been detected in wild boars, information regarding its circulation in this free-living animal species is scarce. To investigate PCV-3 occurrence in free-living wild boars in Brazil, 70 serum samples collected between January 2017 and June 2019 in Paraná state, Brazil were analyzed by PCR assay. Amplicons measuring 330 bp in length were amplified in seven (10.0%) of the serum samples and confirmed to be PCV3-specific by nucleotide (nt) sequencing. As the amplified products from the serum samples yielded only intermediate levels of viral DNA, lung samples from the seven PCR-positive wild boars were also evaluated by PCR. Of these samples, five lung samples were positive and provided high levels of viral DNA. The three lung samples that presented the highest levels of viral DNA were selected for amplification and sequencing of the whole PCV-3 genome. The three full-length sequences obtained were grouped in PCV-3 clade "a", and the sequences exhibited 100% nucleotide similarity among them. The PCV-3 field strains of this study showed nucleotide and amino acid similarities of 98.5-99.8% and 98.8-100%, respectively, with whole-genome PCV-3 sequences from around the world.

Distribution difference of colostrum-derived B and T cells subsets in gilts and sows.

Piglets are highly vulnerable to infections, but colostrum provides them with some protection. The function of colostrum components is unknown, as is if the amount and subsets of leukocytes in colostrum differ between gilts and sows. This study serially characterized leukocyte populations in colostrum for differential leukocyte counts. Differences in humoral and cellular composition of colostrum between 40 gilts and 40 sows (parities orders 3-4) from a commercial herd were examined. Flow cytometry is a useful tool to identify and quantify leukocyte subsets in sow colostrum. Overall, there were no (p ≥ 0.05) parity differences in total macrophages, granulocytes, and T and B cells. However, the sows' colostrum presented significantly higher (p ≤ 0.05) T lymphocyte subsets than gilts, such as central memory CD4+T cells, effector memory CD4+T cells, and central memory CD8+T cells. Among B-lymphocytes, percentages of SWC7+CD5+ cells were significantly higher in sow colostrum than in that of gilts. As expected, IgG concentrations were significantly higher in sows than in gilts. Colostrum from sows had significantly greater mitogenic activity than colostrum from gilts and this fact can be associated with the potential to accelerate the maturation of a newborn's gastrointestinal tract. Our findings suggest that parity order may be one among other factors influencing the cell population and, consequently, the immune adaptive response in piglets that induces neutralizing antibodies and cellular immune responses to antigens.

Applicability of Raman spectroscopy on porcine parvovirus and porcine circovirus type 2 detection.

Porcine parvovirus (PPV) is one of the major infectious causes of reproductive failure of swine. This disease is characterized by embryonic and fetal infection and death, responsible for important economic losses. PPV is also implicated as a trigger in the development of post-weaning multisystemic wasting syndrome (PMWS) caused by Porcine circovirus type 2 (PCV2). Their detection is PCR-based, which is quite sensitive and specific, but laborious, costly and time-demanding. Therefore, this study aimed to assess Raman spectroscopy (RS) as a diagnostic tool for PPV and PCV2 due to its label-free properties and unique ability to search and identify molecular fingerprints. Briefly, swine testis (ST) cells were inoculated with PPV or PCV2 and in vitro cultured (37 °C, 5% CO2) for four days. Fixed cells were then submitted to RS investigation using a 633 nm laser. A total of 225 spectra centered at 1300 cm-1 was obtained for each sample (5 spectra/cell; 15 cells/replicate; 3 replicates) of PPV-, PCV2-infected and uninfected (control) ST cells. Clear statistical discrimination between samples from both virus-infected cells was achieved with a Principal Component - Linear Discriminant Analysis (PCA-LDA) model, reaching sensitivity rates from 95.55% to 97.77%, respectively to PCV2- and PPV-infected cells. These results were then submitted to a Leave-One-Out (LOO) validation algorithm resulting in 99.97% of accuracy. Extensive band assignment was analyzed and compiled for better understanding of PPV and PCV2 virus-cell interaction, demonstrating that specific protein, lipids and DNA/RNA bands are the most important assignments related to discrimination of virus-infected from uninfected cells. In conclusion, these results represent promising bases for RS application on PCV2 and PPV detection for future diagnostic applications.

Investigation of hemotropic Mycoplasmas in fetuses and sows with reproductive failure.

Swine eperythrozoonosis or porcine hemoplasmosis is an infectious disease caused mainly by Mycoplasma suis and is distributed worldwide. This study investigated the occurrence of porcine hemothropic mycoplasmas (PHMs) in fetuses and sows with reproductive failure. Two hundred and seventy-six samples (80 sows' blood and 196 fetal tissue samples) from 27 farms with reproductive disorders were evaluated. The PHMs DNA was detected in 15 out of 80 (18.7%) sows but it was not detected in the fetuses. The bacterial load ranged from 1.32 × 102 to 2.61 × 105 copies/µL. From the 27 tested herds, 11 (40.7%) showed at least one positive sow per farm. The majority of the reproductive problems observed in PMHs positive sows were stillborn fetuses (46.7%) and stillborn associated with fetal mummification (26.7%). So, we evidenced that porcine hemoplasmas circulate among sows in Brazilian herds, however, its real impact on reproductive problems remains unknown.

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.