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.

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.

No molecular evidence for influenza A virus and coronavirus in bats belonging to the families Phyllostomidae, Vespertilionidae, and Molossidae in the state of São Paulo, Brazil.

This study aimed to evaluate, by molecular methods, the presence of influenza A virus (IAV) and coronavirus in non-hematophagous bats collected in the state of São Paulo, Brazil. Samples of lung tissue and small intestine from 105 bats belonging to three families (Phyllostomidae, Vespertilionidae, and Molossidae) were collected in 22 municipalities in the state of São Paulo. Genetic identification of bats species was performed by amplification and sequencing of a fragment of 710 bp of the mitochondrial COI gene. In the detection of IAV, genomes were performed by RT-PCR, aiming at the amplification of a 245-bp fragment of the IAV matrix (M) protein gene. For coronaviruses, two fragments of 602 and 440 bp corresponding to segments along the gene encoding the RNA-dependent RNA polymerase (RdRp) were targeted. The detection limit for each of the PCRs was also determined. All samples analyzed here were negative for both viruses, and the lower limit of detection of the PCRs for the amplification of influenza virus A and coronavirus was estimated at 3.5 × 103 and 4.59 genomic copies per microliter, respectively. Although bats have been shown to harbor a large number of pathogens, the results of the present study support the theory that virus circulation in bats in the wild often occurs at low viral loads and that our understanding of the complex infectious dynamics of these viruses in wild conditions is still limited.

On the influence of the source of porcine colostrum in the development of early immune ontogeny in piglets.

The effects on the ontogeny of serum cytokines and immune cells caused by feeding suckling piglets with sow/gilt colostrum and milk replacer was assessed in the present study. After farrowing, the piglets born were randomized into six groups: GG and SS (n = 10/group): piglets were kept with their dam; GS (n = 10): piglets were changed from gilts to sows; SG (n = 10): piglets were changed from sows to gilts; GMR (n = 6) and SMR (n = 8): piglets from either gilts or sows were isolated from the dams and were bottle-fed ad libitum with commercial formula milk replacer. The piglets remained in the groups during the first 24 h of life and were later returned to their respective mothers. Serum immunoglobulin concentration and lymphocyte proliferation from the blood, spleen, thymus, and mesenteric lymph node of the piglets were assessed at 24 h and at 28 days of age. Serum cytokine concentrations were measured through a cytokine multiplex assay at 24 h. Overall, piglets suckling on sows (SS and GS) had a higher concentration of serum immunoglobulin at 24 h, which was also associated with a rise in plasma cytokine concentration and greater ability of B and T cells from lymphatic organs and blood mononuclear cells to respond to mitogens. We suggest a bias towards Th1-, Th2-, and Th17-cell polarizing and cytokines during the suckling period, which may be influenced by maternal immunological factors in the colostrum, such as dam parity. All findings suggest sow parity having a possible role, which may contribute to exerting a modulating action on immune response development.

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.

One-step multiplex RT-qPCR for the detection and subtyping of influenza A virus in swine in Brazil.

Pandemic H1N1, human-like H1N2 and H3N2 influenza A (IAV) viruses are co-circulating in swine herds in Brazil. The genetic analysis of the Brazilian IAVs has shown that they are genetically distinct from viruses found in swine in other countries; therefore, an update of the diagnostic assays for IAV detection and subtyping is needed. This study describes the development and validation of a TaqMan based - one-step multiplex RT-qPCR to discriminate the hemagglutinin and neuraminidase genes of the three major IAV subtypes circulating in pigs in Brazil. The RT-qPCR assays presented 100% (95.7-100, CI 95%) of diagnostic sensitivity in the analysis of 85 IAVs, previously characterized by sequencing. The limits of detection ranged from 5.09 × 101 to 5.09 × 103 viral RNA copies/µL. For the analytical specificity, 73 pig samples collected during 2017 and 2018 were analyzed, resulting in the identification of the subtype in 74.0% (62.9-82.7, CI 95%) of samples. From these, 46.3% were H3N2, 33.3% were H1N1, 11.1% were H1N2 and 3.7% were HxN1. Mixed viral infections (3.7%) and reassortant viruses (1.9%) were also detected by the test. This multiplex RT-qPCR assay provides a fast and specific diagnostic tool for identification of different subtypes and lineages of IAV in pigs, contributing to the monitoring of influenza in swine.

Influenza A virus infection in Brazilian swine herds following the introduction of pandemic 2009 H1N1.

Influenza A virus (FLUAV) infections are endemic in pork producing countries worldwide but in Brazil it was not considered an important pathogen in pigs. Since the emergence of 2009 pandemic H1N1 (H1N1pdm) FLUAV, many outbreaks of respiratory disease were observed in pig herds. The aim of this study was to evaluate FLUAV infection in swine in 48 pig farms located in seven Brazilian states with previous reports of influenza-like signs by clinical, serological and virological cross-sectional studies. Serological results showed that pigs from all farms had anti-influenza antibodies by NP-ELISA. Antibodies to H3N2, H1N2 and H1N1pdm were detected by HI in pigs from 24 farms. Co-infection with two or more FLUAV subtypes was detected in pigs in seven of those 24 farms. Detection of FLUAV in nasal swabs and oral fluids by RT-qPCR indicated a global concordance >81% for the two biological samples. Moreover, our results show that H1N1pdm, H1N2 and H3N2 viruses are widespread in Brazilian pig herds. The monitoring of FLUAV emergence and evolution in pigs is urgent, as well the study of the pathogenesis of Brazilian isolates, aiming to control influenza in pigs.