Search Results (172)

Surveillance of swine influenza A virus (swIAV) traditionally focuses on respiratory matrices, yet emerging evidence suggests that fecal shedding and secondary environmental contamination may also contribute to viral dissemination. In this study, we collected and analyzed nasal, rectal, environmental, milk, and colostrum samples from naturally infected pigs in a commercial farm in Minas Gerais, Brazil. IAV RNA was detected in 25% of samples, including 42% from asymptomatic animals, with nasal swabs showing higher detection rates (30%) than rectal swabs (20%), though rectal Ct values were consistently higher, indicative of lower viral loads. We successfully isolated viable viruses from feces and effluent samples. Whole-genome sequencing revealed co-circulation of enzootic pH1N1 clade #2 (HA) and pN1 clade #4 (NA), alongside human-origin H3N2 sequences clustering within clade 3C.2a1b.2a.2a.1, and N2 segments related to pre-3C human lineages from 2001 to 2002. Phylogenetic and p-distance analyses support both recent reverse zoonosis and historical transmission events. Detection of complete HA/NA sequences from rectal swabs and treated effluent further emphasizes the surveillance value of non-respiratory matrices. The integration of respiratory and fecal/environmental sampling appears important to achieve more comprehensive IAV monitoring in swine herds and may have significant implications for One Health strategies in Brazil and beyond. Full article

Background/Objectives: Swine influenza A virus (swIAV), a prevalent respiratory pathogen in porcine populations, poses substantial economic losses to global livestock industries and represents a potential threat to public health security. Neuraminidase (NA) has been proposed as an important component for universal influenza vaccine development. NA has potential advantages as a vaccine antigen in providing cross-protection, with specific antibodies that have a broad binding capacity for heterologous viruses. In this study, we evaluated the immunogenicity and protective efficacy of a tetrameric recombinant NA subunit vaccine in a swine model. Methods: We constructed and expressed structurally stable soluble tetrameric recombinant NA (rNA) and prepared subunit vaccines by mixing with ISA 201 VG adjuvant. The protective efficacy of rNA-ISA 201 VG was compared to that of a commercial whole inactivated virus vaccine. Pigs received a prime-boost immunization (14-day interval) followed by homologous viral challenge 14 days post-boost. Results: Both rNA-ISA 201 VG and commercial vaccine stimulated robust humoral responses. Notably, the commercial vaccine group exhibited high viral-binding antibody titers but very weak NA-specific antibodies, whereas rNA-ISA 201 VG immunization elicited high NA-specific antibody titers alongside substantial viral-binding antibodies. Post-challenge, both immunization with rNA-ISA 201 VG and the commercial vaccine were effective in inhibiting viral replication, reducing viral load in porcine respiratory tissues, and effectively mitigating virus-induced histopathological damage, as compared to the PBS negative control. Conclusions: These findings found that the anti-NA immune response generated by rNA-ISA 201 VG vaccination provided protection comparable to that of a commercial inactivated vaccine that primarily induces an anti-HA response. Given that the data are derived from one pig per group, there is a requisite to increase the sample size for more in-depth validation. This work establishes a novel strategy for developing next-generation SIV subunit vaccines leveraging NA as a key immunogen. Full article

The H1N1 swine influenza viruses CQ91 and CQ445, isolated from pigs in China, exhibited distinct virulence in mice despite sharing similar genomic constellations. CQ91 demonstrated higher pathogenicity (MLD₅₀: 5.4 log₁₀ EID₅₀) and replication efficiency in mice compared to CQ445 (MLD₅₀: 6.6 log₁₀ EID₅₀). Through reverse genetics, we found that the attenuation of CQ445 was due to a single substitution of glutamic acid (E) with lysine (K) at position 343 in the PB2 protein. Introducing the CQ445-PB2 (343K) into CQ91 significantly reduced viral replication and pathogenicity in mice, while replacing CQ445-PB2 with CQ91-PB2 (343E) restored virulence. In vitro studies showed that the K343E mutation impaired viral replication in MDCK and A549 cells and reduced polymerase activity in minigenome assays. Mechanistically, the amino acid at position 343 in the PB2 affects the transcription stage of the viral replication process. Structural modeling indicated that the charge reversal caused by E343K altered local electrostatic interactions without major conformational changes. Phylogenetic analysis revealed that PB2-343E is highly conserved (>99.9%) in human and swine H1/H3 influenza viruses, suggesting that PB2-343E confers an adaptive advantage. This study identifies PB2-343E as a critical determinant of influenza virus pathogenicity in mammals, highlighting its role in host adaptation. Full article

Understanding how sample type may influence the probability of influenza A virus (IAV) sequencing and isolation success can help improve the use of diagnostic tests and refine surveillance strategies in swine populations. The objective of this study was to evaluate the probability of success for IAV hemagglutinin (HA) and neuraminidase (NA) Sanger sequencing and virus isolation in Madin–Darby Canine Kidney (MDCK) cells across different porcine sample types submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2018 to 2024. Antemortem and postmortem sample types were selected and analyzed based on reverse transcription real-time polymerase chain reaction (RT-rtPCR) cycle threshold (Ct) values. The Ct values corresponding to 95%, 75%, and 50% probabilities of sequencing or virus isolation success were determined for each sample type. For antemortem samples, a 95% probability of success for HA Sanger sequencing on nasal swabs exhibited a Ct value of 27.8 from 1046 samples and 23.6 for NA sequencing based on 66 nasal swabs. Using oral fluids, HA and NA Sanger sequencing success was at Ct values of 27.3 from 3446 samples and 22.1 from 137 samples, respectively. For postmortem samples, lung tissue had the highest number of sequences for the HA and NA, with Ct values of 25.7 and 21.5, respectively. For a 95% probability of successful virus isolation, nasal swabs demonstrated a Ct value of 21.1 from 647 samples, while lungs had a Ct value of 18.7 from 5892 samples. This study determined that nasal swabs and lung tissue had the highest probability of IAV gene sequencing and virus isolation success, while oral fluids, a common swine diagnostic sample type that is easy to collect and welfare-friendly, can be effective for gene sequencing when using lower IAV RT-rtPCR Ct values, i.e., ≤27.3. These results provide practical expectations for successful IAV HA and NA gene sequencing and virus isolation at 95%, 75%, and 50% probabilities based on sample type and RT-rtPCR Ct values to improve diagnostic testing strategies in swine populations. Full article

Previously, we developed a porcine bronchial epithelial cell line designated as PBE cells and demonstrated that this cell line possesses functional Toll-like receptor 3 (TLR3), triggering the expressions of interferons (IFNs), antiviral factors, and inflammatory cytokines after its stimulation with the synthetic double-stranded ARN poly(I:C). In this work, we aimed to further characterize the PBE cell line as a reliable in vitro model for investigating swine influenza virus (SIV) infection and immunity. We evaluated the capacity of two SIV subtypes, H1N1 and H3N2, to replicate and induce cytopathic effects in PBE cells and to modulate the expressions of IFNs, antiviral factors, inflammatory cytokines, and negative regulators of the TLR signaling. We demonstrated that PBE cells are susceptible to both H1N1 and H3N2. SIV infected PBE cells inducing notable cytopathic effects as shown by the alteration of transepithelial electrical resistance (TEER) and cilia. Both SIV subtypes replicated in PBE cells in similar proportion and altered TEER values in comparable magnitudes. However, SIV H3N2 induced higher alterations of cilia than H1N1. SIV infection induced changes in all the immune factors evaluated in PBE cells. We detected quantitative differences when the subtypes H1N1 and H3N2 were compared. The fold expression changes of IFN-β, Mx1, Mx2, IFITM1, OAS1, OAS2, and OASL were higher in PBE cells infected with H3N2 than in cells challenged with H1N1. In addition, although both subtypes stimulated IL-8 expression, only the H3N2 induced IL-6 in infected PBE cells. SIV H1N1 and H3N2 also upregulated the expressions of the negative regulators A20, BCL-3, and MKP-1, while only H1N1 increased SIGIRR and Tollip. Immortalized respiratory cell lines from pigs can be useful in vitro systems for the study of viral infections and immune responses. These studies are of importance in the context of influenza infections not only for the agricultural field because pigs are natural hosts of these viruses but also because these animals serve as intermediate reservoirs of viruses that can threaten humans’ health. We demonstrated here that the PBE cell line can be a useful in vitro model to study SIV infection and immunity. Full article

Cyanohydrin synthesis, as the simplest preparative method for introducing a carboxyl group into a piperidine molecule, has been used to obtain potentially biologically active piperidinecarboxylic acids, which have alkyl and arylalkyl radicals at the nitrogen atom of the piperidine ring. Hydrochlorides of cyclopropanecarboxylic acid esters based on piperidinecarboxylic acids, as well as hydrochlorides of fluorobenzoic acid esters of N-substituted piperidines, have been synthesized. The purpose of this study was to search for antiviral drugs among new piperidine derivatives. The structure of the synthesized compounds was studied by NMR methods, including COSY (¹H-¹H), HMQC (¹H-¹³C) and HMBC (¹H-¹³C) techniques. The values of chemical shifts, multiplicities, and integrated intensities of ¹H and ¹³C signals in one-dimensional NMR spectra were determined. The results of COSY (¹H-¹H), HMQC (¹H-¹³C), and HMBC (¹H-¹³C) revealed homo- and heteronuclear interactions, confirming the structure of the studied compounds. The antiviral and cytotoxic activities of the synthesized compounds were studied. The antiviral activity in vitro was determined according to the therapeutic regimen against the influenza A/Swine/Iowa/30 (H1N1) virus on the MDCK cell model. The cytotoxicity of the studied substances in vitro was assessed using the MTT test. Based on the results of the antiviral activity against the influenza A virus, it can be concluded that all substances are effective against the influenza A/H1N1 virus compared to the commercial preparations Tamiflu and Rimantadine. Full article

African swine fever virus (ASFV), a highly contagious and lethal virus affecting domestic and wild pigs, has raised global concerns due to its continued spread across Europe and Asia. While traditional transmission pathways involve suids and soft ticks, this study investigates the potential role of freshwater gastropods as environmental reservoirs capable of sustaining ASFV. We analysed ASFV survival in ten gastropod species after long-term co-incubation with the virus. Viral transcriptional activity, particularly of the late gene B646L and members of the multigene family MGF505, was evaluated in snail faeces up to nine weeks post-infection. Results revealed that several gastropods, including Melanoides tuberculata, Tarebia granifera, Physa fontinalis, and Pomacea bridgesii, support long-term persistence of ASFV, accompanied by increased MGF505 gene expression. Notably, the simultaneous activation of MGF5052R and MGF50511R significantly correlated with higher B646L expression and extended viral survival, suggesting a functional role in ASFV maintenance. Conversely, antiviral (AV) activity assays showed that some gastropod faeces reduced replication of the unrelated Influenza virus, hinting at induced host defences. A negative correlation was observed between AV activity and the expression of MGF505 2R/11R, implying that ASFV may suppress antiviral responses to facilitate persistence. These findings suggest that certain gastropods may serve as overlooked environmental hosts, contributing to ASFV epidemiology via long term viral shedding. Further research is needed to clarify the mechanisms underlying ASFV–host interactions and to assess the ecological and epidemiological implications of gastropods in ASFV transmission cycles. Full article

Background/Objectives: Eurasian avian-like (EA) H1N1 swine influenza viruses, with their persistent evolution and zoonotic potential, seriously threaten both swine and human health. The objective was to develop an effective vaccine against these viruses. Methods: A cold-adapted, temperature-sensitive live-attenuated influenza vaccine (LAIV) candidate, GX18ca, was developed. It was derived from the wild-type EA H1N1 strain A/swine/Guangxi/18/2011 (GX18) through serial passaging in embryonated eggs at temperatures decreasing from 33 °C to 25 °C. Its characteristics were studied in mice, including attenuation, immune responses (mucosal IgA, serum IgG, IFN-γ+ CD4⁺/CD8⁺ T-cell responses), and protective efficacy against homologous (GX18), heterologous EA H1N1 (LN972), and human 2009/H1N1 (SC1) viruses. Results: GX18ca showed cold-adapted and temperature-sensitive phenotypes. In mice, it was attenuated, with viral titers in the nasal turbinates and lungs reduced 1000–10,000-fold compared to the wild-type strain, and it cleared by day 5 post infection. Intranasal immunization elicited strong cross-reactive immune responses. Mucosal IgA had broad reactivity, and serum IgG titers reached high levels. IFN-γ+ CD4^+/CD8⁺ T-cell responses were detected against all the tested viruses. A single dose of GX18ca fully protected against GX18 and LN972 challenges, and two doses significantly reduced SC1 lung viral loads, preventing mortality and weight loss. Conclusions: GX18ca is a promising LAIV candidate. It can induce broad immunity, addressing the cross-protection gaps against evolving EA H1N1 SIVs and zoonotic H1N1 variants, which is crucial for swine influenza control and pandemic preparedness. Full article

Educational farms provide students with hands-on experience in agricultural and animal practices. However, the close contact between humans and farm animals creates a significant interface for zoonotic disease transmission, yet research on infectious diseases in such settings remains limited. This study investigates the ongoing spillovers of human-origin influenza A virus (IAV) into swine at an educational farm in central Chile, describing IAV prevalence, outbreak dynamics, and the genomic characterization of detected strains. The Menesianos educational farm, located in Melipilla, central Chile, houses approximately 40 swine alongside other domestic animals, such as horses and cows. As part of an active IAV surveillance project, monthly nasal swab samples were collected from pigs between June 2019 and September 2023 for IAV detection via RT-qPCR targeting the M gene, with positive samples subsequently sequenced. During the study period, monthly IAV prevalence ranged from 0% to 52.5%, with a notable outbreak detected between May and June 2023. The outbreak lasted 5 weeks, peaking at 52.5% prevalence during week 3. Nine IAV strains were isolated over the study period, eight of which were obtained during weeks 2 and 3 of the outbreak. Phylogenetic analysis revealed that all strains were closely related to the pandemic H1N1 2009 influenza virus, with the closest related strains being those circulating in humans in Chile during the same years. These findings highlight the importance of conducting regular IAV surveillance on educational farms, where close interactions between animals and individuals—particularly children and young people—can facilitate viral spillovers and potential reverse zoonosis events. Full article

The genetic diversity of influenza A virus is a major obstacle that makes vaccine effectiveness variable and unpredictable. Objectives: Current vaccines induce strain-specific immunity that oftentimes fail to protect against divergent strains. Our previous research explored synthetic centralized consensus (CC) vaccines to minimize immunogen-strain divergence and focused on the viral glycoprotein hemagglutinin. Methods: Recently, emerging evidence of neuraminidase (NA)-mediated immunity has shifted vaccine strategies, prompting our development of a CC NA type 1 (N1CC) vaccine based on ancestral N1 sequences and delivered using a human adenovirus type 5 vector Results: The N1CC vaccine elicited antibody responses with NA inhibition activity and induced NA-specific T-cell responses. In lethal influenza challenge models, N1CC fully protected mice from death against human, swine, and avian influenza H1N1 and H5N1 strains. Conclusions: These findings support NA as a protective immunogen and demonstrate the power and efficacy of a centralized consensus NA design. Full article

Pigs serve as critical reservoirs and amplifiers for numerous zoonotic viral diseases, presenting substantial public health challenges in India. This study highlights the epidemiology and emerging trends of key zoonotic viruses associated with pigs, emphasizing their role in endemic and emerging disease dynamics. Japanese encephalitis virus (JEV) persists as a major concern, with pigs acting as amplifying host, while hepatitis E virus (HEV) remains a prominent cause of viral hepatitis, transmitted via contaminated water and pork products. Emerging high-fatality viral zoonoses caused by Nipah virus (NiV) and recurrent threats from swine influenza virus (SIV) demonstrate that the zoonotic landscape is evolving. Furthermore, zoonotic viruses like rotavirus, pseudorabies (ADV or SuHV-1), porcine astrovirus (PAstV), and Torque teno sus virus (TTSuV) reflect the expanding diversity of pig-associated pathogens in India. Emerging evidence also implicates viruses such as Chandipura virus (CHPV) in localized outbreaks, indicating broader zoonotic potential. Novel risks such as swine acute diarrhea syndrome coronavirus (SADS-CoV) and SARS-CoV-2 emphasize the role of pigs as potential intermediaries for pandemic-prone viruses. This comprehensive study evaluates the prevalence, outbreak dynamics, and public health implications of zoonotic viral diseases of pigs in India, providing valuable direction for developing effective control measures. Full article

Background/Objectives: Environmental enrichment (EE) devices are required in various countries and markets to promote animal welfare, with dual-purpose devices more likely to encourage adoption. We developed an EE device that allows pigs to self-administer liquids, designed to align with natural and play behaviors, and utilized a maternal pheromone (MP) to attract pigs to the device. This study aimed to evaluate the efficacy of this device in delivering vaccines for Erysipelas, Ileitis, Mycoplasma, and Influenza to growing pigs. Methods: Pigs were assigned to three treatments groups: Control (unvaccinated), Hand-Vaccinated (via oral gavage or intramuscular injection), and Self-Vaccinated using the EE device. Baseline samples were collected to determine initial antibody status, and serum and oral fluids’ IgG and IgA levels were measured post-vaccination to assess immune response. Four studies were conducted with 36 pigs (12 per treatment) over a 49-day period. Results: Self-vaccination pigs receiving the avirulent live Erysipelas vaccine developed oral and serum antibodies comparable to Hand-Vaccinated pigs. Pigs self-administering the avirulent live Lawsonia intracelluaris vaccine developed oral fluid antibodies. In contrast, pigs who received Mycoplasma or Influenza vaccines through self-vaccination exhibited significantly lower antibody levels compared to the Hand-Vaccinated group. Conclusions: These findings demonstrated that self-vaccination using EE devices for the oral administration of avirulent live vaccines offers benefits such as reduced labor and improved animal welfare. However, killed vaccines did not elicit sufficient antibody responses, suggesting the need for modified vaccine formulations or administration strategies to improve self-vaccination efficacy. Full article

Background: Swine hepatitis E (HEV) is a zoonotic infectious disease caused by the swine hepatitis E virus (SHEV). Open reading frame 3 (ORF3) is a key virulence factor in swine HEV, playing a crucial role in the release of viral particles, the modulation of the host innate immune response, and regulation of autophagy and apoptosis, etc. However, its main function and pathogenic mechanism remain incompletely understood. Results: In our study, adenoviruses ADV4-ORF3 and ADV4-GFP were successfully constructed and mediated the overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP in HepG2 cells. A total of 217 differentially expressed messenger RNAs (mRNAs) were screened by high-throughput sequencing, and 27 statistically significant differentially expressed genes were screened for further quantitative real-time reverse transcription (qRT-PCR) verification by functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]). They are mainly involved in six pathways: the cellular response to unfolded protein, inflammatory response, cytokine activity, TNF signaling pathway, influenza A, and pathways in cancer. In a comparative analysis of transcriptome and mRNA expression profiles of lncRNA sequencing, the results showed that 3 mRNAs of GPX1, MDM4, and CLDN and 39 transcripts overlapped and have been identified. Conclusions: Eight differential genes, HSPA1A, HSPA1B, PLD3, RELA, GPI, SAMHD1, RPS6KA4, and PIK3CB, were successfully verified. Comparing and analyzing the results of the two sequencing methods indicated that the 3 mRNAs of GPX1, MDM4, and CLDN and 39 transcripts overlapped and have been identified in SHEV ORF3-expressing HepG2 cells, which has laid a genetic foundation for the physiological function and mechanism of SHEV ORF3. Full article

Vaccination is a common influenza A virus (IAV) control strategy for pigs. Vaccine efficacy depends on strain cross-protection and effective vaccination program implementation. We evaluated a multi-faceted IAV vaccination strategy which included (a) monthly surveillance of pigs at weaning, (b) selection of epidemiologically relevant strains from farms under surveillance, (c) updating IAV strains in custom-made vaccines, and (d) seasonal mass vaccination with custom-made vaccines given to sows in 35 farrow-to-wean farms within an integrated swine farm system. Reduction of IAV in pigs from vaccinated sows was determined by monthly monitoring of farms for 30 months by IAV rRT-PCR (PCR) testing of nasal wipes collected from litters of piglets at weaning. Hemagglutinin (HA) nucleotide and amino acid (AA) sequence homology of the circulating and vaccine strains was determined by pairwise alignment and AA comparison at antigenic sites. Of the 35 farms monitored, 28 (80%) tested positive at least once, and 481 (5.75%) of 8352 PCR tests were IAV positive. Complete HA sequences were obtained from 54 H1 (22 H1-δ_1B.2.1, 28 H1-γ_1A.3.3.3, and 4 H1-pdm_1A.3.3.2 clades) and 14 H3 (12 IV-A 3.1990.4.1 and 2 IV-B 3.1990.4.2 clades) circulating IAV strains. During the study, custom-made vaccines were updated three times (eight strains total) and administered to sows at five distinct time periods. The HA AA similarity between vaccine and circulating strains ranged from 95% to 99%; however, the 0 to 71% similarity at HA antigenic sites prompted the vaccine updates. Herd IAV prevalence decreased from 40% (14/35) to 2.9% (1/35), accompanied by a numerical reduction in IAV-positive samples post-vaccination. Our results support having a comprehensive approach to controlling influenza in swine herds that includes surveillance, vaccination, and careful program implementation to reduce IAV in pigs. Full article

Pigs serve as a mixing vessel for influenza viruses and can independently promote the emergence of pandemic strains in humans. During our surveillance of pig populations from 2021 to 2023 in China, 11 H1 subtype swine influenza viruses (SIVs) were isolated. All viruses were reassortants, possessing internal genes of identical origins (PB2, PB1, PA, NP, M: pdm09/H1N1 origin, NS: North American triple reassortant origin). The H1N1 isolates were all the dominant G4 EA H1N1 viruses in China. Two H1N2 isolates carried early human pdm09/H1N1 HA genes, suggesting a possible pig-to-human transmission route. Mutations that dictate host range specificity were identified in all isolates, a phenomenon which may enhance the affinity to human receptors. These H1 subtype viruses effectively replicated both in vivo and in vitro without prior adaptation and exhibited different pathogenicity and growth characteristics. Some of the H1 viruses were even found to cause lethal infections in mice. Taken together, our study indicates that the H1 subtype SIVs recently circulating in China pose a potential threat to human health and emphasizes the importance of continuing to closely monitor their evolution and spread. Full article