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Abstract In January and November 2008, outbreaks of avian influenza have been reported in 4 provinces of Thailand. Eight Influenza A H5N1 viruses were recovered from these 2008 AI outbreaks and comprehensively characterized and analyzed for nucleotide identity, genetic relatedness, virulence determinants, and possible sites of reassortment. The results show that the 2008 H5N1 viruses displayed genetic drift characteristics (less than 3% genetic differences), as commonly found in influenza A viruses. Based on phylogenetic analysis, clade 1 viruses in Thailand were divided into 3 distinct branches (subclades 1, 1.1 and 1.2). Six out of 8 H5N1 isolates have been identified as reassorted H5N1 viruses, while other isolates belong to an original H5N1 clade. These viruses have undergone inter-lineage reassortment between subclades 1.1 and 1.2 and thus represent new reassorted 2008 H5N1 viruses. The reassorted viruses have acquired gene segments from H5N1, subclade 1.1 (PA, HA, NP and M) and subclade 1.2 (PB2, PB1, NA and NS) in Thailand. Bootscan analysis of concatenated whole genome sequences of the 2008 H5N1 viruses supported the reassortment sites between subclade 1.1 and 1.2 viruses. Based on estimating of the time of the most recent common ancestors of the 2008 H5N1 viruses, the potential point of genetic reassortment of the viruses could be traced back to 2006. Genetic analysis of the 2008 H5N1 viruses has shown that most virulence determinants in all 8 genes of the viruses have remained unchanged. In summary, two predominant H5N1 lineages were circulating in 2008. The original CUK2-like lineage mainly circulated in central Thailand and the reassorted lineage (subclades 1.1 and 1.2) predominantly circulated in lower-north Thailand. To prevent new reassortment, emphasis should be put on prevention of H5N1 viruses circulating in high risk areas. In addition, surveillance and whole genome sequencing of H5N1 viruses should be routinely performed for monitoring the genetic drift of the virus and new reassorted strains, especially in light of potential reassortment between avian and mammalian H5N1 viruses.

Abstract Background Porcine reproductive and respiratory syndrome virus (PRRSV) is a causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS). In this study, the complete nucleotide sequences of the selected two Thai PRRSV isolates, EU (01CB1) and US (01NP1) genotypes were determined since both isolates are the Thai prototypes. Results 01CB1 and 01NP1 contain 14,943 and 15,412 nucleotides, respectively. The viruses compose 2 untranslated regions (5' UTR and 3' UTR) and 8 open reading frames (ORFs) designated as ORF1a, ORF1b and ORF2-7. Phylogenetic analysis of full length of the viruses also showed that the 01CB1 and 01NP1 were grouped into the EU and US genotype, respectively. In order to determine the genetic variation and genetic relatedness among PRRSV isolates, the complete nucleotide sequences of PRRSV isolated in Thailand, 01CB1 and 01NP1 were compared with those of 2 EU strains (Lelystad, and EuroPRRSV), 6 US strains (MLV, VR2332, PA8, 16244B, SP and HUN4). Our results showed that the 01CB1 genome shares approximately 99.2% (Lelystad) and 95.2% (EuroPRRSV) nucleotide identity with EU field strains. While, the 01NP1 genome has 99.9% nucleotide identity with a live vaccine strain (MLV) and 99.5% and 98.5% nucleotide identity with 2 other US isolates, VR2332 and 16244B, respectively. In addition, ORF5 nucleotide sequences of 9 PRRS viruses recovered in Thailand during 2002-2008 were also included in this study. Phylogenetic analysis of ORF5 showed high similarity among EU and US genotypes of the recent Thai PRRS viruses (2007-2008 viruses) with 01CB1 and 01NP1. Conclusion Overall, the results suggested that the Thai EU isolate (01CB1) may evolve from the EU prototype, Lelystad virus, whereas the Thai US isolate (01NP1) may originate and evolve from the vaccine virus or its derivatives. Interestingly, the US-MLV vaccine was not available in the Thai market in 2001. The Vaccine-like virus might have persisted in the imported pigs or semen and later spread in the Thai swine industry. This report is the first report of complete nucleotide sequences of the Thai PRRS viruses both EU and US genotypes.

Duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, is notably associated with neurological disorders and acute egg drop syndrome in ducks. We previously demonstrated that the susceptibility of ducks to DTMUV infection varies significantly with age, with younger ducks (4-week-old) exhibiting more severe disease than older ducks (27-week-old). However, the immunological mechanisms underlying these age-related differences in disease severity remain unclear. In this study, we investigated the dynamics of immune responses following DTMUV infection in adult laying ducks (27-week-old) and compared them to our previous findings on young ducks (4 weeks old). The numbers of T helper, cytotoxic T, B, and non-T and B lymphocytes, as well as neutralizing antibody levels, were measured in parallel with DTMUV loads in the blood and target organs. Our results revealed that the number of non-T and B lymphocytes/myeloid cells in 27-week-old adult laying ducks infected with DTMUV remained consistently stable throughout the observation period, in contrast to findings in 4-week-old younger ducks, where myeloid cell responses were implicated in disease progression. Regarding lymphocyte responses, unlike in 4-week-old younger ducks, only cytotoxic T lymphocyte responses in 27-week-old older ducks showed a significant negative correlation with the reduction of viremia and viral loads in target organs, indicating their role in controlling viral replication in older ducks. Additionally, 27-week-old adult laying ducks infected with DTMUV exhibited high levels of neutralizing antibodies, which were significantly correlated with reduced viral loads in blood and target organs. Overall, the presence of robust DTMUV-specific neutralizing antibody and CTL responses, along with a finely tuned myeloid cell response likely plays a significant role in controlling severe neurological outcomes in 27-week-old adult laying ducks. This study highlights the age-related differences in immune responses following DTMUV infection, which potentially contribute to the varying disease severity among ducks of different ages. Understanding the interplay between the host and DTMUV provides significant implications for disease management strategies and vaccine development., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

While mRNA vaccines have shown their worth, they have the same failing as inactivated vaccines, namely they have limited half-life, are non-replicating, and therefore limited to the size of the vaccine payload for the amount of material translated. New advances averting these problems are combining replicon RNA (RepRNA) technology with nanotechnology. RepRNA are large self-replicating RNA molecules (typically 12-15 kb) derived from viral genomes defective in at least one essential structural protein gene. They provide sustained antigen production, effectively increasing vaccine antigen payloads over time, without the risk of producing infectious progeny. The major limitations with RepRNA are RNase-sensitivity and inefficient uptake by dendritic cells (DCs), which need to be overcome for efficacious RNA-based vaccine design. We employed biodegradable delivery vehicles to protect the RepRNA and promote DC delivery. Condensing RepRNA with polyethylenimine (PEI) and encapsulating RepRNA into novel Coatsome-replicon vehicles are two approaches that have proven effective for delivery to DCs and induction of immune responses in vivo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Rabies vaccination is mandatory in dogs in Thailand. In this study, shelter management quality and rabies immune status were evaluated by questionnaire and rabies virus neutralising antibody (RVNA) measurement. The questionnaire was designed to assess all relevant factors of shelter management, which could impact the rabies vaccine antibody response. Thirteen participating shelters were classified into 4 groups, namely group A (best), B (good), C (fair), and D (require improvement). Sera were collected from randomly selected dogs (n = 113) within 4 weeks after rabies re-vaccination from a representative shelter of group B, C and D. Sample from group A was not included in the study due to time limitation. Both the number of dogs with acceptable response (RVNA ≥ 0.5 IU/ml) and the RVNA titres were significantly higher in group B than group C and D. Our results indicate that the quality of shelter management could affect rabies immune status., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Herein, we provide the first description of a synthetic delivery method for self-replicating replicon RNAs (RepRNA) derived from classical swine fever virus (CSFV) using a Coatsome-replicon vehicle based on Coatsome® SS technologies. This results in an unprecedented efficacy when compared to well-established polyplexes, with up to ∼65 fold-increase of the synthesis of RepRNA-encoded gene of interest (GOI). We demonstrated the efficacy of such Coatsome-replicon vehicles for RepRNA-mediated induction of CD8 T-cell responses in mice. Moreover, we provide new insights on physical properties of the RepRNA, showing that the removal of all CSFV structural protein genes has a positive effect on the translation of the GOI. Finally, we successfully engineered RepRNA constructs encoding a porcine reproductive and respiratory syndrome virus (PRRSV) antigen, providing an example of antigen expression with potential application to combat viral diseases. The versatility and simplicity of modifying and manufacturing these Coatsome-replicon vehicle formulations represents a major asset to tackle foreseeable emerging pandemics., Competing Interests: Declaration of competing interest A patent for the application of delivery vehicles for the delivery of RepRNA vaccines to DCs using replicons derived from classical swine fever virus, as employed in this paper, has been filed in Europe and granted in USA, Canada, and India, with priority date of 4 June 2008. The filing was by Kenneth C. McCullough, Nicolas Ruggli and Jon Duri Tratschin (all three as inventors) (WO 2009146867), and assigned to their employer – the Institute of Virology and Immunology IVI. This does not alter the authors' adherence to the policies of sharing data and materials. Carlos A. Guzmán and Thomas Ebensen are named as inventors in a patent application covering the use of c-di-AMP as adjuvant (PCT/EP 2006010693)., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Veterinary education is the foundation of veterinary services in the country. Starting from the service sector in the army, veterinary education and practice in Thailand have been standardized and progressed toward international veterinary standards. The 6-year Doctor of Veterinary Medicine core curriculum is deployed to develop the curriculum for each Veterinary Education Establishment (VEE). The challenges for veterinary education and practices reflect the country's expectations of veterinary services. With regional and global collaboration, the VEEs have been developing tools and learning platforms for delivering qualified veterinary graduates that fit fast-growing society needs., Competing Interests: The authors declare no conflicts of interest., (© 2022 The Korean Society of Veterinary Science.)

Duck Tembusu virus (DTMUV), an emerging avian pathogenic flavivirus, causes severe neurological disorders and acute egg drop syndrome in ducks. However, the effects of DTMUV on duck immunological components and functions remain largely unknown. In this study, the dynamics of cellular and humoral immune responses of DTMUV-infected ducks were investigated. The numbers of CD4 + and CD8 + T, B and non-T and B lymphocytes as well as the levels of neutralizing antibodies were quantified in parallel with DTMUV loads in blood and target organs. Our results demonstrated that DTMUV infection caused severe losses of non-T and B lymphocyte/myeloid cell subpopulation, and reduction in phagocytic activity during 3-5 days after infection. We also found that the numbers of T and B cells were increased during the first week of DTMUV infection. A significant negative correlation between the levels of CD4 + and CD8 + T, B and non-T and B lymphocytes and viral loads in blood and target organ (spleen) was observed during the early phase of infection. Additionally, DTMUV infection induced an early and robust neutralizing antibody response, which was associated with DTMUV-specific IgM and IgG responses. The presence of neutralizing antibody also correlated with reduction of viremia and viral load in the spleen. Overall, DTMUV elicited both cellular and humoral immune responses upon infection, in which the magnitude of these responses was correlated with reduction of viremia and viral loads in the target organ (spleen). The results suggested the critical role of both cellular and humoral immunity against DTMUV infection. This study expands our understanding of the immunological events following DTMUV infection in ducks., (© 2022 Wiley-VCH GmbH.)

Background: Dermatophagoides farinae (Der f) is a common allergen in dogs with atopic dermatitis (AD). However, the relevant components of Der f require further investigation., Objectives: We aimed to provide data on the immunoglobulin (Ig)E-binding specific components of Der f for further diagnostic and therapeutic applications., Animals: Serum samples were collected from five healthy, nine Der f-allergic atopic and seven non-Der f-allergic atopic dogs identified based on an intradermal skin test., Methods and Materials: We explored the component profiles of Der f extracts through 2D gel electrophoresis and IgE immunoblotting. The IgE-binding components in both groups of atopic dogs were analysed by mass spectrometry., Results: The majority of Der f-allergic atopic dogs recognised Der f Alternaria alternata allergen 10 (Der f Alt a 10), elongation factor 1-alpha (EF1-α), gelsolin-like allergen Der f 16, Der f 28 and Der f 2. Der f 3, Der f 10, Der f 20 and Der f 32 were recognised as minor allergens. Alpha-enolase, serine protease, arginine kinase and a few hypothetical proteins were recognised components in both groups of atopic dogs. Unexpectedly, Der f 15 (chitinase) was found to be a minor component., Conclusions and Clinical Importance: Multiple IgE-binding allergens of Der f were identified in Thai atopic dogs. We propose that the specific antigen set that is bound by IgE, comprising Der f Alt a 10, EF1-α, gelsolin-like Der f 16, Der f 28 and Der f 2, could be used for future diagnostics and immunotherapy platforms., (© 2021 ESVD and ACVD.)

To investigate an outbreak of African horse sickness (AHS) on a horse farm in northeastern Thailand, we used whole-genome sequencing to detect and characterize the virus. The viruses belonged to serotype 1 and contained unique amino acids (95V,166S, 660I in virus capsid protein 2), suggesting a single virus introduction to Thailand.

Background: Nipah virus (NiV) infection causes encephalitis and has > 75% mortality rate, making it a WHO priority pathogen due to its pandemic potential. There have been NiV outbreak(s) in Malaysia, India, Bangladesh, and southern Philippines. NiV naturally circulates among fruit bats of the genus Pteropus and has been detected widely across Southeast and South Asia. Both Malaysian and Bangladeshi NiV strains have been found in fruit bats in Thailand. This study summarizes 20 years of pre-emptive One Health surveillance of NiV in Thailand, including triangulated surveillance of bats, and humans and pigs in the vicinity of roosts inhabited by NiV-infected bats., Methods: Samples were collected periodically and tested for NiV from bats, pigs and healthy human volunteers from Wat Luang village, Chonburi province, home to the biggest P. lylei roosts in Thailand, and other provinces since 2001. Archived cerebrospinal fluid specimens from encephalitis patients between 2001 and 2012 were also tested for NiV. NiV RNA was detected using nested reverse transcription polymerase chain reaction (RT-PCR). NiV antibodies were detected using enzyme-linked immunosorbent assay or multiplex microsphere immunoassay., Results: NiV RNA (mainly Bangladesh strain) was detected every year in fruit bats by RT-PCR from 2002 to 2020. The whole genome sequence of NiV directly sequenced from bat urine in 2017 shared 99.17% identity to NiV from a Bangladeshi patient in 2004. No NiV-specific IgG antibodies or RNA have been found in healthy volunteers, encephalitis patients, or pigs to date. During the sample collection trips, 100 community members were trained on how to live safely with bats., Conclusions: High identity shared between the NiV genome from Thai bats and the Bangladeshi patient highlights the outbreak potential of NiV in Thailand. Results from NiV cross-sectoral surveillance were conveyed to national authorities and villagers which led to preventive control measures, increased surveillance of pigs and humans in vicinity of known NiV-infected roosts, and increased vigilance and reduced risk behaviors at the community level. This proactive One Health approach to NiV surveillance is a success story; that increased collaboration between the human, animal, and wildlife sectors is imperative to staying ahead of a zoonotic disease outbreak.

Among swine genetic markers, the highly polymorphic swine leukocyte antigen (SLA) is one of the key determinants, associated with not only immune responses but also reproductive performance and meat quality. The objective of this study was to characterize the SLA class I and II diversities in the commercial pig populations. In this study, a total number of 158 pigs (126 gilts and 32 boars) were randomly selected from different breeding herds of five major pig-producing companies, which covered ~70% of Thai swine production. The results indicate that a moderate level of SLA diversity was maintained in the Thai swine population, despite the performance-oriented breeding scheme. The highly common SLA class I alleles were SLA-1 * 08:XX, SLA-2 * 02:XX, and SLA-3 * 04:XX at a combined frequency of 30.1, 18.4, and 34.5%, respectively, whereas DRB1 * 04:XX, DQB1 * 02:XX and DQA * 02:XX were the common class II alleles at 22.8, 33.3, and 38.6%, respectively. The haplotype Lr-32.0 (SLA-1 * 07:XX, SLA-2 * 02:XX, and SLA-3 * 04:XX) and Lr-0.23 (DRB1 * 10:XX, DQB1 * 06:XX, DQA * 01:XX) was the most common SLA class I and II haplotype, at 15.5 and 14.6%, respectively. Common class I and II haplotypes were also observed, which Lr-22.15 was the most predominant at 11.1%, followed by Lr-32.12 and Lr-4.2 at 10.8 and 7.9%, respectively. To our knowledge, this is the first report of SLA class I and II diversities in the commercial pigs in Southeast Asia. The information of the common SLA allele(s) in the population could facilitate swine genetic improvement and future vaccine design., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Techakriengkrai, Nedumpun, Golde and Suradhat.)

CRISPR/Cas9 enables dsDNA viral genome engineering. However, the lack of RNA targeting activities limits the ability of CRISPR/Cas9 to combat RNA viruses. The recently identified class II type VI CRISPR/Cas effectors (Cas13) are RNA-targeting CRISPR enzymes that enable RNA cleavage in mammalian and plant cells. We sought to knockdown the viral RNA of porcine reproductive and respiratory syndrome virus (PRRSV) directly by exploiting the CRISPR/Cas13b system. Effective mRNA cleavage by CRISPR/Cas13b-mediated CRISPR RNA (crRNA) targeting the ORF5 and ORF7 genes of PRRSV was observed. To address the need for uniform delivery of the Cas13b protein and crRNAs, an all-in-one system expressing Cas13b and duplexed crRNA cassettes was developed. Delivery of a single vector carrying double crRNAs enabled the simultaneous knockdown of two PRRSV genes. Transgenic MARC-145 cells stably expressing the Cas13b effector and crRNA mediated by lentiviral-based transduction showed a robust ability to splice the PRRSV genomic RNA and subgenomic RNAs; viral infection was almost completely abrogated by the combination of double crRNAs simultaneously targeting the ORF5 and ORF7 genes. Our study indicated that the CRISPR/Cas13b system can effectively knockdown the PRRSV genome in vitro and can potentially be used as a potent therapeutic antiviral strategy.

Impaired innate and adaptive immune responses are evidenced throughout the course of PRRSV infection. We previously reported that interleukin-1 receptor antagonist (IL-1Ra) was involved in PRRSV-induced immunosuppression during an early phase of infection. However, the exact mechanism associated with PRRSV-induced IL-1Ra immunomodulation remains unknown. To explore the immunomodulatory properties of PRRSV-induced IL-1Ra on porcine immune functions, monocyte-derived dendritic cells (MoDC) and leukocytes were cultured with type 2 PRRSV, and the immunological role of IL-1Ra was assessed by addition of anti-porcine IL-1Ra Ab. The results demonstrated that PRRSV - induced IL-1Ra reduced phagocytosis, surface expression of MHC II (SLA-DR) and CD86, as well as downregulation of IFNA and IL1 gene expression in the MoDC culture system. Interestingly, IL-1Ra secreted by the PRRSV-infected MoDC also inhibited T lymphocyte differentiation and proliferation, but not IFN-γ production. Although PRRSV-induced IL-1Ra was not directly linked to IL-10 production, it contributed to the differentiation of regulatory T lymphocytes (Treg) within the culture system. Taken together, our results demonstrated that PRRSV-induced IL-1Ra downregulates innate immune functions, T lymphocyte differentiation and proliferation, and influences collectively with IL-10 in the Treg induction. The immunomodulatory roles of IL-1Ra elucidated in this study increase our understanding of the immunobiology of PRRSV.

The Chinese highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has caused a severe threat to the pig population in Southeast Asian countries. The purpose of this study was to investigate the efficacy of a type 2 PRRSV modified live vaccine (PrimePac™ PRRS, lineage 7) against a Thai HP-PRRSV (10PL01, lineage 8). Three-week-old PRRSV-free pigs were randomly assigned into three groups. Vaccinated challenged group (group 1, n = 16) was immunized with PrimePac™ PRRS vaccine at 3 weeks old. The unvaccinated challenged group (group 2, n = 16) was injected with PBS at 3 weeks old, and unvaccinated unchallenged group (group 3, n = 10) was served as a negative control. At 9 weeks old, all groups, except the negative control group, were challenged with the Thai HP-PRRSV. All pigs were monitored daily during 10 days post-infection (dpi) and were necropsied at 10 and 17 dpi. The results revealed that vaccinated challenged pigs showed significantly lower (p < 0.05) mean rectal temperatures, clinical respiratory scores, lung lesion scores, and levels of virus load in serum and lung tissue compared with the unvaccinated challenged pigs. Moreover, vaccinated challenged pigs exhibited PRRSV-specific serum neutralizing antibodies at the end of the experiment. Our findings indicated that the studied type 2 PRRSV vaccine provided partial protection against the Thai HP-PRRSV infection based on the body temperature, levels of viremia, and the severity of lung lesions. These results demonstrated that partial protection of PrimePac™ PRRS vaccine might be useful for controlling HP-PRRSV infection in the endemic area.

Regulatory T lymphocytes (Treg) residing within the tissues, are known to possess immunosuppressive properties which contribute to immunomodulation within the organs. PRRSV infection usually weakens lung defense mechanisms, leading to porcine respiratory disease complex (PRDC). Induction of circulatory Treg is one of the reported mechanisms involved in PRRSV-induced immunomodulation. However, whether PRRSV can induce tissue-infiltrating Treg in the lungs and lymph nodes is still unclear. To investigate the effect of PRRSV on induction of porcine Treg in the tissues, we isolated mononuclear cells from the lungs and tracheobronchial lymph nodes, and identified the existence of Treg by flow cytometry. The results demonstrated that PRRSV could induce Treg proliferation in the cultured mononuclear cells derived from lungs and tracheobronchial lymph nodes, regardless of the pig's PRRSV infective status. Furthermore, PRRSV-infected pigs exhibited higher numbers of total tissue-infiltrating Treg and PRRSV-specific Treg in the lungs and tracheobronchial lymph nodes than the PRRSV-negative pigs. To determine if the lung Treg could produce an inhibitory cytokine, the numbers of IL-10-producing Treg were determined. Significantly higher numbers of IL-10-producing Treg in the lungs of PRRSV-infected pigs were observed. Altogether, our findings indicate the potent effect of PRRSV on induction of Treg in the lungs and tracheobronchial lymph nodes of the infected pigs. The findings expand our understanding in PRRSV immunopathogenesis within the target organs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is one of the most important swine pathogens, and causes a major economic impact worldwide. Recently, a new variant type 2 PRRSV, highly pathogenic PRRSV (HP-PRRSV) has emerged and continued to circulate in Southeast Asia region. Currently, commercially available PRRSV vaccines, modified live PRRS vaccines (MLV) are not able to provide complete protection against HP-PRRSV and been reported to induce negative immunomodulatory effects. Interestingly, a novel DNA vaccine was developed and successfully used to improve PRRSV-specific immune responses following MLV vaccination. To investigate the efficacy of a heterologous DNA-MLV prime-boost immunization against the HP-PRRSV infection, an experimental vaccinated-challenged study was conducted. Two-week-old, PRRSV-seronegative, crossbred pigs (5-8 pigs/group) were allocated into 5 groups. At day -14 (D-14), the treatment group (DNA-MLV) was immunized with a DNA vaccine encoding PRRSV-truncated nucleocapsid protein (pORF7t), followed by a commercial modified live type 2 PRRS vaccine (MLV) at D0. The other groups included the group that received PBS at D-14 followed by MLV at D0 (MLV), pORF7t at D-14 (DNA), PBS at D0 (PBS) and the negative control group. At D42, all groups, except the negative control group, were challenged with HP-PRRSV (strain 10PL1). The results demonstrated that pigs that received MLV, regardless of the DNA priming, exhibited less clinical signs and faster viral clearance. Following HP-PRRSV challenge, the DNA-MLV group exhibited improved PRRSV-specific immunity, as observed by increased neutralizing antibody titers and PRRSV-specific IFN-γ production, and reduced IL-10 and PRRSV-specific Treg productions. However, neither the prime-boost immunization nor the MLV was able to induce complete clinical protection against HP-PRRSV infection. In conclusion, improved immunological responses, but not clinical protection, were achieved by DNA-MLV prime-boost immunization. This study highlights the potential use of heterologous prime-boost vaccination regimen, where DNA can be incorporated with other vaccine candidates, for improving anti-PRRSV immunity that may eventually lead induction of complete PRRSV protection., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) infection poorly induces pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) and type I IFN production during the early phase of infection. Our microarray analysis indicated strong upregulation of the IL1RA gene in type 2 PRRSV -infected monocyte-derived dendritic cells. Interleukin-1 receptor antagonist (IL-1Ra) is an early inhibitory cytokine that suppresses pro-inflammatory cytokines and T-lymphocyte responses. To investigate the induction of IL-1Ra by PRRSV, monocyte-derived dendritic cells were cultured with type 2 PRRSV or other swine viruses. PRRSV increased both IL1RA gene expression and IL-1Ra protein production in the culture. The enhanced production of IL-1Ra was further confirmed in PRRSV-cultured PBMC and PRRSV-exposed pigs by flow cytometry. Myeloid cell population appeared to be the major IL-1Ra producer both in vitro and in vivo. In contrast to the type 2 PRRSV, the highly pathogenic (HP)- PRRSV did not upregulate IL1RA gene expression in vitro. To determine the kinetics of PRRSV-induced IL1RA gene expression in relation to other pro-inflammatory cytokine genes, PRRSV-negative pigs were vaccinated with a commercially available type 2 modified-live PRRS vaccine or intranasally inoculated with HP-PRRSV. In modified-live PRRS vaccine pigs, upregulation of IL1RA, but not IL1B and IFNA, gene expression was observed from 2 days post- vaccination. Consistent with the in vitro findings, upregulation of IL1RA gene expression was not observed in the HP-PRRSV-infected pigs throughout the experiment. This study identified IL-1Ra as an early immunomodulatory mediator that could be involved in the immunopathogenesis of PRRSV infections.

In vitro derivation of dendritic cells (DCs) is an alternative approach to overcome the low frequency of primary DCs and the difficulty of isolation techniques for studying DC immunobiology. To date, the conventional culture protocol of porcine monocyte-derived DCs (MoDCs) has been widely used. However, this protocol is not practical due to the requirement of a substantial number of blood monocytes, and the process often interferes with DC maturation. To improve in vitro porcine MoDC generation, we modified the previous conventional DC generation protocol, based on the human and mouse primary DC culture system, and compared phenotypic and functional features of MoDCs derived from the modified protocol to the conventional protocol. The modified protocol consumed fewer monocytes but generated higher CD1 + cells with DC-like morphology and the ability of maturation. In addition, MoDCs from the modified protocol exhibited increased antigen uptake and IFN-γ production in response to LPS stimulation. Our findings indicate that the modified protocol is expedient and reliable for generating potent MoDCs that substitute for primary DCs. This will be a valuable platform for future research in antigen delivery, vaccines and immunotherapy in pigs, as well as relevant veterinary species., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Porcine Reproductive and Respiratory Syndrome virus (PRRSV) induces several immunomodulatory mechanisms that resulted in delayed and ineffective anti-viral immune responses. Recently, it has been shown that intradermal immunization of plasmid encoding truncated nucleocapsid protein (pORF7t) could reduce PRRSV-induced immunomodulatory activities and enhances anti-PRRSV immunity in vaccinated pigs. However, intradermal immunization may not be practical for farm setting. Currently, there are several transdermal delivery systems available in the market, although they were not originally designed for plasmid delivery., Objectives: To investigate the potential use of a transdermal delivery system for delivering of pORF7t and its immunological outcomes., Method: The immunomodulatory effects induced by transdermal delivery of pORF7t were compared with intradermal immunization in an experimental pig model. In addition, immunomodulatory effects of the DNA vaccine were determined in the fattening pigs kept in a PRRSV-positive farm environment, and in the experimental pigs receiving heterologous prime-boost, pORF7t-modified live vaccine (MLV) immunization., Result: The patterns of PRRSV-specific cellular responses induced by transdermal and intradermal immunizations of pORF7t were similar. Interestingly, the pigs transdermally immunized with pORF7t exhibited higher number of PRRSV-specific CD8(+)IFN-γ(+) cells. Pigs immunized with pORF7t and kept at PRRSV-positive environment exhibited enhanced PRRSV-specific IFN-γ(+) production, reduced numbers of regulatory T lymphocytes (Tregs) and lower lung scores at the end of the finishing period. In the heterologous prime-boost experiment, priming with pORF7t prior to MLV vaccination resulted in significantly higher numbers of CD3(+)IFN-γ(+) subpopulations, lower numbers of PRRSV-specific CD3(+)IL-10(+) cells and Tregs, and rapid antibody responses in immunized pigs., Conclusion: Transdermal immunization with pORF7t reduced PRRRSV-induced immunomodulatory effects and enhanced long-term PRRSV-specific cellular responses in vaccinated pigs. Furthermore, heterologous DNA-MLV prime-boost immunization significantly improved the quality of PRRSV-specific cellular and humoral immunity. The information could benefit the future development of PRRSV management and control strategies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Viral-induced interleukin (IL)-10 and regulatory T lymphocytes (Tregs) are believed to play a major role in shaping the immunological and clinical outcomes following Porcine Reproductive and Respiratory Syndrome virus (PRRSV) infection. Recently, it has been shown that PRRSV nucleocapsid (N) protein can induce IL-10 production which is essential for induction of PRRSV-specific Tregs. We hypothesized that immunity to N protein should reduce PRRSV-induced negative immunomodulatory effects which will be essential for establishing proper anti-PRRSV immunity in infected pigs., Objectives: To investigate the immunomodulatory effects of DNA vaccine encoding a linearized, truncated form of PRRSV-N protein (pORF7t) which was designed to preferentially induce cell-mediated immunity against PRRSV N protein., Method: Immunomodulatory effects of the novel DNA vaccine were investigated in an experimental vaccinated-challenged model. In addition, long-term immunomodulatory effects of the DNA vaccine were investigated in vaccinated pigs kept at the PRRSV-positive environment until the end of the fattening period. Pigs were vaccinated either prior to or following natural PRRSV infection., Result: The results indicated that pORF7t could modulate the anti-PRRSV immune responses and promote the control of viral replication in the vaccinated-challenged pigs. Immunized pigs exhibited increased numbers of PRRSV-specific activated CD4(+)CD25(+) lymphocytes, reduced numbers of PRRSV-specific Tregs, and rapid viral clearance following infection. In a long-term study, regardless of the time of vaccination, DNA vaccine could modulate the host immune responses, resulted in enhanced PRRSV-specific IFN-γ producing cells, and reduced numbers of PRRSV-specific Tregs, without evidence of enhanced antibody responses. No vaccine adverse reaction was observed throughout the study., Conclusion: This study revealed the novel concept that PRRSV-specific immunity can be modulated by induction of cell-mediated immunity against the nucleocapsid protein. This concept could potentially benefit the development of PRRSV management and control strategies., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In January 2012, several clinical cases of dogs with flu-like symptoms, including coughing, sneezing, nasal discharge, and fever, were reported in a small-animal hospital located in Bangkok, Thailand. One influenza A virus was identified and characterized as an avian-like influenza virus H3N2. The virus was named A/canine/Thailand/CU-DC5299/12. A phylogenetic analysis indicated that the canine virus belonged to an avian Eurasian lineage and was genetically related to the canine influenza viruses H3N2 from China and Korea. This canine virus displays a unique genetic signature with two amino acid insertions in the NA protein, which is similar to the canine influenza viruses from eastern China (Zhejiang and Jiangsu). This study constitutes the first report of H3N2 canine influenza virus infection in a small-animal hospital in Thailand.

The outbreak of highly pathogenic H5N1 avian influenza, with its international spread, confirmed that emerging infectious disease control must be underpinned by effective laboratory services. Laboratory results are the essential data underpinning effective surveillance, case diagnosis, or monitoring of responses. Importantly, laboratories are best managed within national and international networks of technological support rather than in isolation. A well planned laboratory network can deliver both a geographical spread of testing capacity and also a cost effective hierarchy of capability. Hence in the international context regional networks can be particularly effective. Laboratories are an integral part of a country's veterinary services and their role and function should be clearly defined in the national animal health strategy and supporting government policies. Not every laboratory should be expected to deliver every possible service, and integration into regional and broader international networks should be a part of the overall strategy. The outputs required of each laboratory should be defined and then ensured through accredited quality assurance. The political and scientific environment in which laboratories operate changes continuously, not only through evolving national and regional animal health priorities but also through new test technologies and enhancements to existing technologies. Active networks help individual laboratories to monitor, evaluate, and respond to such challenges and opportunities. The end result is enhanced emerging infectious disease preparedness across the region.

Porcine circovirus-2 (PCV-2) serology is commonly used for PCV-2 herd status determination and optimal timing of PCV-2 vaccination programs. The objectives of the current study were to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) using a recombinant nuclear localization signal truncated capsid (rntCap) protein expressed in an Escherichia coli system and to determine the diagnostic performance of the developed rntCap indirect ELISA in comparison with immunoperoxidase monolayer assays (IPMAs). Based on a receiver operating characteristic (ROC) curve analysis of the rntCap indirect ELISA (n = 90), an optimum cutoff optical density (OD) of 0.330 was determined, which resulted in diagnostic sensitivity, diagnostic specificity, and accuracy of 98.33%, 93.33%, and 96.67%, respectively. Average OD values of the positive (n = 8) and negative sera (n = 8) tested by either purified glutathione-S-transferase (GST) protein or the rntCap protein as the coating antigen revealed that the mean OD values tested by the rntCap indirect ELISA were significantly different from using GST alone (P < 0.005). The correlation between the established rntCap indirect ELISA and the IPMA results demonstrated as the linear regression (Spearman correlation coefficient = 0.772, P < 0.005) indicated that the OD ratio obtained from the rntCap indirect ELISA could be used to predict the levels of the IPMA titers. More samples are needed for enhancing the diagnostic sensitivity, specificity and accuracy. In conclusion, the establishment of the rntCap indirect ELISA could be used as a serodiagnostic assay for large-scale detection of PCV-2 antibodies in swine and has the capability to be produced commercially for routine use in diagnostic laboratories.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection induces interleukin (IL)-10 production and increased numbers of PRRSV-specific regulatory T-lymphocytes in infected pigs. In the present study, the roles of the nucleocapsid (N) protein in induction of IL-10 and CD4(+)CD25(+)Foxp3(+) lymphocytes (T(reg)) were investigated. Transfection of porcine monocyte-derived dendritic cells (MoDCs) and pulmonary alveolar macrophages (PAMs) with a plasmid encoding N protein resulted in significant upregulation of IL-10 gene expression in the gene-transfected cells. Structural conformation, but not nuclear localization, of the expressed N protein was indicated to be essential for the ability to induce IL-10. Furthermore, the presence of recombinant N proteins in cultured PBMCs increased the number of IL-10-producing lymphocytes. Strong induction of IL-10-producing cells and T(reg) was observed when using N protein-pulsed MoDCs, suggesting an important role of MoDCs in induction of IL-10 and T(reg) by the N protein. Neutralization of IL-10 by addition of an anti-IL-10 antibody in the culture system resulted in marked reduction of PRRSV-induced T(reg) in the cultured PBMCs. Together, the data demonstrate the immunomodulatory properties of the PRRSV N protein and the linkage between IL-10 production and development of PRRSV-induced T(reg). Our results reveal an immunomodulatory function of the PRRSV N protein that may contribute to the unique immunological outcome observed following PRRSV infection.

For the past 10 years, endemic swine influenza H1 viruses in Thailand have been characterized as reassortants of swine virus genes from swine influenza viruses (SIV) in US and European pigs. Here the authors report the emergence of a novel reassorted H1N1 (rH1N1) virus consisted of human, avian, and swine virus genes from the pandemic H1N1 2009 (pH1N1) virus with a neuraminidase (NA) gene from a Thai swine H1N1 (ThH1N1) isolate. The rH1N1 virus was detected in nursery pigs during a respiratory disease outbreak in central Thailand in early 2010. The rH1N1 virus was repeatedly isolated from infected pigs, suggesting that it can transmit efficiently among the pig population. The appearance of rH1N1 virus in the field occurred within months of the introduction of pH1N1 virus into the Thai swine population in late 2009. The finding highlights the role of pig in generating newly reassorted influenza A viruses and also the significance of continuing disease surveillance and genetic characterization of SIV in pigs.

The purpose of this study was to investigate the diversity of yeast associated with the degree of canine seborrheic dermatitis (SD) by anatomical sites. Fifty-seven samples were divided as 17 healthy skin, 20 with primary seborrheic dermatitis (PSD), and 20 with secondary seborrheic dermatitis (SSD). Yeast isolation and characterization were carried out based on microscopical features and biochemical properties. DNA analysis at the internal transcribed spacer I of 26S rDNA region was utilized for species confirmation. Four species of yeast consisting Malassezia pachydermatis, Malassezia furfur, Candida parapsilosis and Candida tropicalis recovered from examined dogs. M. pachydermatis and C. parapsilosis were isolated from all dogs, but C. tropicalis and M. furfur were recovered from 3 healthy dogs and one diseased dog, respectively. The number of M. pachydermatis and C. parapsilosis in diseased dogs was higher than that of healthy specimens (P<0.01). High frequency and population size of C. parapsilosis were closely associated to PSD, while those of M. pachydermatis were associated with both PSD and SSD (P<0.01). C. parapsilosis were predominant at the perianal area. This study demonstrated the co-colonization of M. pachydermatis and C. parapsilosis in large amounts and frequency associated with stage of disease and anatomical site., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We investigated influenza interspecies transmission in two commercial swine farms in Thailand. Sera from swine-exposed workers (n=78), age-matched non-swine-exposed healthy people (n=60) and swine populations in both farms (n=85) were studied. Hemagglutination-inhibition (HI) assay was performed on Thai swine H1 viruses (swH1N1 and swH1N2) isolated from both farms. Thai human H1N1 (huH1N1) and pandemic H1N1 2009 (pH1N1) were also used as test antigens. The hemagglutinin (HA) 1 genes of swH1N1 and swH1N2 viruses were sequenced and shown to be genetically distinct from the Thai huH1N1 and pH1N1 viruses. Evidence of pig-to-human influenza virus transmission was found in farm workers with increased odds of elevated antibody titers to both swH1N1 (OR 42.63, 95% CI, 14.65-124) and swH1N2 (OR 58, 95% CI, 13.12-256.3) viruses. No evidence of human-to-pig influenza virus transmission was detected in this study., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, causing economically significant impacts on the swine industry worldwide. In this article, we share the information related to the Thai PRRSV and review the available options for PRRS control strategies. Unfortunately, the traditional control strategies and conventional vaccines fail to provide sustainable disease control, as they suffer from both antigenic heterogeneity and various immune evasion strategies of PRRSV. Induction of interleukin (IL)-10 following PRRSV infection is believed to be a focal mechanism leading to the unique immunological outcomes and interference of PRRS vaccine efficacy. It is likely that the nucleocapsid protein plays an important role in induction of IL-10 following PRRSV infection. We propose that removal or reduction of the PRRSV-induced, negative immunomodulatory effects especially during the first 2 weeks following infection is essential to establish proper anti-PRRSV immunity. In other word, incorporation of the "taming strategy" will be needed to reduce PRRSV-induced immunomodulatory effects, and to ensure maximal vaccine-induced immunity in the face of viral exposure. Any PRRSV vaccine that can induce cross-protective immunity and simultaneously eliminate the immunoinhibitory effects of PRRSV would be ideal. In addition, tracking of the inhibitory parameters, following the PRRSV challenge should be included in the vaccine testing protocol. Therefore, the future of PRRSV vaccine development relies tremendously on the basic knowledge of host-virus interactions and the communication between the basic and clinical PRRSV research fields., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A swine influenza outbreak occurred on a commercial pig farm in Thailand. Outbreak investigation indicated that pigs were co-infected with pandemic (H1N1) 2009 virus and seasonal influenza (H1N1) viruses. No evidence of gene reassortment or pig-to-human transmission of pandemic (H1N1) 2009 virus was found during the outbreak.

A surveillance program for influenza A viruses (H5N1) was conducted in live bird and food markets in central Thailand during July 2006-August 2007. Twelve subtype H5N1 viruses were isolated. The subtype H5N1 viruses circulating in the markets were genetically related to those that circulated in Thailand during 2004-2005.

Three major outbreaks of avian influenza (AI) occurred in Thailand. During the third episode in October 2005, we have isolated H5N1 viruses from one human case and three poultry cases. The whole genomes of AI viruses from human, chickens and quail from the outbreaks were characterized. Sequence analysis of eight gene segments revealed that the 2005 H5N1 viruses isolated in October 2005 were closely related to those recovered from chicken, tiger(s) and human(s) in January and July 2004. In addition, the genetic changes of the AI isolates at the HA cleavage site have been observed.

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.

In this report, the genome of the Thai avian influenza virus A (H5N1); A/Chicken/Nakorn-Pathom/Thailand/CU-K2/04, isolated from the Thai avian influenza A (AI) epidemic during the early of 2004 was sequenced. Phylogenetic analyses were performed in comparison to AI viruses from Hong Kong 1997 outbreaks and other AI (H5N1) isolates reported during 2001-2004. Molecular characterization of the Thai AI (H5N1) HA gene revealed a common characteristic of a highly pathogenic AI (HPAI), a 20-codon deletion in the neuraminidase gene, a 5-codon deletion in the NS gene and polymorphisms of the M2 and PB2 genes. Moreover, the HA and NA genes of the Thai AI displayed high similarity to those of the AI viruses isolated from human cases during the same epidemic. Finally, our results demonstrated that the Thai AI emerged as a member of 2000's AI lineage with most of the genetic sequences closely related to the Influenza A/Duck/China/E319.2/03 (H5N1).

Recent studies suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system by upregulating interleukin (IL)-10 gene expression. To determine the effect of PRRSV on porcine cytokine gene expression in vivo, we infected pigs with either the European or North American strain of PRRSV and monitored cytokine gene expression in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) using a multiplex PCR assay. Our results showed that both European and North American strains of PRRSV significantly upregulated IL-10 gene expression in PBMC of infected pigs from 5 days post-infection (p.i.). In addition, upregulation of IL-10 and interferon (IFN)-gamma gene expression was observed in BALC starting from 9 days p.i. The upregulation of cytokine gene expression in BALC was observed concurrent with an increased percentage of lymphocytes in the BALC population, suggesting a role for peripheral leukocytes in cytokine production in lungs. Our results showed that PRRSV infection resulted in an upregulation of IL-10 gene expression in vivo and that both European and North American strains induced comparable levels of IL-10 gene expression in the infected pigs, despite differences in the clinical signs. Our data support the notion that induction of IL-10 production may be one of the strategies used by PRRSV to modulate the host's immune responses, and this may contribute to the unique clinical picture observed following PRRSV infection.

In Thailand, where vaccination is routinely employed, there has been an increased incidence of chronic classical swine fever (CSF) outbreaks during the past decade. The major causative virus has been identified to be the moderate virulence, classical swine fever virus (CSFV) of the genogroup 2.2. An investigation was made into the efficacy of a CSF vaccine against this genogroup 2.2 challenge. Five-week-old pigs, grouped by their level of passive antibody titer were immunized with lapinized Chinese-strain CSF vaccine and challenged with CSFV genogroup 2.2, 13 days after vaccination. The group containing passive titers of lower than 64 at the time of immunization, had significantly higher number of CSFV-specific IFN-gamma secreting cells and was completely protected against the challenge. Interestingly, both cellular and antibody responses were inhibited in the pigs with the higher passive titer. Furthermore, following challenge, CSFV could be isolated from 50% of the pigs in this group. It was demonstrated that the CSF vaccine could induce complete protection in pigs, provided that the maternal derived titer at the time of vaccination was lower than 64. The result implied that an increase in CSFV outbreaks might be due to the inappropriate timing of vaccination as well as the nature of the CSFV genogroup 2.2.

Several lines of evidence suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system. To determine the effect of PRRSV on cytokine production, a multiplex PCR was established. This allowed a semi-quantitative analysis of IFN-gamma, IL-2, IL-4, IL-10 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression levels from porcine peripheral blood mononuclear cells (PBMCs). These results showed that both live and inactivated PRRSV predominantly upregulated IL-10 gene expression in porcine PBMCs. In addition, when PBMCs from pigs immunized previously with classical swine fever virus (CSFV) vaccine were cultivated with the recall antigen, CSFV, in the presence of PRRSV, significant upregulation of IL-10 gene expression and reduction of IFN-gamma gene expression were observed. These findings indicated that the presence of PRRSV in the culture could affect recall antigen response. This study implies that the induction of IL-10 production may be one of the strategies used by PRRSV to modulate host immune responses.

Immunization with naked plasmid DNA effectively induces both humoral and cell-mediated immunity to vaccine antigens and can confer protection against numerous infectious diseases. To explore the potential use of DNA immunization to induce rotavirus-specific immune responses, we used plasmid DNA encoding the VP4 gene of bovine rotavirus (BRV). Intrasmuscular injection of the plasmid encoding the VP4 gene into C57BI/6 mice induced cell-mediated immunity as measured by cytokine production. Although DNA immunization did not induce a detectable BRV-specific antibody response, DNA-immunized animals were primed for antibody production and a cellular immune response. Following viral inoculation, the immunized animals displayed an enhanced number of BRV-specific antibody-secreting cells and cytotoxic activity. The immune response induced by DNA immunization alone or followed by viral inoculation was biased toward IFN-gamma production (Th1-like). CD4+ lymphocytes were the major source of IFN-gamma production in the spleen following DNA immunization. In contrast, a balanced cytokine production was observed in the spleens of animals receiving whole virus. These experiments showed that DNA immunization with a gene encoding the VP4 protein of BRV stimulated a Th1-like immune response in mice, and this bias in the immune response persisted following exposures to whole virus.