New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome.

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.

Impacts of shelter management on canine rabies immune status.

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.

Coatsome-replicon vehicles: Self-replicating RNA vaccines against infectious diseases.

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.

Perspectives on veterinary education in Thailand.

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.

Dynamics of cellular and humoral immune responses following duck Tembusu virus infection in ducks.

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.

Two decades of one health surveillance of Nipah virus in 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.

African Horse Sickness Virus Serotype 1 on Horse Farm, Thailand, 2020.

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.

Diversity of the Swine Leukocyte Antigen Class I and II in Commercial Pig Populations.

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.

Allergen components of Dermatophagoides farinae recognised by serum immunoglobulin (Ig)E in Thai dogs with atopic dermatitis.

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.

Abrogation of PRRSV infectivity by CRISPR-Cas13b-mediated viral RNA cleavage in mammalian cells.

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.

Negative Immunomodulatory Effects of Type 2 Porcine Reproductive and Respiratory Syndrome Virus-Induced Interleukin-1 Receptor Antagonist on Porcine Innate and Adaptive Immune Functions.

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.

Efficacy of a type 2 PRRSV modified live vaccine (PrimePac™ PRRS) against a Thai HP-PRRSV challenge.

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.

Induction of porcine reproductive and respiratory syndrome virus (PRRSV)-specific regulatory T lymphocytes (Treg) in the lungs and tracheobronchial lymph nodes of PRRSV-infected pigs.

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.

Positive immunomodulatory effects of heterologous DNA vaccine- modified live vaccine, prime-boost immunization, against the highly-pathogenic PRRSV infection.

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.

Interleukin-1 receptor antagonist: an early immunomodulatory cytokine induced by porcine reproductive and respiratory syndrome virus.

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.

Generation of potent porcine monocyte-derived dendritic cells (MoDCs) by modified culture protocol.

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.

Transdermal delivery of plasmid encoding truncated nucleocapsid protein enhanced PRRSV-specific immune responses.

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.

A novel DNA vaccine for reduction of PRRSV-induced negative immunomodulatory effects: A proof of concept.

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.

Development of veterinary laboratory networks for avian influenza and other emerging infectious disease control: the southeast asian experience.

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.

Genetic characterization of canine influenza A virus (H3N2) in Thailand.

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.