We have conjugated the S9 peptide, a mimic of the group B streptococcal type III capsular polysaccharide, to different carriers in an effort to elicit an optimal immune response. As carriers, we utilized the soluble protein keyhole limpet hemocyanin and virus-like particles (VLPs) from two plant viruses, Cowpea Chlorotic Mottle Virus and Cowpea Mosaic Virus. We have found that coupling the peptide to the soluble protein elicits a Th2 immune response, as evidenced by the production of the peptide-specific IgG1 antibody and IL-4/IL-10 production in response to antigen stimulation, whereas the peptide conjugated to VLPs elicited a Th1 response (IgG2a, IFN-γ). Because the VLPs used as carriers package RNA during the assembly process, we hypothesize that this effect may result from the presence of nucleic acid in the immunogen, which affects the Th1/Th2 polarity of the response.

Nervous necrosis virus (NNV), the etiological agent of viral nervous necrosis, has a high mortality rate of 100% in hatchery-reared larvae and juveniles. At present, there are still no effective vaccines available for NNV. Pichia pastoris surface display of viral capsid proteins was generated in hopes of developing an oral vaccine against red-grouper-nervous-necrosis virus (RGNNV) in fish. Fingerlings or juveniles that showed clinical signs of NNV infection were proved by RT-PCR for the appearance of expected length of 198 bpcDNA and further analysis by DNA sequencing. The DNA fragment containing AGα1 linked to RG-NNVRNA2, 2100 bp in length, was inserted into pPIC9K vector. Linearlized plasmids were electroporated into P. pastoris GS115 (mut + His −) and yeast isolates that had Muts − His + and resistance phenotype at 4 mg/mL geniticin were selected to determine the integration of the target gene by PCR reaction. The extracted cell walls from the yeasts cultured in buffered-methanol-complex medium (BMMY) through an induction of 0.5% methanol for 6 days, were investigated for the fusion proteins by western blot. A protein band of 73 kDa predicted to be the fusion protein and a non-specific one of 56 kDa were detected. Staining of the fusion proteins expressing cells with corresponding antibodies revealed their presence of NNVRNA2, but varied the intensity of detected signals from cell to cell by confocal laser scanning fluorescence microscopy. The predicted fusion proteins tertiary structure also confirmed exposed conformation of the fusion protein on the cell wall. In this study, the capsid proteins from the red-spotted grouper nervous necrosis virus were successfully expressed on the cell surface of P. pastoris but still low levels of fusion protein expression. Further studies

Hepatitis A virus (HAV) contamination in food can lead to major health problems. We developed a combination reverse transcription (RT) PCR method plus enzyme-linked immunosorbent assay (ELISA) to detect HAV in fresh oysters harvested along the east coast of the Gulf of Thailand. Viral nucleic acid was extracted via the glycine-arginine-polyethylene glycol method followed by RT-PCR amplification with specifically designed primers against HAV and an ELISA to detect the digoxigenin-labeled RT-PCR products. The ELISA in concert with the RT-PCR protocol further increased the detection sensitivity by 100-fold for the HAV genome and 10-fold in artificially contaminated oysters. The overall sensitivity of the RT-PCR in combination with the ELISA was 31.88 pg and 16 PFU/g, respectively. The ELISA increases the specificity of the RT-PCR assay for detecting naturally occurring HAV in oysters. This combined RT-PCR-ELISA approach is a practical and sensitive method for HAV detection and can be utilized in routine screening for HAV in shellfish.

Rotavirus remains a major cause of diarrhea in infants and young children especially in developing countries. Monitoring changes in rotavirus strains is necessary to assess the potential effectiveness of vaccines in specific geographic locations. This project aimed at monitoring the epidemiological status of rotavirus among Thai children. A total of 73 positive fecal samples for rotavirus were collected from hospitalized infants and children with acute gastroenteritis or diarrhea between 2012 and 2014 and analyzed for G-and P-genotypes by semi-nested multiplex PCR or DNA sequencing. The most prevalent genotype was G1P[8] (61.64%) followed by G8P[8] (21.92%), G2P[4] (8.22%), G3P[8] (4.11%), G9P [8] (2.74%), and G2P [8] (1.37%), Interestingly, the unusual genotypes, G8P[8] and G2P [8] were identified in this study. A phylogenetic analysis of VP7 and VP4 genes of strains detected in the present study revealed that G1 belonged to G1-Ic, and G1-IIc ; G2 belonged to G2-IIa; G3 belonged to G3-Ia; G8 belonged to G8-I; and G9 belonged to G9-III lineages, while P[4] and P[8] were identified as P[4]-Vb and P[8]-III lineages, respectively. The distribution of unusual genotypes that possess neither VP7 nor VP4 specificity with the available rotavirus vaccine currently in use may represent a challenge to the outcome and success of vaccination.

Point mutation and abrupt changes of segmented genomes of rotavirus group A (RVAs) from mixed infection are critical factors for generating genetically divergent strains. Our preceding study of rotavirus infections in 2012 - 2014 in Thailand indicated the high prevalence of G8P[8] strains. This study aimed to identify the origin of each segment of the genome of these G8P[8] RVA strains by using phylogenetic analysis. All gene segments from the 5 selected G8P[8] were sequenced partially and aligned with locally circulating rotavirus strains to estimate the phylogenic relationship. Based on the genome's constellation of the genome, these 5 strains’ genetic backgrounds possessed genotype constellation 2 or called the DS-1-like genotype. Ten genes, except for VP6, were clustered within the same phylogenic tree of the cognate genes of Thai circulating G8P[8]strains isolated between 2013 - 2014. Their 5 genes VP2, VP3, NSP1, NSP3, and NSP5, also exhibited high nucleotide similarities with the first unusual Japanese DS-1-like strains G1P[8] NT004.  Besides, the VP4 shared a high nucleotide sequence similarity with the cognate gene of circulating Thai human G1P[8] Wa-like and G1P[8] NT004. However, the remaining 4 gene segments (VP1, VP7, NSP2, and NSP4) were clustered with other human or animal RVA strains within the same monophyletic. All of these G8P[8] strains shown to be an intragenogroup reassortment. Thus, characterization of the genomes is crucial to monitor the evolutionary dynamics and relationships of co-circulating RVAs in predicting the effectiveness of current vaccines and future vaccine development strategies. HIGHLIGHTS G8P[8] strains’ genetic backgrounds possessed genotype constellation 2 ( DS-1-like genotype) G8P[8] strains might originate from a sequential process of multiple reassortment events involving intra-genogroups from humans and animal species Ten genes, except for VP6, were clustered within the same phylogenic tree of the cognate genes of Thai circulating G8P[8]strains isolated between 2013 - 2014 GRAPHICAL ABSTRACT