Development of Differentiating Infected from Vaccinated Animals (DIVA) Real-Time PCR for African Horse Sickness Virus Serotype 1.

African horse sickness (AHS) is a highly infectious and often fatal disease caused by 9 serotypes of the orbivirus African horse sickness virus (AHSV). In March 2020, an AHS outbreak was reported in Thailand in which AHSV serotype 1 was identified as the causative agent. Trivalent live attenuated vaccines serotype 1, 3, and 4 were used in a targeted vaccination campaign within a 50-km radius surrounding the infected cases, which promptly controlled the spread of the disease. However, AHS-like symptoms in vaccinated horses required laboratory diagnostic methods to differentiate infected horses from vaccinated horses, especially for postvaccination surveillance. We describe a real-time reverse transcription PCR-based assay for rapid characterization of the affecting field strain. The development and validation of this assay should imbue confidence in differentiating AHS-vaccinated horses from nonvaccinated horses. This method should be applied to determining the epidemiology of AHSV in future outbreaks.

Characterization of Fowlpox virus in chickens and bird-biting mosquitoes: a molecular approach to investigating Avipoxvirus transmission.

Avian pox is a highly contagious avian disease, yet relatively little is known about the epidemiology and transmission of Avipoxviruses. Using a molecular approach, we report evidence for a potential link between birds and field-caught mosquitoes in the transmission of Fowlpox virus (FWPV) in Singapore. Comparison of fpv167 (P4b), fpv126 (VLTF-1), fpv175-176 (A11R-A12L) and fpv140 (H3L) gene sequences revealed close relatedness between FWPV strains obtained from cutaneous lesions of a chicken and four pools of Culex pseudovishnui, Culex spp. (vishnui group) and Coquellitidea crassipes caught in the vicinity of the study site. Chicken-derived viruses characterized during two separate infections two years later were also identical to those detected in the first event, suggesting repeated transmission of closely related FWPV strains in the locality. Since the study location is home to resident and migratory birds, we postulated that wild birds could be the source of FWPV and that bird-biting mosquitoes could act as bridging mechanical vectors. Therefore, we determined whether the FWPV-positive mosquito pools (n=4) were positive for avian DNA using a polymerase chain reaction-sequencing assay. Our findings confirmed the presence of avian host DNA in all mosquito pools, suggesting a role for Cx. pseudovishnui, Culex spp. (vishnui group) and Cq. crassipes mosquitoes in FWPV transmission. Our study exemplifies the utilization of molecular tools to understand transmission networks of pathogens affecting avian populations, which has important implications for the design of effective control measures to minimize disease burden and economic loss.

Detection and characterization of a novel marine birnavirus isolated from Asian seabass in Singapore.

BACKGROUND: Lates calcarifer, known as seabass in Asia and barramundi in Australia, is a widely farmed species internationally and in Southeast Asia and any disease outbreak will have a great economic impact on the aquaculture industry. Through disease investigation of Asian seabass from a coastal fish farm in 2015 in Singapore, a novel birnavirus named Lates calcarifer Birnavirus (LCBV) was detected and we sought to isolate and characterize the virus through molecular and biochemical methods. METHODS: In order to propagate the novel birnavirus LCBV, the virus was inoculated into the Bluegill Fry (BF-2) cell line and similar clinical signs of disease were reproduced in an experimental fish challenge study using the virus isolate. Virus morphology was visualized using transmission electron microscopy (TEM). Biochemical analysis using chloroform and 5-Bromo-2'-deoxyuridine (BUDR) sensitivity assays were employed to characterize the virus. Next-Generation Sequencing (NGS) was also used to obtain the virus genome for genetic and phylogenetic analyses. RESULTS: The LCBV-infected BF-2 cell line showed cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells observed at 3 to 5 days' post-infection. The propagated virus, when injected intra-peritoneally into naïve Asian seabass under experimental conditions, induced lesions similar to fish naturally infected with LCBV. Morphology of LCBV, visualized under TEM, revealed icosahedral particles around 50 nm in diameter. Chloroform and BUDR sensitivity assays confirmed the virus to be a non-enveloped RNA virus. Further genome analysis using NGS identified the virus to be a birnavirus with two genome segments. Phylogenetic analyses revealed that LCBV is more closely related to the Blosnavirus genus than to the Aquabirnavirus genus within the Birnaviridae family. CONCLUSIONS: These findings revealed the presence of a novel birnavirus that could be linked to the disease observed in the Asian seabass from the coastal fish farms in Singapore. This calls for more studies on disease transmission and enhanced surveillance programs to be carried out to understand pathogenicity and epidemiology of this novel virus. The gene sequences data obtained from the study can also pave way to the development of PCR-based diagnostic test methods that will enable quick and specific identification of the virus in future disease investigations.

Rabbit haemorrhagic disease virus 2 from Singapore 2020 outbreak revealed an Australian recombinant variant.

Rabbit haemorrhagic disease (RHD) is a significant and debilitating viral disease affecting lagomorphs. In September 2020, Singapore reported its first cases of RHD virus (RHDV) infection in domesticated rabbits. The initial findings reported that the outbreak strain belonged to genotype GI.2 (RHDV2/RHDVb), and epidemiological investigations could not identify the definitive source of the virus origin. Further recombination detection and phylogenetic analyses of the Singapore outbreak strain revealed that the RHDV was a GI.2 structural (S)/GI.4 non-structural (NS) recombinant variant. Sequence analyses on the National Centre for Biotechnology Information (NCBI) database showed high homology to recently emerged Australian variants, which were prevalent in local Australian lagomorph populations since 2017. Time-structured and phylogeographic analyses for the S and NS genes revealed a close genetic relationship between the Singapore RHDV strain and the Australian RHDV variants. More thorough epidemiological inquiries are necessary to ascertain how an Australian RHDV was introduced into the Singapore rabbit population, and opportune development of RHDV diagnostics and vaccines will be important to safeguard lagomorphs from future RHDV infection and disease management.

Serological prevalence and molecular characterization of hepatitis E virus in imported pigs in Singapore (2000-2019).

Hepatitis E is a significant liver disease caused by infection with hepatitis E virus (HEV). The risk factors for hepatitis E in developed countries include blood transfusion and ingestion of undercooked meat or meat products derived from HEV-infected animals. Since 2000, there has been increased human hepatitis E incidence reported in Singapore. Although the causes of this increase have not been established, several studies have linked zoonotic HEV infections in humans to pork consumption. It is therefore important to closely monitor the presence of HEV in food sources for the prevalence and virulence. In this study, we demonstrated the presence of HEV in pigs imported into Singapore for consumption through serological and molecular investigation of live pig and post-slaughter samples collected between 2000 and 2019. Among imported pigs, anti-HEV antibody prevalence remained at a level around 35% until 2017, with a statistically significant increase in 2018. HEV RNA was detected in 8.40% (34/405) of the faecal samples, indicative of an active infection in the pigs. HEV RNA was also detected in 6.67% (4/60) of liver samples obtained post-slaughter. We also report the development of an RT-PCR-based next-generation sequencing (NGS) method that enabled full sequencing of the HEV genome in HEV RNA-positive samples in a relatively short span of time. Phylogenetic analysis identified the HEV in one of the imported pigs (HEV-S28) as genotype 3a, which clustered together with the human HEV strains previously identified in Singapore. We found that the HEV-S28 strain exhibited amino acid substitutions that are associated with reduced HEV replication efficiency. The increase in anti-HEV seroprevalence in the pig population from 2018 is worth further exploration. We will continue to monitor the prevalent HEV strains and assess the genetic diversity of HEV in the imported pigs to confirm the potential association with human infections.

First detection of rabbit haemorrhagic disease virus (RHDV2) in Singapore.

Rabbit haemorrhagic disease (RHD) is a significant viral disease caused by infection with Rabbit haemorrhagic disease virus (RHDV). The first documented cases of RHDV in Singapore occurred in adult pet European rabbits (Oryctolagus cuniculus) in September 2020. Rabbits presented with acute hyporexia, lethargy, huddled posture, and varying degrees of pyrexia and tachypnoea. Clinical pathology consistently reflected markedly elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALKP). Hepatic lobe torsion was ruled out using ultrasonography and colour Doppler studies in all patients. A total of 11 rabbits owned by 3 families were presented to the clinics; 8/11 rabbits died within 48 hr of presentation, while the remaining two rabbits had recovered after prolonged hospitalization and one rabbit was aclinical. Histopathology revealed acute, marked diffuse hepatocellular necrosis and degeneration, findings which were suggestive for RHDV infection and prompted the undertaking of further molecular diagnostics. Subsequent polymerase chain reaction of the liver samples detected RHDV RNA. Molecular characterization of viral genomes by whole genome sequencing revealed that the outbreak strain was of the genotype GI.2 (RHDV2/RHDVb). Nucleotide sequences of the VP60 gene were compared with various RHDV variants using phylogenetic analysis. The sample genome shared highest sequence identity with a GI.2-genotyped virus from GenBank (RHDV isolate Algarve 1 polyprotein and minor structural protein (VP10) genes, GenBank accession KF442961). The combination of clinical, histopathological, molecular and sequencing technologies enabled rapid detection and detailed genetic characterization of the RHDV virus causing the present outbreak for prompt implementation of disease control measures in Singapore. Further epidemiological investigations of potential virus introduction into Singapore are ongoing.

Use of nanopore sequencing to characterize african horse sickness virus (AHSV) from the African horse sickness outbreak in thailand in 2020.

African horse sickness (AHS) is a highly infectious and deadly disease despite availability of vaccines. Molecular characterization of African horse sickness virus (AHSV) detected from the March 2020 Thailand outbreak was carried out by whole-genome sequencing using Nanopore with a Sequence-Independent Single Primer Amplification (SISPA) approach. Nucleotide sequence of the whole genome was compared with closest matching AHSV strains using phylogenetic analyses and the AHSV-1 virus shared high sequence identity with isolates from the same outbreak. Substitution analysis revealed non-synonymous and synonymous substitutions in the VP2 gene as compared to circulating South African strains. The use of sequencing technologies, such as Nanopore with SISPA, has enabled rapid detection, identification and detailed genetic characterization of the AHS virus for informed decision-making and implementation of disease control measures. Active genetic information sharing has also allowed emergence of AHSV to be better monitored on a global basis.

Isolation and characterization of equine influenza virus (H3N8) from an equine influenza outbreak in Malaysia in 2015.

Equine influenza is a major cause of respiratory infections in horses and can spread rapidly despite the availability of commercial vaccines. In this study, we carried out molecular characterization of Equine Influenza Virus (EIV) isolated from the Malaysian outbreak in 2015 by sequencing of the HA and NA gene segments using Sanger sequencing. The nucleotide and amino acid sequences of HA and NA were compared with representative Florida clade 1 and clade 2 strains using phylogenetic analysis. The Florida clade 1 viruses identified in this outbreak revealed numerous amino acid substitutions in the HA protein as compared to the current OIE vaccine strain recommendations and representative strains of circulating Florida sub-lineage clade 1 and clade 2. Differences in HA included amino acids located within antigenic sites which could lead to reduced immune recognition of the outbreak strain and alter the effectiveness of vaccination against the outbreak strain. Detailed surveillance and genetic information sharing could allow genetic drift of equine influenza viruses to be monitored more effectively on a global basis and aid in refinement of vaccine strain selection for EIV.

Efficacy evaluation of Der p 1 DNA vaccine for allergic asthma in an experimental mouse model.

Der p 1 is a major allergen that reacts with IgE in more than 70% allergic sera, hence a good candidate for DNA vaccine development. Previous study demonstrated that intramuscular injection of plasmid DNA containing wild type Der p 1 gene induced specific Th1 skewed immune response. The aim of this study is to further optimize the DNA construct to achieve preventive and therapeutic efficacies. An optimized construct, containing mouse Igkappa light chain leader sequence followed by codon optimized mature Der p 1 gene, was generated and used to prevent or treat Der p1 induced allergic asthma in an experimental model. Results showed that this optimized construct was effective in the inhibition of Der p 1 specific IgE levels, attenuation of Th2 response and reduction of airway hyperresponsiveness in terms of prophylactic efficacy. It also showed therapeutic efficacy in IgE inhibition and up-regulation of IgG2a and IFN-gamma. The information obtained here could facilitate the design of DNA vaccines for allergy in general.

DNA vaccines and allergic diseases.

1. Allergic diseases are characterized by inappropriate immune responses to common environmental antigens. The prevalence of these diseases has been increasing worldwide for reasons that are not exactly clear. 2. Current treatment is largely symptomatic. Because the initial observation that simple plasmid DNA injections resulted in in vivo protein expression and induction of adaptive immune responses to the encoded antigen, the potential of modifying the allergic immune responses by DNA vaccination so as to treat and prevent these diseases has been explored extensively. 3. In the present paper we review preclinical studies using animal models of allergic diseases, with an emphasis on DNA vaccine design, for house dust mite allergens-related allergic asthma.

Expression and immunogenicity of the major house dust mite allergen Der p 1 following DNA immunization.

The mite protein Der p 1 is a major trigger of allergy and atopic asthma world-wide, and thus, a good vaccine candidate for allergy prevention. Since it is a cysteine protease, the catalytic effects of Der p 1 vaccination may be unpredictable. One approach to reduce this risk is to vaccinate with DNA encoding enzymatically inactive forms of Der p 1. Here we show that Der p 1 DNA without its native pre-pro sequences potently induced Der p 1-specific antibodies, as long as its pre-sequence was substituted by another leader sequence. Without any pre-pro sequence, the same DNA fragment was well expressed but failed to induce significant level of anti-Der p 1 antibodies, without further boosting by protein.