Discovery, Pathogenesis, and Complete Genome Characterization of Lates calcarifer Herpesvirus.

In 2015 and 2016, two Barramundi (Lates calcarifer) farms in Singapore reported a disease outbreak characterized by lethargic behavior, pronounced inappetence, generalized skin lesions, erosions of the fins and tail, and ultimately high mortality in their fish. Next-generation sequencing and PCR confirmed presence of a novel virus belonging to the Alloherpesviridae family, Lates calcarifer herpesvirus (LCHV), which was subsequently isolated and cultured. We characterize, for the first time, the complete genome of two cultured LCHV isolates. The genome contains a long unique region of approximately 105,000 bp flanked by terminal repeats of approximately 24,800 bp, of which the first 8.2 kb do not show any similarity to described genomes in the Alloherpesviridae family. The two cultured isolates share 89% nucleotide identity, and their closest relatives are the viruses belonging to the genus Ictalurivirus. Experimental infections using one of the cultured LCHV isolates resulted in identical clinical signs as originally described in the index farm, both in intraperitoneal-injection infected fish and cohabitant fish, with mortality in both groups. Histopathological analysis showed pronounced abnormalities in the gills. Virus culture and PCR analysis confirmed the replication of LCHV in the infected fish, and thus Koch's postulates were fulfilled.

Detection of a novel Babesia sp. in Amblyomma javanense, an ectoparasite of Sunda pangolins.

BACKGROUND: Babesia is a protozoal, tick-borne parasite that can cause life-threatening disease in humans, wildlife and domestic animals worldwide. However, in Southeast Asia, little is known about the prevalence and diversity of Babesia species present in wildlife and the tick vectors responsible for its transmission. Recently, a novel Babesia species was reported in confiscated Sunda pangolins (Manis javanica) in Thailand. To investigate the presence of this parasite in Singapore, we conducted a molecular survey of Babesia spp. in free-roaming Sunda pangolins and their main ectoparasite, the Amblyomma javanense tick. METHODS: Ticks and tissue samples were opportunistically collected from live and dead Sunda pangolins and screened using a PCR assay targeting the 18S rRNA gene of Babesia spp. DNA barcoding of the cytochrome oxidase subunit I (COI) mitochondrial gene was used to confirm the species of ticks that were Babesia positive. RESULTS: A total of 296 ticks and 40 tissue samples were obtained from 21 Sunda pangolins throughout the 1-year study period. Babesia DNA was detected in five A. javanense ticks (minimum infection rate = 1.7%) and in nine different pangolins (52.9%) located across the country. Phylogenetic analysis revealed that the Babesia 18S sequences obtained from these samples grouped into a single monophyletic clade together with those derived from Sunda pangolins in Thailand and that this evolutionarily distinct species is basal to the Babesia sensu stricto clade, which encompasses a range of Babesia species that infect both domestic and wildlife vertebrate hosts. CONCLUSIONS: This is the first report documenting the detection of a Babesia species in A. javanense ticks, the main ectoparasite of Sunda pangolins. While our results showed that A. javanense can carry this novel Babesia sp., additional confirmatory studies are required to demonstrate vector competency. Further studies are also necessary to investigate the role of other transmission pathways given the low infection rate of ticks in relation to the high infection rate of Sunda pangolins. Although it appears that this novel Babesia sp. is of little to no pathogenicity to Sunda pangolins, its potential to cause disease in other animals or humans cannot be ruled out.

Anthropogenic Transmission of SARS-CoV-2 from Humans to Lions, Singapore, 2021.

In Singapore, 10 captive lions tested positive for SARS-CoV-2 by real-time PCR. Genomic analyses of nanopore sequencing confirmed human-to-animal transmission of the SARS-CoV-2 Delta variant. Viral genomes from the lions and zookeeper shared a unique spike protein substitution, S:A1016V. Widespread SARS-CoV-2 transmission among humans can increase the likelihood of anthroponosis.

Detection of African Swine Fever Virus from Wild Boar, Singapore, 2023.

We detected African swine fever virus (ASFV) from a wild boar in Singapore. In <72 hours, we confirmed and reported ASFV p72 genotype II, CD2v serogroup 8, and IGR-II variant by using a combination of real-time PCR and whole-genome sequencing. Continued biosurveillance will be needed to monitor ASFV in Singapore.

Suspected clinical toxoplasmosis in a 12-week-old puppy in Singapore.

BACKGROUND: Toxoplasma gondii is traditionally known as a parasite of felids, with possible infection in intermediate hosts such as dogs and humans, and thus a disease of public health significance. Published data on the prevalence of toxoplasmosis in dogs and cats in Singapore is scanty, and this paper documents a suspect clinical case of toxoplasmosis in a free-roaming puppy trapped from an offshore island of Singapore. CASE PRESENTATION: A 12-week-old puppy presented with hindlimb weakness and sarcopenia, with rapidly progressing ascending paralysis and respiratory distress, one week after trapping. Toxoplasmosis was suspected after indirect fluorescence antibody testing (IFAT) revealed anti-T. gondii antibodies. The puppy responded quickly to clindamycin treatment and was discharged from hospital after 10 days. CONCLUSION: While rare and undocumented, veterinary clinicians in Singapore are advised to also include toxoplasmosis infection as a differential diagnosis in dogs presenting with similar clinical signs. This is especially so for dogs which have access to the outdoors.

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.

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.

Resolution of rabbit haemorrhagic disease virus 2 (RHDV2; Lagovirus europeus GI.2) outbreak in Singapore.

Rabbit haemorrhagic disease (RHD) is a highly contagious viral disease affecting lagomorphs. The first documented cases of RHD in Singapore occurred in adult pet European rabbits in September 2020. Singapore subsequently declared the outbreak resolved in December 2020. Epidemiological investigations ruled out introductions via importation of infected rabbits and contaminated feed. The source could not be definitively determined. However, the findings suggested that the incident involved both inter- and intra-household transmission and veterinary clinic-household transmission. This incident demonstrated the importance of sustained application of biosecurity measures, epidemiological investigations including active case finding, control measures such as expedient vaccine dissemination and risk communications. It showed that even without a wild lagomorph population, an urbanized city-state like Singapore could still encounter emerging diseases such as RHD. Given its social impact on rabbit owners, the National Parks Board, Singapore and private veterinarians worked together to communicate with rabbit owners in order to urge them to adopt biosecurity measures and to address their concerns.

A Novel Virus Causes Scale Drop Disease in Lates calcarifer.

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch's postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.

Differential transcriptomic response in the spleen and head kidney following vaccination and infection of Asian seabass with Streptococcus iniae.

Vaccination is an important strategy in the protection of aquaculture species from major diseases. However, we still do not have a good understanding of the mechanisms underlying vaccine-induced disease resistance. This is further complicated by the presence of several lymphoid organs that play different roles when mounting an immune response. In this study, we attempt to elucidate some of these mechanisms using a microarray-based approach. Asian seabass (Lates calcarifer) were vaccinated against Streptococcus iniae and the transcriptomic changes within the spleen and head kidney at one and seven days post-vaccination were profiled. We subsequently challenged the seabass at three weeks post-vaccination with live S. iniae and similarly profiled the transcriptomes of the two organs after the challenge. We found that vaccination induced an early, but transient transcriptomic change in the spleens and a delayed response in the head kidneys, which became more similar to one another compared to un-vaccinated ones. When challenged with the pathogen, the spleen, but not the head kidneys, responded transcriptomically at 25-29 hours post-challenge. A unique set of genes, in particular those involved in the activation of NF-κB signaling, was up-regulated in the vaccinated spleens upon pathogen challenge but not in the un-vaccinated spleens. A semi-quantitative PCR detection of S. iniae using metagenomic DNA extracted from the water containing the seabass also revealed that vaccination resulted in reduction of pathogen shedding. This result indicated that vaccination not only led to a successful immune defense against the infection, but also reduced the chances for horizontal transmission of the pathogen. In conclusion, we have provided a transcriptomic analysis of how the teleost spleen and head kidneys responded to vaccination and subsequent infection. The different responses from the two organs are suggestive of their unique roles in establishing a vaccine-induced disease resistance.

Prevention and control of avian influenza in Singapore.

The highly pathogenic avian influenza (HPAI) H5N1 virus was first detected in 1996 in Guangdong, China. Since 2003, H5N1 outbreaks have been reported in parts of Asia, Europe, the Middle East, and Africa. It is currently entrenched among poultry in parts of Asia and poses a major challenge to animal and human health. Singapore is free from HPAI. Given Singapore's need to import food, the Agri-Food and Veterinary Authority (AVA) has adopted a pro-active risk management system to prevent the introduction of HPAI. AVA's approach maybe described as a multi-layered control strategy for the prevention and control of HPAI. The strategy includes control measures at source, border control measures, local control measures and emergency preparedness.

Determination of the complete nucleotide sequences of RNA1 and RNA2 from greasy grouper (Epinephelus tauvina) nervous necrosis virus, Singapore strain.

The complete nucleotide sequences of RNA1 and RNA2 from greasy grouper (Epinephelus tauvina) nervous necrosis virus (GGNNV), Singapore strain, were determined. 5' RACE and RNA ligation were used to obtain the complete nucleotide sequences of the 5' and 3' non-coding regions (NCRs). GGNNV RNA1 was determined to be 3103 nt long, containing an ORF of 982 aa, while GGNNV RNA2 was determined to be 1433 nt long, containing an ORF of 338 aa. Both GGNNV RNAs are longer than those of other published betanodavirus sequences and the additional nucleotides were located within the NCRs. Analysis of GGNNV RNA2 revealed that it is closely related to red-spotted grouper nervous necrosis virus and that both grouper viruses share the same neutralization epitope. Predicted domains for six RNA-dependent RNA polymerase motifs and two putative ORFs (proteins B1 and B2) were confirmed by sequence analysis of GGNNV RNA1.