Spike S2 Subunit: Possible Target for Detecting Novel SARS-CoV-2 Variants with Multiple Mutations.

Novel SARS-CoV-2 variants have multiple mutations that may impact molecular diagnostics. The markedly conserved S2 subunit may be utilized to detect new variants. A comparison of 694 specimens (2019-2022) in Thailand using a commercial RT-PCR kit and the kit in combination with S2 primers and a probe was performed. Delayed amplification in ORF1ab was detected in one BA.4 omicron, whereas no amplification problem was encountered in the S2 target. There were no statistically significant differences in mean Ct value between the target genes (E, N, ORF1ab, and S2) and no significant differences in mean Ct value between the reagents. Furthermore, 230,821 nucleotide sequences submitted by 20 representative counties in each region (Jan-Oct 2022) have been checked for mutations in S2 primers and probe using PrimerChecker; there is a very low chance of encountering performance problems. The S2 primers and probe are still bound to the top five currently circulating variants in all countries and Thailand without mismatch recognition (Jun-Nov 2023). This study shows the possible benefits of detecting S2 in combination with simultaneously detecting three genes in a kit without affecting the Ct value of each target. The S2 subunit may be a promising target for the detection of SARS-CoV-2 variants with multiple mutations.

Behavioral-biological surveillance of emerging infectious diseases among a dynamic cohort in Thailand.

BACKGROUND: Interactions between humans and animals are the key elements of zoonotic spillover leading to zoonotic disease emergence. Research to understand the high-risk behaviors associated with disease transmission at the human-animal interface is limited, and few consider regional and local contexts. OBJECTIVE: This study employed an integrated behavioral-biological surveillance approach for the early detection of novel and known zoonotic viruses in potentially high-risk populations, in an effort to identify risk factors for spillover and to determine potential foci for risk-mitigation measures. METHOD: Participants were enrolled at two community-based sites (n = 472) in eastern and western Thailand and two hospital (clinical) sites (n = 206) in northeastern and central Thailand. A behavioral questionnaire was administered to understand participants' demographics, living conditions, health history, and animal-contact behaviors and attitudes. Biological specimens were tested for coronaviruses, filoviruses, flaviviruses, influenza viruses, and paramyxoviruses using pan (consensus) RNA Virus assays. RESULTS: Overall 61/678 (9%) of participants tested positive for the viral families screened which included influenza viruses (75%), paramyxoviruses (15%), human coronaviruses (3%), flaviviruses (3%), and enteroviruses (3%). The most salient predictors of reporting unusual symptoms (i.e., any illness or sickness that is not known or recognized in the community or diagnosed by medical providers) in the past year were having other household members who had unusual symptoms and being scratched or bitten by animals in the same year. Many participants reported raising and handling poultry (10.3% and 24.2%), swine (2%, 14.6%), and cattle (4.9%, 7.8%) and several participants also reported eating raw or undercooked meat of these animals (2.2%, 5.5%, 10.3% respectively). Twenty four participants (3.5%) reported handling bats or having bats in the house roof. Gender, age, and livelihood activities were shown to be significantly associated with participants' interactions with animals. Participants' knowledge of risks influenced their health-seeking behavior. CONCLUSION: The results suggest that there is a high level of interaction between humans, livestock, and wild animals in communities at sites we investigated in Thailand. This study highlights important differences among demographic and occupational risk factors as they relate to animal contact and zoonotic disease risk, which can be used by policymakers and local public health programs to build more effective surveillance strategies and behavior-focused interventions.

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.

Multiple clades of SARS-CoV-2 were introduced to Thailand during the first quarter of 2020.

In early January 2020, Thailand became the first country where a coronavirus disease 2019 (COVID-19) patient was identified outside China. In this study, 23 whole genomes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from patients who were hospitalized from January to March 2020 were analyzed, along with their travel histories. Six lineages were identified including A, A.6, B, B.1, B.1.8, and B.58, among which lineage A.6 was dominant. Seven patients were from China who traveled to Thailand in January and early February. Five of them were infected with the B lineage virus, and the other two cases were infected with different lineages including A and A.6. These findings present clear evidence of the early introduction of diverse SARS-CoV-2 clades in Thailand.

Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia.

Among the many questions unanswered for the COVID-19 pandemic are the origin of SARS-CoV-2 and the potential role of intermediate animal host(s) in the early animal-to-human transmission. The discovery of RaTG13 bat coronavirus in China suggested a high probability of a bat origin. Here we report molecular and serological evidence of SARS-CoV-2 related coronaviruses (SC2r-CoVs) actively circulating in bats in Southeast Asia. Whole genome sequences were obtained from five independent bats (Rhinolophus acuminatus) in a Thai cave yielding a single isolate (named RacCS203) which is most related to the RmYN02 isolate found in Rhinolophus malayanus in Yunnan, China. SARS-CoV-2 neutralizing antibodies were also detected in bats of the same colony and in a pangolin at a wildlife checkpoint in Southern Thailand. Antisera raised against the receptor binding domain (RBD) of RmYN02 was able to cross-neutralize SARS-CoV-2 despite the fact that the RBD of RacCS203 or RmYN02 failed to bind ACE2. Although the origin of the virus remains unresolved, our study extended the geographic distribution of genetically diverse SC2r-CoVs from Japan and China to Thailand over a 4800-km range. Cross-border surveillance is urgently needed to find the immediate progenitor virus of SARS-CoV-2.

Evaluating the efficiency of specimen pooling for PCR-based detection of COVID-19.

In the age of a pandemic, such as the ongoing one caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the world faces a limited supply of tests, personal protective equipment, and factories and supply chains are struggling to meet the growing demands. This study aimed to evaluate the efficacy of specimen pooling for testing of SARS-CoV-2 virus, to determine whether costs and resource savings could be achieved without impacting the sensitivity of the testing. Ten previously tested nasopharyngeal and throat swab specimens by real-time polymerase chain reaction (PCR), were pooled for testing, containing either one or two known positive specimens of varying viral concentrations. Specimen pooling did not affect the sensitivity of detecting SARS-CoV-2 when the PCR cycle threshold (Ct) of original specimen was lower than 35. In specimens with low viral load (Ct > 35), 2 of 15 pools (13.3%) were false negative. Pooling specimens to test for Coronavirus Disease 2019 infection in low prevalence (≤1%) areas or in low risk populations can dramatically decrease the resource burden on laboratory operations by up to 80%. This paves the way for large-scale population screening, allowing for assured policy decisions by governmental bodies to ease lockdown restrictions in areas with a low incidence of infection, or with lower-risk populations.