Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis.

BACKGROUND: RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key pre-analytical step for SARS-CoV2 detection which often achieved using commercial RNA-extraction kits. However, due to the COVID-19 pandemic, bulk production and the supply chains for the commercial RNA-extraction kit have been seriously compromised. The shortage of commercial RNA-extraction kit is even more acute in developing country. Furthermore, use of one-off design RNA-columns can generate plastic wastes that have an environmental pollution effect. METHODS AND RESULTS: To address these issues, in this study, we used warm alkaline solution containing Triton X-100 for the complete removal of the residual SARS-CoV2 RNA from the used RNA-binding silica column. Columns regenerated using the alkaline solution have the viral RNA purification capability that is comparable to the fresh silica columns. We also demonstrated that RNA-binding silica columns can be regenerated and reused for a minimum of five-times. CONCLUSIONS: Therefore, the use of the RNA-column regeneration method may benefits several SARS-CoV2 diagnostic laboratories throughout the world by cutting down the requirement of commercial RNA-purification column.

Complete coding region sequence analyses and antigenic characterization of emerging lineage G-IX of foot- and-mouth disease virus serotype Asia1.

Foot-and-mouth disease Virus (FMDV) serotype Asia1 is prevalent in the Indian subcontinent, with only G-III and G-VIII reported in India until 2020. However, in 2019, a novel genetic group within serotype Asia1, designated as G-IX, emerged in Bangladesh, followed by its detection in India in 2020. This report presents analyses of the complete coding region sequences of the G-IX lineage isolates. The length of the open reading frame (ORF) of the two G-IX isolates was 6990 nucleotides without any deletion or insertion. The G-IX isolates showed the highest sequence similarity with an isolate of G-III at the ORF, L, P2, and P3 regions, and with an isolate of G-VIII at the P1 region. Phylogenetic analysis based on the capsid region (P1) supports the hypothesis that G-VIII and G-IX originated from a common ancestor, as speculated earlier. Further, VP1 region-based phylogenetic analyses revealed the re-emergence of G-VIII after a gap of 3 years. One isolate of G-VIII collected during 2023 revealed a codon insertion in the G-H loop of VP1. The vaccine matching studies support the suitability of the currently used Indian vaccine strain IND63/1972 to contain outbreaks due to viruses belonging to G-IX.

Foot-and-Mouth Disease Virus Serotype O Exhibits Phenomenal Genetic Lineage Diversity in India during 2018-2022.

In India, widespread foot-and-mouth disease (FMD) outbreaks occurred in 2021. The objective of this study was to identify genetic lineages and evaluate the antigenic relationships of FMD virus (FMDV) isolates gathered from outbreaks reported between 2019 and 2022. Our study shows that the lineages O/ME-SA/Ind2001e and the O/ME-SA/Cluster-2018 were both responsible for the FMD outbreaks on an epidemic scale during 2021. This observation is in contrast to earlier findings that suggested epidemic-scale FMD outbreaks in India are often connected to a single genetic lineage. Additionally, we report here the identification of the O/ME-SA/PanAsia-2/ANT10 sub-lineage in India for the first time, which was connected to two intermittent outbreaks in Jammu and Kashmir. The current study demonstrates that the O/ME-SA/ind2001e lineage has a strong presence outside of the Indian subcontinent. Furthermore, the O/ME-SA/Cluster-2018 was observed to have a wider geographic distribution than previously, and like the O/ME-SA/Ind2001d and O/ME-SA/Ind2001e lineages in the past, it may eventually spread outside of its geographic niche. For O/ME-SA/Ind2001e and O/ME-SA/Cluster-2018, the predicted substitution rate for the VP1 region was 6.737 × 10-3 and 8.257 × 10-3 nt substitutions per site per year, respectively. The time of the most recent common ancestor of the O/ME-SA/Ind2001e and O/ME-SA/Cluster-2018 strains suggests that the viruses possibly emerged during 2003-2011 and 2009-2017, respectively. Recent sightings of the O/ME-SA/PanAsia2/ANT10 virus in India and the O/ME-SA/Ind2001e virus in Pakistan point to possible cross-border transit of the viruses. The results of a two-dimensional viral neutralization test revealed that all of the field isolates were antigenically matched to the currently used Indian vaccine strain O INDR2/1975. These results suggest that the serotype O vaccine strain can protect against outbreaks brought on by all three circulating lineages.

A reverse transcription-multiplex PCR strategy devised for concomitant detection and differentiation of foot and mouth disease virus serotypes O, A and Asia 1 in India.

Serotype identification occupies the central part of foot and mouth disease (FMD) diagnosis workflow and vaccination decision tree. In this study, a reverse transcription-multiplex PCR (RT-mPCR) strategy wherein three assays with unique combinations of serotype specific primers targeting the VP1 region was developed to differentiate FMD virus serotypes O, A and Asia 1 based on differential size of the PCR amplicons on agarose gel. Their diagnostic performance relative to the mPCR assay in use in India was evaluated on 169 clinical samples and 210 cell culture grown virus isolates. The relative diagnostic sensitivity was found to be 99.69%, 98.78% and 99.08% for primer combinations 1, 2 and 3, respectively. These assays proved their worth by detecting serotype in three FMD suspected specimens that went undiagnosed in the existing mPCR and also by identifying multiple serotypes in the same sample. Their detection limits varied from log10 2 to log10 4 viral RNA dilution and from 100 to 0.1 TCID50 virus depending on the serotype. The validated novel mPCR assays show promise to be included in the routine diagnostic tool-box to augment the efficiency of diagnosis of FMD virus serotypes that display extreme genetic diversity and a tendency of transboundary dispersal.

Emergence of a novel genetic lineage 'A/ASIA/G-18/2019' of foot and mouth disease virus serotype A in India: A challenge to reckon with.

Foot and mouth disease (FMD) has engendered large scale socioeconomic crises on numerous occasions owing to its extreme contagiousness, transboundary nature, complicated epidemiology, negative impact on productivity, trade embargo, and need for intensive surveillance and expensive control measures. Emerging FMD virus variants have been predicted to have originated and spread from endemic Pool 2, native to South Asia, to other parts of the globe. In this study, 26 Indian serotype A isolates sampled between the year 2015 and 2022 were sequenced for the VP1 region. BLAST and maximum likelihood phylogeny suggest emergence of a novel genetic group within genotype 18, named here as 'A/ASIA/G-18/2019' lineage, that is restricted so far only to India and its eastern neighbour, Bangladesh. The lineage subsequent to its first appearance in 2019 seems to have displaced all other prevalent strains, in support of the phenomenon of 'genotype/lineage turnover'. It has diversified into two distinct sub-clusters, reflecting a phase of active evolution. The rate of evolution of the VP1 region for the Indian serotype A dataset was estimated to be 6.747 × 10-3 substitutions/site/year. India is implementing a vaccination centric FMD control programme. The novel lineage showed good antigenic match with the proposed vaccine candidate A IND 27/2011 when tested in virus neutralization test, while the existing vaccine strain A IND 40/2000 showed homology with only 31% of the isolates. Therefore, in order to combat this challenge of antigenic divergence, A IND 27/2011 could be the preferred strain in the Indian vaccine formulations.

Foot-and-mouth disease status in India during the second decade of the twenty-first century (2011-2020).

Foot-and-mouth disease (FMD) is a major disease of livestock in India and causes huge economic losses. The formal FMD control program started in 2003-04 in selected districts and was gradually expanded. The present study provides a descriptive review of the FMD outbreaks, prevalent serotypes, and genetic and antigenic features of the FMD virus (FMDV) that circulated in the country between 2011 and 2020. FMD outbreaks were regularly reported in cloven-hoofed domestic livestock and wildlife, with three serotypes including O, A, and Asia1. During the study period, a total of 2226 FMD outbreaks were documented and serotypes confirmed. FMDV serotype O dominated the outbreak scenario, accounting for about 92% of all outbreaks, followed by Asia1 (5% of all outbreaks) and A (3% of all outbreaks). Two major epidemics of FMD on an unprecedented scale during the years 2013 and 2018 by serotype O were recorded. The spatial distribution of FMD was characterized by a larger number of outbreaks in the southern region of the country. In an annual-scale analysis, 2020 was the year with the lowest outbreaks, and 2013 was the year with the highest. The month-scale analysis showed that outbreaks were reported throughout the year, with the highest numbers between October and March. The emergence of three major lineages (O/ME-SA/Ind2001d, O/ME-SA/Ind2001e, and O/ME-SA/Ind2018) of serotype O was observed during the period. In the cases of serotype A and Asia1, the appearance of at least one novel lineage/genetic group, including A/G-18/non-deletion/2019 and Asia1/Group-IX, was documented. While serotype A showed the advent of antigenic variants, serotypes O and Asia1 did not show any antigenic diversity. It was noticed during the course of an outbreak that animal movement contributes significantly to disease transmission. Except for 2018, when numerous FMD outbreaks were recorded, the number of annual outbreaks reported after 2016 has been lower than in the first half of the decade, probably due to mass vaccination and COVID-19 pandemic-linked movement restrictions. Even during outbreaks, disease symptoms in ruminant populations, including cattle, were found to be less severe. Regular six-monthly immunization certainly has a positive impact on the reduction of disease burden and should be followed without fail and delay, along with intensive disease surveillance.

Pathological and molecular investigations of systemic form of camelpox in naturally infected adult male dromedary camels in India.

Camelpox is a wide-spread infectious viral disease of camelids. An outbreak of camelpox was reported in 15 adult male dromedary camels aged between 10 to 16 years of an organized herd in winter season. The infected camels showed clinical signs of fever, anorexia, lachrymation, pendulous lips, excessive salivation and pock lesions on the skin of head, neck, mouth, lips, extremities, thigh, abdomen, scrotum and inguinal region. Mortalities were recorded in three infected camels after 10-12 days of infection and showed systemic pox lesions characterized by vesicles, papules, ulcerations and raised pock lesions in the mucous membranes of the mouth, tongue, tracheal mucosa, lung, abomasum and liver. Histopathology study revealed characteristic pox lesions with intracytoplasmic eosinophilic inclusion bodies in tongue. Lung showed lesion of interstitial pneumonia (n = 2) and bronchointerstitial pneumonia (n = 1). Liver showed infiltration of mononuclear cells around central veins and degenerative changes of hepatocytes. The abomasum and intestine showed ulcerations, marked capillary congestion and areas of lymphocyte infiltration in mucosa and submucosa. The presence of camelpox virus (CMLV) was confirmed in viral DNA isolated from formalin fixed paraffin embedded (FFPE) tissues of tongue, lung, abomasum, liver, heart and intestine of infected camels by C18L gene PCR. The sequencing of viral DNAs showed phylogenetic relatedness with other CMLV isolates from India and other countries. Thus, our study confirmed the rare severe form of systemic camelpox outbreak in adult male dromedary camels hence future attention should be given for studies on virulence, strain identification and molecular epidemiology of CMLV for planning of effective preventive and control strategies.

Genetic characterization of foot-and-mouth disease virus serotype O isolates collected during 2014-2018 revealed dominance of O/ME-SA/Ind2001e and the emergence of a novel lineage in India.

Foot-and-mouth disease (FMD) is endemic in India with a preponderance of outbreaks caused by FMD virus (FMDV) serotype O. Out of the 11 global topotypes of serotype O, only ME-SA topotype has been reported in the country so far. Lineage O/ME-SA/Ind2001 and O/ME-SA/PanAsia are documented as the most dominant ones in terms of the number of outbreaks caused by them. To understand the distribution of topotype/lineages in India and their antigenic behaviour during the year 2014-2018, a total of 286 FMDV serotype O viral isolates were sequence determined at the VP1 region, and 109 isolates were characterized antigenically. All the isolates grouped in the ME-SA topotype, being distributed in lineage O/ME-SA/Ind2001 (within sub-lineages O/ME-SA/Ind2001d and O/ME-SA/Ind2001e), and a new group designated here as O/ME-SA/2018 cluster. The sub-lineage O/ME-SA/Ind2001e reported for the first time in India during the year 2015, replaced sub-lineage O/ME-SA/Ind2001d gradually, which was dominating since 2008. During the years 2014-2018, the sub-lineage O/ME-SA/Ind2001e was found to be the most predominant one whose mean evolutionary rate was observed to be faster than that of the sub-lineage O/ME-SA/Ind2001d. The codon sites 45 and 85 of VP1 were found to be under diversifying selection in a large proportion of trees. The common ancestor predicted for sub-lineages O/ME-SA/Ind2001e and O/ME-SA/2018 dates back to 2012 and 2016, respectively. The sustenance and spread of the new O/ME-SA/2018 cluster need to be assessed by continued surveillance. The Indian vaccine strain O/INDR2/1975 was found to provide adequate antigenic coverage to the emerging and prevalent serotype O lineages. The trait association tests showed frequent virus exchange among different states, which could be an important confounder in the region-specific assessment of effectiveness of FMD control programme.