Porcine circovirus type 3 (PCV3) is an emerging pathogen linked with reproductive failure, respiratory disease, dermatitis, nephropathy, and multisystemic inflammation, posing growing threats to the swine industry worldwide. Although molecular evidence of PCV3 circulation has been reported in India, there is no published information on its seroprevalence, leaving the extent of exposure and population immunity largely unknown. Serological tools are critical for epidemiological surveillance and monitoring vaccine responses; however, validated assays for PCV3 remain scarce. In the present study, we developed and evaluated an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of PCV3-specific antibodies, based on a recombinant truncated Cap protein expressed in Escherichia coli. The antigenic fragment of the Cap gene, excluding the N-terminal nuclear localization signal, was used as coating antigen and assay parameters like optimal antigen concentration, serum dilution, and conjugate conditions, were standardized. The developed ELISA exhibited high specificity, with no cross-reactivity to antisera against other common porcine viruses, and the relative diagnostic sensitivity and specificity were estimated to be 97.0 % and 94.5 %, respectively. The assay also demonstrated strong repeatability and reproducibility and was validated at four different laboratories with κ- values indicating perfect agreement Application of the assay to field sera revealed widespread seropositivity (64.25 %), underscoring its utility for epidemiological surveillance and sero-monitoring of PCV3. This study provides the first indigenous ELISA with potential application in India, and is the first report indicating widespread seropositivity of PCV3 in India.

The lumpy skin disease (LSD) virus poses a significant threat to bovine health and productivity in endemic regions, highlighting the need for reliable and standardized reagents for serological surveillance. However, the availability of standardized recombinant antigens for serological assays is limited in clinical settings. This study describes a bioinformatics-guided strategy for developing recombinant LSDV P32 and B5R antigens for serodiagnostic applications. Despite their notable immunogenicity, the P32 and B5R proteins, which are hydrophobic envelope proteins with transmembrane regions, present considerable challenges in terms of recombinant expression and purification. Initial attempts to express ectodomain-only constructs (P32Tr and B5RTr) using the pET-33b(+) system resulted in detectable proteins but faced significant challenges during purification. Guided by in silico predictions regarding solubility and aggregation propensity, the expression strategy was modified by employing a thioredoxin fusion system [pET-32a(+)], which facilitated expression and successful purification via Ni-NTA chromatography under denaturing conditions, followed by refolding. The purified recombinant proteins were confirmed using SDS-PAGE and immunoblotting. Preliminary indirect ELISA demonstrated the specific seroreactivity of both P32 and B5R proteins, confirming their antigenicity by showing reactivity with LSDV-positive sera without any cross-reactivity with sera from other bovine viruses. This study establishes a practical workflow to overcome the expression and purification challenges associated with aggregation-prone or hydrophobic proteins, thereby demonstrating the potential of recombinant P32 and B5R proteins as standardized biological reagents for use in serological assays.

AbstractThe present study includes molecular confirmation and understanding the phylogenetic relationships of circulating goat pox virus (GTPV) strains in clinical cases of goat pox in Uttar Pradesh. Viral DNA was amplified for P32 gene using the polymerase chain reaction and sequenced. To evaluate genetic relatedness, the phylogenetic tree construction and multiple sequence alignment were carried out. The examined sequences exhibited high nucleotide identity (>98%) and confirmed the genetic conservation and regional distribution of GTPV strains. The examined sequences were closely clustered within recognised GTPV clades, suggesting little sequence divergence. These results contribute to the understanding of the molecular epidemiology of GTPV and also support the development of an indigenous virus-based vaccine to prevent and control GTPV.

Capripoxviruses, including the goatpox virus (GTPV), sheeppox virus (SPPV), and lumpy skin disease virus (LSDV), cause substantial economic losses in domestic ruminants in Asia, Africa, the Middle East, and Europe. The identification of viral antigens that elicit protective immunity, along with the characterization of antigen-specific humoral responses, is crucial for the rational development of vaccines and serodiagnostic assays. L1R is an essential membrane protein of the intracellular mature virion (IMV) and a known neutralizing antibody target for poxviruses. In this study, a truncated L1R ectodomain (rL1RΔTM; 191 aa; 20.6 kDa) was expressed in Escherichia coli, purified under denaturing conditions via affinity chromatography, and subsequently refolded to yield an antigenically functional protein. The expression of the recombinant protein was confirmed using SDS-PAGE and Western blotting. Immunization of rabbits with purified rL1RΔTM (250 μg per dose) elicited antigen-specific IgG responses and generated cross-neutralizing antibodies against capripoxviruses. In contrast, the rL1RΔTM-based indirect ELISA, utilizing sera from naturally infected or vaccinated animals, demonstrated that L1R-specific antibodies represent only a minor component of the capripoxvirus-specific humoral response. Consequently, the rL1RΔTM ELISA exhibited high diagnostic specificity (98.7-100%) but limited sensitivity (26-39% in infected animals and 8-22% in vaccinated animals). Additionally, it showed a weak correlation with neutralization titers (R2 = 0.11-0.18). These findings demonstrate that the subdominant contribution of L1R to the natural humoral immune response limits its effectiveness as a standalone antigen for serodiagnosis. Nonetheless, L1R is a conserved immunogen capable of inducing cross-neutralizing antibodies against capripoxviruses, supporting its potential utility in the development of pan-capripoxvirus subunit vaccines and monoclonal antibody production.

Lumpy skin disease (LSD) poses a significant threat to cattle populations in India and worldwide, owing to its rapid spread, high morbidity rate, and continuous emergence of new variants. Reliable in vitro systems for LSD virus (LSDV) isolation are essential for diagnostic applications, virological studies, and vaccine development. Primary ruminant cells are commonly used for initial virus isolation; however, they suffer from limited availability and poor reproducibility. In this study, we systematically evaluated the susceptibility of five cell lines, Vero, MA-104, MDBK, BHK-21, and goat testis (GT) cells for the direct isolation and propagation of LSDV. Viral replication and infectivity were assessed using cytopathic effect (CPE) monitoring, conventional multi-target PCR, 50% tissue culture infectious dose (TCID₅₀) assay, and an indirect immunoperoxidase test (IPT) for antigen detection. Direct virus isolation from clinical scab material was successful only in MA-104 and GT cells. In contrast, Vero, MDBK, and BHK-21 cells did not support primary isolation but readily propagated LSDV after prior adaptation to GT cells. Subsequent one-step growth kinetics, performed at multiplicities of infection (MOI) of 0.1 and 0.01, further elucidated the distinct cell line-dependent replication patterns. Vero cells yielded the highest titers (106.33 TCID50/mL), whereas MA-104 cells supported sustained replication with peak titers of 105.5 TCID50/mL, demonstrating their strong capacity for propagation, comparable to other permissive continuous cell lines (MDBK and BHK-21 cells), which required prior viral adaptation. Unlike primary GT cells, MA-104 is a novel, permissive continuous cell line offering benefits such as reproducibility, ready availability, and reduced ethical concerns, making it a robust alternative for primary cell isolation. This study provides practical insights into the selection of appropriate cell lines and determination of optimal harvesting time points for efficient LSDV isolation and propagation, as well as downstream applications, such as pathogenesis studies and vaccine development.

Detailed spatiotemporal pathogenesis data for virulent Indian Goatpox virus (GTPV) isolates in the natural host remain limited. This study characterized systemic dissemination and tissue tropism of the GTPV Mukteshwar strain following experimental intradermal inoculation (106 SRD50) in goats. Sixteen GTPV-seronegative goats (12-21 months) were monitored longitudinally, with replicated serial necropsies conducted from 3 to 35 days post-infection (dpi). Viral dissemination was quantified by real-time PCR targeting the RPO147 gene, and tissue lesions and antigen localization were evaluated by gross pathology, histopathology, immunohistochemistry, and special staining. Infected goats developed fever, lymphadenopathy, and characteristic cutaneous lesions after an incubation period of 3-11 days. Primary replication was detected at the inoculation site by 3 dpi, followed by viremia between 5 and 14 dpi, with peak blood viral loads of 1.64-3.55 log10 DNA copies/μL during 8-11 dpi. Systemic dissemination involved skin, lymphoid organs, respiratory and gastrointestinal tissues, urinary system, and male reproductive organs, while central nervous system tissues remained negative. Viral shedding occurred predominantly via conjunctival and nasal secretions between 8 and 21 dpi, peaking at 5.04 and 4.24 log10 DNA copies/μL, respectively, at 11 dpi, with intermittent urinary detection reaching 2.11 log10 DNA copies/μL at 14 dpi. Persistent antigen and/or viral DNA detection in respiratory and gastrointestinal tissues beyond clinical recovery indicates tissue-specific viral maintenance. Defined viremia peaks and persistence windows establish a kinetic framework for systemic dissemination and provide quantitative benchmarks for vaccine efficacy assessment, surveillance, and control strategies in endemic regions.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV) within the family Poxviridae. These transboundary and highly infectious viruses cause substantial economic losses by affecting the productivity of both small and large ruminants. Clinical manifestations include cutaneous lesions (skin nodules and pustular lesions), lymphadenopathy, pneumonia, reduced milk yield, mastitis, infertility and abortion. The diagnosis is based on a combination of clinical signs, virus isolation, serology and PCR/real-time PCR. Recent advancements have significantly improved the sensitivity and specificity of CaPV detection and differentiation. These include multiplexed serological assays, isothermal DNA amplification methods such as recombinase polymerase reaction, CRISPR-Cas12a fluorescence assays and advanced DNA sequencing platforms. In enzootic regions, strategic control measures should include public awareness, vector control, early detection, vaccination, use of ethnoveterinary formulations, veterinary care, strict biosecurity and movement restrictions. The live attenuated vaccines remain the most suitable option for these regions owing to their efficacy. Cross-protective CaPV vaccine strains also support heterologous vaccination strategies. Emerging multivalent and recombinant vaccines offer promising avenues for providing broad protection and simplifying disease management. Overall, it is essential to break the viral transmission cycle to mitigate the economic losses.

Recent genomic studies have focused on understanding the genetic basis of high-altitude adaptation, a classic example of evolutionary response to extreme environments. While most of these studies have concentrated on autosomal regions, the role of sex chromosomes, particularly the X chromosome, in this adaptation has been relatively unexplored. So, in this study, we performed an intensive scan of the X chromosome of Changthangi sheep as well as other Indian and exotic sheep to understand the indications of high-altitude adaptation by exploring population structure, genetic relationships, and signatures of selection on X-chromosome. Our findings reveal a close genetic affinity between Changthangi and Tibetan sheep, suggesting a shared evolutionary history that is similar to the findings of autosomal study. Additionally, we uncovered evidence of gene flow from Deccani and Australian Merino breeds into the Changthangi gene pool. Through a comprehensive analysis of selection signatures, we identified 14 candidate genes and several unannotated loci, out of which nine genes were potentially associated with high-altitude adaptation and these genes are implicated in various biological processes, including fatty acid metabolism, muscle function, potassium channel regulation, transcription, glycogen metabolism, neuronal function, RNA modification, mRNA splicing, synaptic transmission, and hypoxia response. Our study was the first to provide valuable insights into X chromosome genetic mechanisms underlying highaltitude adaptation in Changthangi sheep. These, along with autosomal findings have implications for future breeding strategies aimed at improving the resilience and productivity of Changthangi sheep in harsh environments.

Baculovirus expression system has become the most widely used eukaryotic system for recombinant protein production due to its ability to perform post-translational modifications and produce proteins with native-like conformation and antigenicity. In this study, two major immunodominant intracellular mature virion genes of Goatpox virus were amplified into three constructs: A27L, full-length P32, and truncated P32 (tr-P32), respectively. The constructs were cloned into the pFastBac HTA vector and expressed in Trichoplusiani (TN5) insect cells using the baculovirus expression system. Recombinant A27L was purified under native conditions, however full-length P32 and tr-P32 required denaturing conditions. tr-P32 showed markedly higher expression and solubility than full-length P32. All recombinant proteins were immunoreactive with hyperimmune GTPV sera, as confirmed by western blotting. Native PAGE analysis of rA27L revealed concentration-dependent oligomerization into dimers, trimers and tetramers. Furthermore, in indirect ELISA, both A27L and P32 exhibited strong reactivity with sera from GTPV and SPPV infected or vaccinated animals at 1:50 dilution and did not show any cross-reactivity with related viruses. These results quantitatively demonstrated that baculovirus-expressed A27L and P32 proteins are superior diagnostic antigens, providing higher sensitivity and specificity for Capripoxviruses serodiagnosis and offering potential as improved candidates for both serological and prophylactic applications.

Lumpy Skin Disease Virus (LSDV), a Capripoxvirus of significant veterinary and economic importance, has been reported to manipulate host cellular processes, including autophagy, to enhance its replication and persistence. However, the precise mechanisms by which LSDV interacts with autophagy remain unclear. This study investigates the effect of LSDV infection on host autophagic pathways in MDBK cells, focusing on autophagic flux modulation and its implications for viral replication. Western blot and immunofluorescence analyses demonstrated that LSDV does not robustly induce autophagy but interferes with autophagic flux by suppressing lysosomal degradation, as indicated by reduced LAMP2 expression at later stages of infection. Pharmacological modulation of autophagy using activators (Rapamycin, Torin 2) and inhibitors (Bafilomycin A1, MRT68921) revealed that increased autophagy enhanced LSDV replication, whereas inhibition of autophagy significantly impaired viral propagation, underscoring the virus's reliance on autophagic processes for efficient replication. Notably, cells infected with BEI-inactivated LSDV exhibited significantly higher LC3B II accumulation than those infected with live LSDV, suggesting that active viral replication is required for autophagy modulation. Additionally, dose-dependent analysis revealed that LSDV-mediated suppression of LC3B II was most prominent at higher viral titers, further confirming the virus's capacity to regulate host autophagic responses. These findings suggest that LSDV strategically modulates autophagy by maintaining basal autophagic levels while suppressing lysosomal degradation, allowing for optimal viral replication. Understanding the interplay between LSDV and autophagy provides novel insights into its pathogenesis and identifies potential antiviral targets to mitigate LSDV infections in cattle.

Orf or contagious ecthyma is a highly contagious, zoonotic, and economically important global viral disease of small ruminants and is endemic in India. Vaccination of susceptible goats/sheep along with suitable recombinant protein-based serological assay will be useful in the control of the infection. In this study, the full-length and truncated versions of F1L encoding gene (ORF 059) of orf virus were cloned into pFasBac HT A vector, transformed in DH10Bac cells, and expressed in insect cells. The full-length and truncated recombinant F1L proteins were expressed as a 6 × histidine-tagged fusion protein for ease of purification by Ni–NTA affinity chromatography under denaturing conditions. A protein with 40 kDa and 35 kDa for full-length and truncated F1L protein, respectively, were expressed and confirmed by SDS-PAGE and western blot. The protein reactivity evaluated by western blot analysis and indirect ELISA using ORFV hyperimmune serum was also found to be reactive. The results of the present study showed that the purified recombinant F1L protein can be used as a diagnostic antigen in sero-surveillance of ORFV infection in small ruminants. To the best of authors’ knowledge, this is the first report on the expression of ORFV F1L in insect cells using a baculovirus vector and its successful purification to use as the potential diagnostic antigen in ELISA.

Goatpox and sheeppox are highly contagious and economically important viral diseases of small ruminants. Due to the risk they pose to animal health, livestock production, and international trade, capripoxviruses are a considerable threat to the livestock economy. In this study, we expressed two core proteins (A4L and A12L) and one extracellular enveloped virion protein (A33R) of goatpox virus in a baculovirus expression vector system and evaluated their use as diagnostic antigens in ELISA. Full-length A4L, A12L, and A33R genes of the GTPV Uttarkashi strain were amplified, cloned into the pFastBac HT A donor vector, and introduced into DH10Bac cells containing a baculovirus shuttle vector plasmid to generate recombinant bacmids. The recombinant baculoviruses were produced in Sf-21 cells by transfection, and proteins were expressed in TN5 insect cells. The recombinant proteins were analysed by SDS-PAGE and confirmed by western blot, with expected sizes of 30 kDa, 31 kDa, and 32 kDa for A4L, A12L, and A33R, respectively. The recombinant proteins were purified, and the immunoreactivity of the purified proteins was confirmed by western blot using anti-GTPV serum. The antigenic specificity of the expressed proteins as diagnostic antigens was evaluated by testing their reactivity with infected, vaccinated, and negative GTPV/SPPV serum in indirect ELISA, and the A33R-based indirect ELISA was optimized. The diagnostic sensitivity and specificity of the A33R-based indirect ELISA were found to be of 89

AIMS:Japanese encephalitis (JE) is endemic in India. Although pigs are considered important hosts and sentinels for JE outbreaks in people, limited information is available on JE virus (JEV) surveillance in pigs. METHODS AND RESULTS:We investigated the spatio-temporal distribution of JEV seroprevalence and its association with climate variables in 4451 samples from pigs in 10 districts of eastern Uttar Pradesh, India, over 10 years from 2013 to 2022. The mean seroprevalence of IgG (2013-2022) and IgM (2017-2022) was 14% (95% CI 12.8-15.2) and 10.98% (95% CI 9.8-12.2), respectively. Throughout the region, higher seroprevalence from 2013 to 2017 was observed and was highly variable with no predictable spatio-temporal pattern between districts. Seroprevalence of up to 60.8% in Sant Kabir Nagar in 2016 and 69.5% in Gorakhpur district in 2017 for IgG and IgM was observed, respectively. IgG seroprevalence did not increase with age. Monthly time-series decomposition of IgG and IgM seroprevalence demonstrated annual cyclicity (3-4 peaks) with seasonality (higher, broader peaks in the summer and monsoon periods). However, most variance was due to the overall trend and the random components of the time series. Autoregressive time-series modelling of pigs sampled from Gorakhpur was insufficiently predictive for forecasting; however, an inverse association between humidity (but not rainfall or temperature) was observed. CONCLUSIONS:Detection patterns confirm seasonal epidemic periods within year-round endemicity in pigs in eastern Uttar Pradesh. Lack of increasing age-associated seroprevalence indicates that JEV might not be immunizing in pigs which needs further investigation because models that inform public health interventions for JEV could be inaccurate if assuming long-term immunity in pigs. Although pigs are considered sentinels for human outbreaks, sufficient timeliness using sero-surveillance in pigs to inform public health interventions to prevent JEV in people will require more nuanced modelling than seroprevalence and broad climate variables alone.

Lumpy skin disease (LSD) was reported for the first time in India in 2019 and since then, it has become endemic. Since a homologous (LSD-virus based) vaccine was not available in the country, goatpox virus (GPV)-based heterologous vaccine was authorized for mass immunization to induce protection against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed by using an Indian field strain, isolated in 2019 from cattle. The virus was attenuated by continuous passage (P = 50) in Vero cells. The vaccine (50(th) LSDV passage in Vero cells, named as Lumpi-ProVac( Ind )) did not induce any local or systemic reaction upon its experimental inoculation in calves (n = 10). At day 30 post-vaccination (pv), the vaccinated animals were shown to develop antibody- and cell-mediated immune responses and exhibited complete protection upon virulent LSDV challenge. A minimum Neethling response (0.018% animals; 5 out of 26,940 animals) of the vaccine was observed in the field trials conducted in 26,940 animals. There was no significant reduction in the milk yield in lactating animals (n = 10108), besides there was no abortion or any other reproductive disorder in the pregnant animals (n = 2889). Sero-conversion was observed in 85.18% animals in the field by day 30 pv.

Members of the genus capripoxvirus (CaPV) encode more than 100 putative proteins and CaPV ORF 095 and 103 genes encode two major immunogenic core proteins that are partly exposed to the host immune system. In this study, full-length genes of these two core proteins of goatpox virus (GTPV) were over-expressed in E. coli BL21-codonPlus (DE3)-RIPL strain using pET32a vector and purified under denaturing conditions and characterized by SDS-PAGE. Expressed proteins were confirmed as 37 kDa (ORF 095) and 39 kDa (ORF 103) proteins in western blot. Both the proteins have shown a good immunoreactivity with anti-GTPV and -SPPV antibodies in optimized ELISA and they did not show any cross-reactivity with other viruses of sheep and goats. However, the ORF095 protein has shown better reactivity than ORF103 to a standard known positive serum when tested in optimized ELISA. Therefore, capripoxvirus-specific rORF095 alone or in combination with other CaPV proteins has the immense potential in developing ELISA as a coating antigen for sero-evaluation of capripox in sheep and goats.

Lumpy skin disease (LSD) is an economically important poxviral disease endemic to Asia, Europe, and Africa. Recently, LSD has spread to naive countries, including India, China, Bangladesh, Pakistan, Myanmar, Vietnam, and Thailand. Here, we describe the complete genomic characterization of LSDV from India, LSDV-WB/IND/19 isolated from an LSD affected calf in 2019 determined by Illumina next-generation sequencing (NGS). The LSDVWB/IND/19 has a genome size of 150,969 bp encoding 156 putative ORFs. Phylogenetic analysis based on complete genome sequence suggested that LSDV-WB/IND/19 is closely related to Kenyan LSDV strains with 10-12 variants with non-synonymous changes confined to LSD_019, LSD_049, LSD_089, LSD_094, LSD_096, LSD_140, and LSD_144 genes. In contrast to complete kelch-like proteins in Kenyan LSDV strains, LSDV-WB/IND/ 19 LSD_019 and LSD_144 genes were found to encode truncated versions (019a, 019b, and 144a, 144b). LSD_019a and LSD_019b proteins of LSDV-WB/IND/19 resemble that of wild-type LSDV strains based on SNPs and the C-terminal part of LSD_019b except for deletion at K229, whereas the LSD_144a and LSD_144b proteins resemble that of Kenyan LSDV strains based on SNPs, however, C-terminal part of LSD_144a resembles that of vaccine-associated LSDV strains due to premature truncation. The NGS findings were confirmed by Sanger sequencing of these genes in Vero cell isolate as well as in the original skin scab along with similar findings in another Indian LSDV from scab specimen. LSD_019 and LSD_144 genes are thought to modulate virulence and host range in capripoxviruses. This study demonstrates the circulation of unique LSDV strains in India and highlights the importance of constant monitoring of the molecular evolution of LSDV and associated factors in the region in light of the emergence of recombinant LSDV strains.

Sheeppox virus (SPPV) is responsible for a significant economic loss to sheep husbandry in enzootic regions of Africa, the Middle East, and Asia including the Indian subcontinent. In this study, we present the complete genome sequence of SPPV vaccine strain SPPV-Srin38/00 from India determined by next-generation sequencing (NGS) using Illumina technology. The attenuated Srinagar vaccine strain of SPPV (SPPV-Srin38/00) was developed by serial passaging the virus initially in lamb testes (LT) cells followed by Vero cell line. The SPPV-Srin38/00 virus has a genome size of 150, 103 bp, which encodes for 147 functional putative genes and consists of a central coding region flanked by two identical 2353 bp inverted terminal repeats (ITRs). Comparative phylogenetic analysis based on complete genome sequences of Capripoxviruses formed three distinct groups each for SPPV, GTPV, and LSDV with clustering of SPPV-Srin38/00 strain with SPPV-A strain. Nine ORFs of SPPV-Srin38/00 namely SPPV-Srin_002/SPPV-Srin_155, SPPV-Srin_004/SPPV-Srin_153, SPPV-Srin_009, SPPV-Srin_013, SPPV-Srin_026, SPPV-Srin_132, and SPPV-Srin_136 were found to be fragmented as compared to LSDV, whereas only one ORF (such as SPPV-Srin_136) was found to be fragmented as compared to GTPV. SPPV genomes, including the SPPV-Srin38/00 strain, shared 99.78-99.98% intraspecies nucleotide identity, indicating that SPPV strains have extremely low genetic diversity. The strain shared 96.80-97.08% and 97.11-97.61% nt identity with GTPV and LSDV strains, respectively. Its ORFs 016, 021, 022, 130 and 138 are the least identical ORFs among three species of the genus Capripoxvirus with 72.5-93% aa identity to GTPV and LSDV strains and may be potentially used for differentiation of CaPV species. This study may contribute to a better understanding of the epidemiology and evolution of capripoxviruses as well as the development of specific detection methods, better expression vectors, and vaccines with improved safety and efficacy.

The orf virus (ORFV) is an epitheliotropic virus causing a highly contagious skin disease mainly in sheep and goats. Several diagnostics including molecular tools like Loop mediated isothermal amplification (LAMP) assay are available to detect ORFV in affected species. However, the carry-over contamination associated with LAMP as open tube format prevents the assay applicability as point of care test in field diagnostic settings. In this study, the B2L gene based LAMP assay was optimized in a closed tube format using hydroxynaphthol blue (HNB) and calcein as pre-addition dyes and it has shown a clear positive and negative signal at 60 °C using 4 and 5 mM concentrations of MgSO4 respectively for these dyes. Optimitimzed assay that could reveal the result within one hour is highly specific and senstive with a limit of detection at 12.5 femtogram of viral genomic DNA or ~85 virus genome equivalent. This improved method prevented the cross-contamination of future LAMP reactions in the laboratory without compromising diagnostic sensitivity (100%) and specificity (100%) when compared to open tube system. This closed tube LAMP method has potential to act as a simple visual detection assay for the rapid and specific diagnosis of ORFV in sheep and goats.

Look into the nature, and then you will understand it better. – Albert Einstein, quoted by Margo Einstein, in a letter to Carl Seelig, May 8, 1955 Tiwari et al.[1] review the impairment of olfactory and gustatory sensations in severe acute respiratory syndrome – Coronavirus-2 disease in June 2021 issue of the journal. They scan relevant literature, analyse it comprehensively, piece together important common points, and draw some useful and pragmatic conclusion there. There is a table in the properly drafted article which summarizes essential points at a single place. However, we want to highlight two related points in the write up which we are unable to reconcile. First, therein under a heading of 'Introduction', the authors write that the (novel) virus severe acute respiratory syndrome - coronavirus - 2 (SARS-CoV-2) mainly spreads via droplets from an infected patient but can also spread through direct contact and orofaecal route. But fact of the matter is that now we recognize another important route of its transmission and that is by aerosol. When an infected person speaks or simply breathes, these invisible particles make a gaseous emission akin to smoke around and when a healthy person inhales it, one gets infected. Torjsen wrote early last year that it was for this reason that improving ventilation in hospitals was necessary so that concentration of viral particles was reduced in the ambience and healthcare staff derived more safety. As the particles of the aerosol are in micrometre range, stay afloat, move around in enclosed spaces, surgical mask may not provide adequate protection and hospital staff outside the intensive care unit too need to use high-end respirators to get protection besides those inside.[2] Baraniuk wrote that high-efficiency particulate air filters or specialized ventilation systems might prevent airborne transmission by removing the virus particles in first case and diluting it in the next scenario. And by this logic, outdoor spaces are relatively safer than indoors provided all the other denominators are same. Therefore, while opening up the economy in unlock phase, preference and priority should be given to opening systems outdoors, for example, public parks and open-air restaurants, rather than indoor dining halls or cinema halls.[3] If doing so is economically unsuitable, risky, and disingenuous, we need to remember that shutting economy down altogether hurts everybody more anyway when the number of cases surge and hospitals gets crowded. US Center for Disease Control and Prevention now acknowledges that this virus is an airborne threat. The agency now states explicitly in large, bold lettering that airborne virus can be inhaled even when one is more than 6 ft away from an infected individual. The new language, posted online, is a change from the agency's previous position that most infections were acquired through 'close contact, not airborne transmission'.[4] Second point is related to Table 1 of the article. The table enlists several studies which the authors explored to highlight symptomatology of the infected patients. There all sorts of patients are visible presenting with a wide spectrum of the illness. But in our working experience in our corona virus disease - 2019 (COVID) hospital, one of the common symptoms in severe cases is chest pain. World Health Organization mentions this symptom on its concerned webpage.[5] Rather surprisingly this one is not visible on that table. Only once in a study at the top, chest tightness is complained by patients as depicted in first row. But amazingly as this one of the common symptoms – which patients describe when they land up in our hospital – is missing, the fact demands further investigation into the matter. Moreover, when patients visit our hospital and are treated with drugs, many times their symptoms are relieved. If that is the case here too, that means somehow the symptoms are noted after administration of the drugs; the point should be noted concurrently. Nevertheless, if we are conducting a study to record various symptoms when get infected, absence of one bewilders us. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. We accessed all the webpages at the time of submission of this letter to the editor.

Lumpy skin disease (LSD) is an economically important poxviral disease endemic to Asia, Europe, and Africa. Recently, LSD has spread to naïve countries, including India, China, Bangladesh, Pakistan, Myanmar, Vietnam, and Thailand. Here, we describe the complete genomic characterization of LSDV from India, LSDV-WB/IND/19 isolated from a calf in Vero cells determined by Illumina next-generation sequencing (NGS). The LSDV-WB/IND/19 has a genome size of 150969 bp encoding 156 putative ORFs. Phylogenetic analysis based on complete genome sequence suggested that LSDV-WB/IND/19 is closely related to Kenyan LSDV strains with 10-12 variants with non-synonymous changes confined to LSD_019, LSD_049, LSD_089, LSD_094, LSD_096, LSD_140, and LSD_144 genes. In contrast, to complete kelch-like proteins in Kenyan LSDV strains, LSDV-WB/IND/19 LSD_019 and LSD_144 genes were found to encode truncated versions (019a, 019b, and 144a, 144b). LSD_019a and LSD_019b proteins of LSDV-WB/IND/19 resemble that of wild-type LSDV strains based on SNPs and the C-terminal part of LSD_019b except for deletion at K229, whereas the LSD_144a and LSD_144b proteins resemble that of Kenyan LSDV strains based on SNPs, however, C-terminal part of LSD_144a resembles that of vaccine-associated LSDV strains due to premature truncation. The NGS findings were confirmed by Sanger sequencing of these genes in Vero cell isolate as well as in the original skin scab along with similar findings in another Indian LSDV from scab specimens. LSD_019 and LSD_144 genes are thought to modulate virulence and host range in capripoxviruses. This study demonstrates the circulation of unique LSDV strains in India and highlights the importance of constant monitoring of the molecular evolution of LSDV and associated factors in the region in light of the emergence of recombinant LSDV strains.