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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 against LSD in cattle. This study describes the evaluation of safety, immunogenicity and efficacy of a new live-attenuated LSD vaccine developed using an Indian field strain (LSDV/India/2019/Ranchi). The virus was attenuated by continuous passage (P=50) in Vero cells. The vaccine (50thLSDV passage in Vero cells, named as Lumpi-ProVacInd) 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 response and exhibited complete protection upon virulent LSDV challenge. We observed a minimum Neethling response (0.018% animals; 5 out of 26940 animals) of the vaccine in field trials among 26940 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.

Host-dependency factors have increasingly been targeted to minimize antiviral drug resistance. In this study, we have demonstrated that inhibition of p38 mitogen-activated protein kinase (a cellular protein) suppresses buffalopox virus (BPXV) protein synthesis by targeting p38-MNK1-eIF4E signaling pathway. In order to provide insights into the evolution of drug resistance, we selected resistant mutants by long-term sequential passages (P; n = 60) in the presence of p38 inhibitor (SB239063). The P60-SB239063 virus exhibited significant resistance to SB239063 as compared to the P60-Control virus. To provide mechanistic insights on the acquisition of resistance by BPXV-P60-SB239063, we generated p38-α and p38-ϒ (isoforms of p38) knockout Vero cells by CRISPR/Cas9-mediated genome editing. It was demonstrated that unlike the wild type (WT) virus which is dependent on p38-α isoform, the resistant virus (BPXV-P60-SB239063) switches over to use p38-ϒ so as to efficiently replicate in the target cells. This is a rare evidence wherein a virus was shown to bypass the dependency on a critical cellular factor under selective pressure of a drug.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly evolved to generate several antigenic variants. These variants have raised concerns whether pre-existing immunity to vaccination or prior infection would be able to protect against the newly emerging SARS-CoV-2 variants or not. We isolated SARS-CoV-2 from the coronavirus disease 2019 (COVID-19)-confirmed patients in the beginning of the first (April/May 2020) and second (April/May 2021) waves of COVID-19 in India (Hisar, Haryana). Upon complete nucleotide sequencing, the viruses were found to be genetically related with wild-type (WT) and Delta variants of SARS-CoV-2, respectively. The Delta variant of SARS-CoV-2 produced a rapid cytopathic effect (24–36 h as compared to 48–72 h in WT) and had bigger plaque size but a shorter life cycle (~6 h as compared to the ~8 h in WT). Furthermore, the Delta variant achieved peak viral titers within 24 h as compared to the 48 h in WT. These evidence suggested that the Delta variant replicates significantly faster than the WT SARS-CoV-2. The virus neutralization experiments indicated that antibodies elicited by vaccination are more efficacious in neutralizing the WT virus but significantly less potent against the Delta variant. Our findings have implications in devising suitable vaccination, diagnostic and therapeutic strategies, besides providing insights into understanding virus replication and transmission.

Our study suggests that methylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is essential for its optimal replication in the target cells. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1, an RNA-binding protein) was shown to mediate deposition of N6-methyladenosine (m6A) in internal SARS-CoV-2 RNA. The levels of hNRNPA1 expression and extent of methylation varied, depending on the course of SARS-CoV-2 life cycle. The recruitment of eIF4E (translational initiation factor) facilitated viral RNA translation at 1 hour post infection (1 hpi). However, at 2 hpi, methylation of internal SARS-CoV-2 RNA recruited hNRNPA1 which facilitated viral RNA transcription but resulted in translational repression, a phenomenon contributing in understanding the early translation to replication switch in the viral life cycle. Besides, the abrogation of methylation also produced a defective 5’ cap of viral RNA which failed to interact with eIF4E, thereby resulting in a decreased synthesis of viral proteins. To conclude, methylation of the internal and 5’ cap of SARS-CoV-2 RNA was shown to regulate transcription and translation of SARS-CoV-2 in a time dependent manner.IMPORTANCERNA modifications are found in all life forms and have been linked to development, health and diseases. Our study reveals that internal SARS-CoV-2 RNA methylation (m6A) is essential for interaction with hNRNPA1 to effectively synthesize viral genome. Besides, m6A-marked RNA and hRNPA1 interaction was also shown to regulate early translation to replication switch in SARS-CoV-2 life cycle. Blocking SARS-CoV-2 RNA methylation resulted in reduced virus yield, suggesting epitranscriptomic machinery (methylation) facilitates SARS-CoV-2 replication and might represent potential target for new antiviral drugs against COVID-19.

Mitogen-activated protein kinases (MAPKs) play a key role in complex cellular processes such as proliferation, development, differentiation, transformation and apoptosis. Mammals express at least four distinctly regulated groups of MAPKs which include extracellular signal-related kinases (ERK)-1/2, p38 proteins, Jun amino-terminal kinases (JNK1/2/3) and ERK5. p38 MAPK is activated by a wide range of cellular stresses and modulates activity of several downstream kinases and transcription factors which are involved in regulating cytoskeleton remodeling, cell cycle modulation, inflammation, antiviral response and apoptosis. In viral infections, activation of cell signalling pathways is part of the cellular defense mechanism with the basic aim of inducing an antiviral state. However, viruses can exploit enhanced cell signalling activities to support various stages of their replication cycles. Kinase activity can be inhibited by small molecule chemical inhibitors, so one strategy to develop antiviral drugs is to target these cellular signalling pathways. In this review, we provide an overview on the current understanding of various cellular and viral events regulated by the p38 signalling pathway, with a special emphasis on targeting these events for antiviral drug development which might identify candidates with broad spectrum activity.

Emetine is a FDA-approved drug for the treatment of amebiasis. In the recent times we had also demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. Following emergence of the COVID-19, we further evaluated thein vitroantiviral activity of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The therapeutic index of emetine was determined to be 10910.4, at a cytotoxic concentration 50 (CC50) of 1603.8 nM and effective concentration 50 (EC50) of 0.147 nM.Besides, we also demonstrated the protective efficacy of emetine against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment was shown to decrease viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding.In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 RNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation ofprotein translation). Further, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathwayfor its effective replication in the target cells. To conclude, emetine targets SARS-CoV-2 protein synthesis which is mediated via inhibiting the interaction of SARS-CoV-2 RNA with eIF4E. This is a novel mechanistic insight on the antiviral efficacy of emetine.In vitroantiviral efficacy against SARS-CoV-2 and its ability to protect chicken embryos against IBV suggests that emetine could be repurposed to treat COVID-19.

Emetine is a FDA-approved drug for the treatment of amebiasis. Previously we demonstrated the antiviral efficacy of emetine against some RNA and DNA viruses. In this study, we evaluated the in vitro antiviral efficacy of emetine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and found it to be a low nanomolar (nM) inhibitor. Interestingly, emetine exhibited protective efficacy against lethal challenge with infectious bronchitis virus (IBV; a chicken coronavirus) in the embryonated chicken egg infection model. Emetine treatment led to a decrease in viral RNA and protein synthesis without affecting other steps of viral life cycle such as attachment, entry and budding. In a chromatin immunoprecipitation (CHIP) assay, emetine was shown to disrupt the binding of SARS-CoV-2 mRNA with eIF4E (eukaryotic translation initiation factor 4E, a cellular cap-binding protein required for initiation of protein translation). Further, molecular docking and molecular dynamics simulation studies suggested that emetine may bind to the cap-binding pocket of eIF4E, in a similar conformation as m7-GTP binds. Additionally, SARS-CoV-2 was shown to exploit ERK/MNK1/eIF4E signalling pathway for its effective replication in the target cells. Collectively our results suggest that further detailed evaluation of emetine as a potential treatment for COVID-19 may be warranted., Graphical abstract Image 1

Antiviral drugs have traditionally been developed by directly targeting essential viral components. However, this strategy often fails due to the rapid generation of drug-resistant viruses. Recent genome-wide approaches, such as those employing small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeats (CRISPR) or those using small molecule chemical inhibitors targeting the cellular "kinome," have been used successfully to identify cellular factors that can support virus replication. Since some of these cellular factors are critical for virus replication, but are dispensable for the host, they can serve as novel targets for antiviral drug development. In addition, potentiation of immune responses, regulation of cytokine storms, and modulation of epigenetic changes upon virus infections are also feasible approaches to control infections. Because it is less likely that viruses will mutate to replace missing cellular functions, the chance of generating drug-resistant mutants with host-targeted inhibitor approaches is minimized. However, drug resistance against some host-directed agents can, in fact, occur under certain circumstances, such as long-term selection pressure of a host-directed antiviral agent that can allow the virus the opportunity to adapt to use an alternate host factor or to alter its affinity toward the target that confers resistance. This review describes novel approaches for antiviral drug development with a focus on host-directed therapies and the potential mechanisms that may account for the acquisition of antiviral drug resistance against host-directed agents.

We describe herein that Apigenin, which is a dietary flavonoid, exerts a strong in vitro and in ovo antiviral efficacy against buffalopox virus (BPXV). Apigenin treatment was shown to inhibit synthesis of viral DNA, mRNA and proteins, without affecting other steps of viral life cycle such as attachment, entry and budding. Although the major mode of antiviral action of Apigenin was shown to be mediated via targeting certain cellular factors, a modest inhibitory effect of Apigenin was also observed directly on viral polymerase. We also evaluated the selection of drug-resistant virus variants under long-term selection pressure of Apigenin. Wherein Apigenin-resistant mutants were not observed up to ~ P20 (passage 20), a significant resistance was observed to the antiviral action of Apigenin at ~ P30. However, a high degree resistance could not be observed even up to P60. To the best of our knowledge, this is the first report describing in vitro and in ovo antiviral efficacy of Apigenin against poxvirus infection. The study also provides mechanistic insights on the antiviral activity of Apigenin and selection of potential Apigenin-resistant mutants upon long-term culture.

Bovine herpesvirus 1 (BHV1) and 5 (BHV5) are genetically and antigenically related alphaherpesviruses. Infection with one virus induces protective immunity against the other. However, disease associated with BHV1 and BHV5 varies significantly; whereas BHV1 infection is usually associated with rhinotracheitis and abortion, BHV5 causes encephalitis in cattle. BHV5 outbreaks are sporadic and mainly restricted to the South American countries. We report BHV5 infection for the first time from aborted cattle in India. Based on the characteristic cytopathic effects in MDBK cells, amplification of the viral genome in PCR, differential PCR for BHV1/BHV5, nucleotide sequencing and restriction endonuclease patterns, identity of the virus was confirmed as BHV5 subtype A. Serum samples from the aborted cattle strongly neutralized both BHV1 and BHV5 suggesting an active viral infection in the herd. Upon UL27, UL44 and UL54 gene-based sequence and phylogenetic analysis, the isolated virus clustered with BHV5 strains and showed highest similarity with the Brazilian BHV5 strains.Author SummaryBHV5 causes fatal meningoencephalitis that may result in a mortality rate of up to 100% in cattle. BHV5 is rarely associated with abortion and its distribution is restricted to South American countries. Only a few cases of this disease have been reported from other countries. For the first time, we provide a strong evidence of BHV5 infection from aborted cattle in India. The finding may necessitate inclusion of BHV5 test protocol in testing of semen for sexually transmitted diseases. Also, the isolated virus would be useful for developing diagnostic, prophylactic and therapeutic agents to combat BHV5 disease in the country.

The Glass, rayon based carbon, PAN based carbon and high silica fabric reinforcements were impregnated with standard resole type phenolic resin in a vertical impregnating plant. The heating zones of prepreg plant converts resin impregnated fabric into partially polymerized state is called B-stage. The required volatile content and chang’s index are achieved by adjusting plant parameter based on online testing of prepreg, and it was noticed resin content varying depend on reinforcement used. The rayon carbon/phenolic prepregs had high wet resin content and glass/phenolic prepregs had low wet resin content

The ecosystem is continuously exposed to a wide variety of antimicrobials through waste effluents, agricultural run-offs and animal-related and anthropogenic activities, which contribute to the spread of antibiotic resistance genes (ARGs). The contamination of ecosystems with ARGs may create increased opportunities for their transfer to naive microbes and eventually lead to entry into the human food chain. Transduction is a significant mechanism of horizontal gene transfer in natural environments, which has traditionally been underestimated as compared to transformation. We explored the presence of ARGs in environmental bacteriophages in order to recognize their contribution in the spread of ARGs in environmental settings. Bacteriophages were isolated against environmental bacterial isolates, purified and bulk cultured. They were characterized, and detection of ARG and intI genes including bla TEM, bla OXA-2, intI1, intI2, intI3, tetA and tetW was carried out by PCR. This study revealed the presence of various genes [tetA (12.7 %), intI1 (10.9 %), intI2 (10.9 %), intI3 (9.1 %), tetW (9.1 %) and bla OXA-2 (3.6 %)] and bla TEM in a significantly higher proportion (30.9 %). bla SHV, bla OXA-1, tetO, tetB, tetG, tetM and tetS were not detected in any of the phages. Soil phages were the most versatile in terms of ARG carriage. Also, the relative abundance of tetA differed significantly vis-a-vis source. The phages from organized farms showed varied ARGs as compared to the unorganized sector, although bla TEM ARG incidences did not differ significantly. The study reflects on the role of phages in dissemination of ARGs in environmental reservoirs, which may provide an early warning system for future clinically relevant resistance mechanisms.

Chicken astroviruses (CAstVs) infect young chicks and are associated with gastroenteritis, stunted growth or visceral gout (gout). True incidence and distribution of CAstVs as well as virus variants circulating in India is not well understood. In this study, 80 gout-affected broiler chicken flocks from Haryana, a north-western state of India, were tested for the presence of astroviruses by targeting the polymerase gene of both CAstV and avian nephritis virus (ANV) and capsid gene of CAstV. Of these, 22 (27.5%) flocks were found positive for CAstV, 7(8.75%) for ANV and 2 (2.5%) for both CAstV and ANV genome by reverse-transcription-polymerase chain reaction. CAstV was isolated by inoculating tissue (kidney) homogenate from gout-affected birds into specific-pathogen free embryonated chicken eggs where the infected embryos showed stunted growth with necrosis of liver and enlarged kidney with urate deposits. Capsid gene-based phylogenetic analysis revealed the clustering of CAstV strains from this study with Indian strains of serogroup Biii suggesting their antigenic relatedness. Thus the present study reveals the presence of chicken astroviruses in broiler chickens affected with gout.

Lumpy skin disease (LSD) has devastating economic impact. During the last decade, LSD had spread to climatically new and previously disease-free countries, which also includes its recent emergence in the Indian subcontinent (2019). This study deals with the LSD outbreak(s) from cattle in Ranchi (India). Virus was isolated from the scabs (skin lesions) in the primary goat kidney cells. Phylogenetic analysis based on nucleotide sequencing of LSD virus (LSDV) ORF011, ORF012 and ORF036 suggested that the isolated virus (LSDV/Bos taurus-tc/India/2019/Ranchi) is closely related to Kenyan LSDV strains. Further, we adapted the isolated virus in Vero cells, an alternative cell line that was found suitable for LSDV propagation. In addition, kinetics of viral DNA synthesis and one-step growth curve analysis suggested that LSDV initiates synthesizing its genome at ∼24 hours post-infection (hpi) with a peak level at ∼96 hpi whereas evidence of progeny virus particles was observed at 36-48 hours (h) with a peak titre at ∼120 h. This study reports the first successful isolation of LSDV in India, besides describing alternative cells for virus isolation (primary goat kidney cells) and in vitro propagation (Vero cells) as well as providing some insights on LSDV life cycle.Author SummaryThis study describes the first successful isolation of LSDV in India. Nucleotide sequencing of LSDV ORF011, ORF012 and ORF036 suggested that the isolated virus is closely related to Kenyan LSDV strains. This is in accordance with the previous (only) study, thereby suggesting that a single LSDV strain is circulating in India. For the first time, we employed primary goat kidney (PGK) cells for the isolation of LSDV, where PGK cells were found more sensitive for LSDV isolation as compared to the primary lamb testicle (PLT) and primary goat testicle (PGT) cells. We also adapted the isolated virus in Vero cells and demonstrated that Vero cells are also suitable for in vitro propagation of LSDV. Besides, we also provide some insights on the LSDV life cycle.

A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS-PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp23 gene sequence with previously reported phages showed similarity with T4-like phages infecting Enterobacteriaceae instead of Aeromonas spp. Thus, to our knowledge, this report points toward the fact that a novel/evolved phage might exist in equine environment against A. hydrophila, which can be potentially used as a biocontrol agent.

Systems biology refers to system-wide changes in biological components such as RNA/DNA (genomics), protein (proteomics) and lipids (lipidomics). In this review, we provide comprehensive information about morbillivirus replication. Besides discussing the role of individual viral/host proteins in virus replication, we also discuss how systems-level analyses could improve our understanding of morbillivirus replication, host-pathogen interaction, immune response and disease resistance. Finally, we discuss how viroinformatics is likely to provide important insights for understanding genome-genome, genome-protein and protein-protein interactions.

Ethnopharmacological relevance Synthetic antiviral drugs have several limitations including high cost. Thus research on antiviral property of medicinal plants is continuously gaining importance. Polyalthia longifolia possesses several medicinal properties and has been used in traditional ayurvedic medicine for treatment of dermatological ailments as kushta, visarpa/herpes virus infection and also to treat pyrexia of unknown origin as mentioned in Visarpa Chikitsa. Aim of the study Keeping in view the cytotoxic, anti-cancer activity and antiviral efficacy of Polyalthia longifolia against herpes, present study was undertaken to evaluate the in vitro antiviral activity of methanolic extract of Polyalthia longifolia leaves, if any, and to unravel the possible target(s)/mechanism of action. Material and methods Antiviral activity of Polyalthia longifolia methanolic extract was studied using Vero cell lines against paramyxoviruses, namely-peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV). Cytotoxicity of the test extract was evaluated employing MTT assay. Virucidal activity, and viral-attachment, virus entry and release assays were determined in Vero cells using standard experimental protocols. The viral RNA in the virus-infected cells was quantified by qRT-PCR. Results At non-cytotoxic concentration, methanolic extract of Polyalthia longifolia leaves was found to inhibit the replication of PPRV and NDV at viral entry and budding level, whereas other steps of viral life cycle such as attachment and RNA synthesis remained unaffected. Conclusions Polyalthia longifolia leaves extract possesses promising antiviral activity against paramyxoviruses and acts by inhibiting the entry and budding of viruses; and this plant extract evidently possesses excellent and promising potential for development of effective herbal antiviral drug.

SUMMARY Coinfections involving viruses are being recognized to influence the disease pattern that occurs relative to that with single infection. Classically, we usually think of a clinical syndrome as the consequence of infection by a single virus that is isolated from clinical specimens. However, this biased laboratory approach omits detection of additional agents that could be contributing to the clinical outcome, including novel agents not usually considered pathogens. The presence of an additional agent may also interfere with the targeted isolation of a known virus. Viral interference, a phenomenon where one virus competitively suppresses replication of other coinfecting viruses, is the most common outcome of viral coinfections. In addition, coinfections can modulate virus virulence and cell death, thereby altering disease severity and epidemiology. Immunity to primary virus infection can also modulate immune responses to subsequent secondary infections. In this review, various virological mechanisms that determine viral persistence/exclusion during coinfections are discussed, and insights into the isolation/detection of multiple viruses are provided. We also discuss features of heterologous infections that impact the pattern of immune responsiveness that develops.

Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is a membrane-bound cytosolic enzyme which is known to regulate the uptake of calcium into the sarco/endoplasmic reticulum. Herein, we demonstrate for the first time that SERCA can also regulate virus replication. Treatment of Vero cells with SERCA-specific inhibitor (Thapsigargin) at a concentration that is nontoxic to the cells significantly reduced Peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV) replication. Conversely, overexpression of SERCA rescued the inhibitory effect of Thapsigargin on virus replication. PPRV and NDV infection induced SERCA expression in Vero cells, which could be blocked by Thapsigargin. Besides inducing enhanced formation of cytoplasmic foci, Thapsigargin was shown to block viral entry into the target cells as well as synthesis of viral proteins. Furthermore, NDV was shown to acquire significant resistance to Thapsigargin upon long-term passage (P) in Vero cells. As compared to the P0 and P70-Control, the fusion (F) protein of P70-Thapsigargin virus exhibited a unique mutation at amino acid residue 104 (E104K), whereas no Thapsigargin-associated mutations were observed in HN gene. To the best of our knowledge, this is the first report describing the virus-supportive role of SERCA and a rare report suggesting that viruses may acquire resistance even in the presence of an inhibitor that targets a cellular factor.

A small molecule chemical inhibitor CGP57380 that blocks activation of MAPK interacting kinase 1 (MNK1) was found to significantly suppress buffalopox virus (BPXV) replication. BPXV infection was shown to induce MNK1 activation. Depletion of MNK1 by small interfering RNA (siRNA), blocking activation of extracellular regulated kinase (ERK, an upstream activator of MNK1) and disruption of eIF4E/eIF4G interaction (downstream substrate of MNK1 which plays a central role in cap-dependent translation initiation), resulted in reduced BPXV replication, suggesting that ERK/MNK1/eIF4E signaling is a prerequisite for BPXV replication. With the help of time-of-addition and virus step-specific assays, CGP57380 treatment was shown to decrease synthesis of viral genome (DNA). Disruption of ERK/MNK1/eIF4E signaling resulted in reduced synthesis of viral proteins, suggesting that BPXV utilizes cap-dependent mechanism of translation initiation. Therefore, we concluded that decreased synthesis of viral genome in presence of MNK1 inhibitor is the result of reduced synthesis of viral proteins. Furthermore, BPXV was sequentially passaged (P = 40) in presence of CGP57380 or vehicle control (DMSO). As compared to P0 and P40-control viruses, P40-CGP57380 virus replicated at significantly higher (∼10-fold) titers in presence of CGP57380, although a complete resistance could not be achieved. In a BPXV egg infection model, CGP57380 was found to prevent development of pock lesions on chorioallantoic membrane (CAM) as well as associated mortality of the embryonated chicken eggs. We for the first time demonstrated in vitro and in ovo antiviral efficacy of CGP57380 against BPXV and identified that ERK/MNK1 signaling is a prerequisite for synthesis of viral proteins. Our study also describes a rare report about generation of drug-resistant viral variants against a host-targeting antiviral agent.

Sarco/endoplasmic reticulum calcium-ATPase (SERCA) is a membrane bound cytosolic enzyme that is known to regulate the uptake of calcium into the sarco/endoplasmic reticulum. Herein, we demonstrate for the first time that SERCA can also regulate paramyxovirus [Peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)] replication. Treatment of Vero cells with SERCA specific inhibitor (Thapsigargin) at a concentration that is nontoxic to the cells significantly reduced virus replication. Conversely, overexpression of SERCA rescued the inhibitory effect of Thapsigargin on virus replication. PPRV/NDV infection induced SERCA expression in Vero cells which could be blocked by Thapsigargin. With the help of time-of-addition and virus step-specific assays, it was observed that Thapsigargin specifically inhibits viral entry and subcellular localization of the viral proteins. Furthermore, NDV, but not PPRV acquired a significant resistance to Thapsigargin on long-term passage (P=70) in Vero cells. To the best of our knowledge, this is the first report describing virus supportive role of SERCA and a rare report suggesting that viruses may acquire resistance even in the presence of an inhibitor that targets a cellular factor. This study will contribute in understanding paramyxovirus replication and development of antiviral therapeutics using SERCA (host factor) as a candidate drug target.

Highlights • Viruses are known to exploit cellular signaling pathways. • MAPK is a major cell signaling pathway activated by diverse group of viruses. • MNK1 regulates both cap-dependent and IRES-mediated mRNA translation. • This review discuss the role of MAPK, particularly the role of MNK1 in virus replication., Viruses are obligate intracellular parasites; they heavily depend on the host cell machinery to effectively replicate and produce new progeny virus particles. Following viral infection, diverse cell signaling pathways are initiated by the cells, with the major goal of establishing an antiviral state. However, viruses have been shown to exploit cellular signaling pathways for their own effective replication. Genome-wide siRNA screens have also identified numerous host factors that either support (proviral) or inhibit (antiviral) virus replication. Some of the host factors might be dispensable for the host but may be critical for virus replication; therefore such cellular factors may serve as targets for development of antiviral therapeutics. Mitogen activated protein kinase (MAPK) is a major cell signaling pathway that is known to be activated by diverse group of viruses. MAPK interacting kinase 1 (MNK1) has been shown to regulate both cap-dependent and internal ribosomal entry sites (IRES)-mediated mRNA translation. In this review we have discuss the role of MAPK in virus replication, particularly the role of MNK1 in replication and translation of viral genome.

At a noncytotoxic concentration, emetine was found to inhibit replication of DNA viruses [buffalopoxvirus (BPXV) and bovine herpesvirus 1 (BHV-1)] as well as RNA viruses [peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)]. Using the time-of-addition and virus step-specific assays, we showed that emetine treatment resulted in reduced synthesis of viral RNA (PPRV and NDV) and DNA (BPXV and BHV-1) as well as inhibiting viral entry (NDV and BHV-1). In addition, emetine treatment also resulted in decreased synthesis of viral proteins. In a cell free endogenous viral polymerase assay, emetine was found to significantly inhibit replication of NDV, but not BPXV genome, suggesting that besides directly inhibiting specific viral polymerases, emetine may also target other factors essentially required for efficient replication of the viral genome. Moreover, emetine was found to significantly inhibit BPXV-induced pock lesions on chorioallantoic membrane (CAM) along with associated mortality of embryonated chicken eggs. At a lethal dose 50 (LD50) of 126.49 ng/egg and at an effective concentration 50 (EC50) of 3.03 ng/egg, the therapeutic index of the emetine against BPXV was determined to be 41.74. Emetine was also found to significantly delay NDV-induced mortality in chicken embryos associated with reduced viral titers. Further, emetine-resistant mutants were not observed upon long-term (P = 25) sequential passage of BPXV and NDV in cell culture. Collectively, we have extended the effective antiviral activity of emetine against diverse groups of DNA and RNA viruses and propose that emetine could provide significant therapeutic value against some of these viruses without inducing an antiviral drug-resistant phenotype.

Cancer is a leading cause of human deaths worldwide. Besides inherited genetic disorders, a diverse range of physical, chemical and biological agents may induce cancer. About 15-20% of cancers are known to be originated due to pathogens. Viruses are considered to be the second (after smoking) most important risk factor in inducing human cancer. Viruses may either harbour a copy of oncogene or have an ability to alter the expression of cellular copy of the oncogenes. Both RNA and DNA viruses are can induce oncogenesis. Most of the DNA tumour viruses either integrate their genome (complete or part of it) into the host genome or express early genes that are required for early event of virus replication. These early genes are responsible for oncogenic transformation of host cells. Based upon the mechanism involved, oncogenic RNA viruses are divided into two groups-transforming and non-transforming RNA viruses. Transforming RNA viruses carry viral oncogenes that are homologous to the host oncogene, their expression in infected cells results in oncogenic transformation of the cell. Non-transforming RNA viruses induce oncogenesis similar to the DNA viruses. Contrary, oncolytic viruses selectively replicate in cancerous cells and induce cell death without any damage to the normal tissues. Typically, oncolytic viruses are nonpathogenic to humans that can naturally replicate in cancer cells by exploiting oncogenic cell signalling pathways. Pathogenic viruses can also be genetically manipulated which allow them to replicate in cancerous but not in normal cells. This review review describes the molecular mechanisms associated with virus induced oncogenesis and oncolysis.

A role for bacteriophage therapy was envisaged early last century; however, due to discovery of the antimicrobials, it fell out of research interest. Currently, bacteriophages are resurfacing as an alternative to antimicrobials in order to overcome the increasing incidence of antimicrobial resistance. Here, we report isolation of bacteriophages against Escherichia coli, Shigella spp., Aeromonas hydrophila and Citrobacter sedlakii isolates of equine origin. Phages were isolated from equine farm soil and sewage samples. For enrichment, sample aliquot was incubated overnight with host bacteria at 37 C with vigorous shaking. The crude lysate obtained was centrifuged and filtered and the presence of any phage in the suspension was detected by agar overlay technique. Appropriate dilutions of enriched samples were plated to obtain individual plaques and themost dominant plaquewas transferred into SM buffer, serially diluted and plated for plaque re-isolation three times to ensure purity, followed by large scale preparation of phage stocks. Any host nucleic acids was degraded using pancreatic DNaseI and RNase and bacteriophage particles were precipitated using PEG8000. Phage titre was determined by plaque assay and phage concentrates were accessioned in the Veterinary Type Culture Collection (VTCC) repository. The phage concentrates were visualized by transmission electron microscopy (TEM). The temperature stability of bacteriophages was checked after incubating phage concentrates over the range of 4 C 80 C temperature for one hour. A clear single plaque was obtained on nutrient agar against Shigella spp. and after purification and concentration, its analysis by electron microscopy revealed presence of multiple phages belonging to families Myoviridae, Siphoviridae and Podoviridae. However in case of Citrobacter sedlakii, a Siphoviridae phage (VTCCBPA61) with dimensions: 60 nm x 650 nm was observed. Against a pathogenic A. hydrophila isolate of equine origin (expressing aerolysin gene), a Myoviridae phage (VTCCBPA6) was isolated with dimensions: 62 nm x 138 nm. Against E. coli of equine origin, a Myoviridae phage (VTCCBPA9) of dimensions: 86 nm x 100 nm was obtained. Bacteriophage VTCCPBA61 against C. sedlakiiwas found to completely lose its biological activity at 65 C in vitro however the group of phages against Shigella spp. were found to be stable upto temperature as high as 80 C. Thus we demonstrated the basic biological characteristics of phages, and some novel ones (such as against A. hyrophila and C. sedlakii) bacterial isolates of equine originwhich have never been reported till now. These lytic phages could find potential in phage therapy, as biocides, in biosensors and in phage ligand technology and are being explored by us further to depict their therapeutic value in small animal model. As more studies are reporting safety, tolerance and efficacy of phage therapy in humans and animals, their use in phage therapy has a promising future as an emerging alternative to chemical agents. Posters 006 Microbiological investigation into the cause of Equine Grass Sickness

Swinepox virus (SWPV), a member of the genus Suipoxvirus causes generalized pock-like lesions on the body of domestic and wild pigs. Although outbreak has been reported in India since 1987, virus isolation and genetic characterization remained elusive. In September 2013, an outbreak of acute skin infection occurred in piglets in a commercial piggery unit at Rohtak district in Haryana, India. The presence of SWPV in scab samples collected from piglets succumbed to infection was confirmed by virus isolation, PCR amplification of SWPV-specific gene segments and nucleotide sequencing. Phylogenetic analysis of host-range genes of the SWPV revealed that the Indian isolate is genetically closely related to reference isolate SWPV/pig/U.S.A/1999/Nebraska. To the best of our knowledge this is the first report on isolation and genetic characterization of SWPV from pigs in India.

Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum causes fowl typhoid (FT), which results in huge economic losses to poultry farmers in India. We report the draft genome sequence of Salmonella biovar Gallinarum strain VTCCBAA614, isolated from a chicken in an FT affected broiler flock.

Camelpox virus (CMLV), a close variant of variola virus (VARV) infects camels worldwide. The zoonotic infections reported from India signify the need to study the host-range genes—responsible for host tropism. We report sequence and phylogenetic analysis of five host-range genes: cytokine response modifier B (crmB), chemokine binding protein (ckbp), viral schlafen-like (v-slfn), myxomavirus T4-like (M-T4-like) and b5r of CMLVs isolated from outbreaks in India. Comparative analysis revealed that these genes are conserved among CMLVs and shared 94.5–100 % identity at both nucleotide (nt) and amino acid (aa) levels. All genes showed identity (59.3–98.4 %) with cowpox virus (CPXV) while three genes—crmB, ckbp and b5r showed similarity (92–96.5 %) with VARVs at both nt and aa levels. Interestingly, three consecutive serine residue insertions were observed in CKBP protein of CMLV-Delhi09 isolate which was similar to CPXV-BR and VACVs, besides five point mutations (K53Q, N67I, F84S, A127T and E182G) were also similar to zoonotic OPXVs. Further, few inconsistent point mutation(s) were also observed in other gene(s) among Indian CMLVs. These indicate that different strains of CMLVs are circulating in India and these mutations could play an important role in adaptation of CMLVs in humans. The phylogeny revealed clustering of all CMLVs together except CMLV-Delhi09 which grouped separately due to the presence of specific point mutations. However, the topology of the concatenated phylogeny showed close evolutionary relationship of CMLV with VARV and TATV followed by CPXV-RatGer09/1 from Germany. The availability of this genetic information will be useful in unveiling new strategies to control emerging zoonotic poxvirus infections.

A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS-PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp23 gene sequence with previously reported phages showed similarity with T4-like phages infecting Enterobacteriaceae instead of Aeromonas spp. Thus, to our knowledge, this report points toward the fact that a novel/evolved phage might exist in equine environment against A. hydrophila, which can be potentially used as a biocontrol agent.

Phage therapy has been previously tried for treatment of diarrhoea in calves, pigs and lambs but those trials were conducted without any detailed information of used phages. Here, we report isolation of a broad-spectrum phage which showed bactericidal activity against 47.3 % of calf diarrhoeal isolates of Escherichia coli, in vitro. The isolated phage resembled the characteristics of Myoviridae family and showed ~97 % similarity with earlier reported bacteriophages of sub family-Tevenvirinae, genus-T4-like virus, based on nucleotide sequence of major head protein-gp23 gene. The phage exhibits the potential to be used as drug substitute tool against E. coli causing diarrhoea in cattle in farm environments.

The Pasteurella multocida subsp. multocida B:2 serotype causes hemorrhagic septicemia in bubalines with high morbidity and mortality in the Indian subcontinent. We report the draft genome sequence of Pasteurella multocida strain VTCCBAA264 isolated from the small-intestine of a buffalo calf that died of high fever.

Background Despite repeated outbreaks of poxvirus infections unique to the Indian subcontinent region and veterinary research work in this field, much less diagnostic awareness with resultant treatment protocols have been formulated in the human medical field. Aims With this objective in mind, a combined human medical and veterinary study was conducted on a recent outbreak of buffalopox infection in a village in northern India. Methods A team of doctors did the clinical examination and collected swab and serum samples from both cattle and humans, and these were subjected to viral isolation, cell culture, plaque reduction neutralization test, polymerase chain reaction, and partial genome sequencing. Results A clustered foci of 12 human patients aged 11–60 years, 12 buffaloes, and 10 cows were found to be affected with buffalopox infection with some atypical features. Conclusion Awareness, diagnosis, education, early intervention, and formulation of disaster guidelines are needed in view of the potential epidemiologic outbreak, if this happens in the future.

Successful purification of multiple viruses from mixed infections remains a challenge. In this study, we investigated peste des petits ruminants virus (PPRV) and foot-and-mouth disease virus (FMDV) mixed infection in goats. Rather than in a single cell type, cytopathic effect (CPE) of the virus was observed in cocultured Vero/BHK-21 cells at 6th blind passage (BP). PPRV, but not FMDV could be purified from the virus mixture by plaque assay. Viral RNA (mixture) transfection in BHK-21 cells produced FMDV but not PPRV virions, a strategy which we have successfully employed for the first time to eliminate the negative-stranded RNA virus from the virus mixture. FMDV phenotypes, such as replication competent but noncytolytic, cytolytic but defective in plaque formation and, cytolytic but defective in both plaque formation and standard FMDV genome were observed respectively, at passage level BP8, BP15 and BP19 and hence complicated virus isolation in the cell culture system. Mixed infection was not found to induce any significant antigenic and genetic diversity in both PPRV and FMDV. Further, we for the first time demonstrated the viral interference between PPRV and FMDV. Prior transfection of PPRV RNA, but not Newcastle disease virus (NDV) and rotavirus RNA resulted in reduced FMDV replication in BHK-21 cells suggesting that the PPRV RNA-induced interference was specifically directed against FMDV. On long-term coinfection of some acute pathogenic viruses (all possible combinations of PPRV, FMDV, NDV and buffalopox virus) in Vero cells, in most cases, one of the coinfecting viruses was excluded at passage level 5 suggesting that the long-term coinfection may modify viral persistence. To the best of our knowledge, this is the first documented evidence describing a natural mixed infection of FMDV and PPRV. The study not only provides simple and reliable methodologies for isolation and purification of two epidemiologically and economically important groups of viruses, but could also help in establishing better guidelines for trading animals that could transmit further infections and epidemics in disease free nations.

The neuraminidase (NA) gene sequences of four Indian equine influenza viruses (EIVs) isolated from epizootic in 2008 and 2009 were analyzed. The phylogenetic relationship and selection pressure of NA genes were established in comparison to other EIVs circulating worldwide along with the domains and motifs of the encoded protein to find out the significance of mutational changes. Among Indian isolates, two amino acid (aa) changes each in Mysore/12/08 (Asn67Tyr & Asp396Gly), Gopeshwar/1/09 (Ile49Val & Asp396Gly), and Uttarkashi/1/09 (Ile49Val & Asp396Gly) isolates were observed in respect to Jammu-Katra/06/08 isolate. Amino acid (aa) sequence analysis also revealed five consistent aa residue changes viz, Gly/Arg40Glu, Tyr66His, Val191Ile, Val209Ile and Asp235Asn in Asian including Indian isolates, Spain/07 and Spain/09 isolates in comparison to other EIVs circulating worldwide. The topology of the phylogenetic tree revealed that the Indian, Chinese, Mongolian and Kazakhstan isolates together formed a subgroup with Yokohama/10 isolate. Spain/07 & Spain/09 isolates showed closest clustering with Asian isolates. This indicates that non-synonymous mutations in Asian isolates with temporal pattern originating from Spain/07, led to the subgroup of the Asian isolates within Florida clade 2 sublineage. The analysis of the predicted secondary structure has not shown any significant difference in the NA proteins of all Indian isolates. Fixed-effects likelihood (FEL) analysis of the selection pressure revealed three codons (43, 355 & 434) under positive selection pressure. The overall evolutionary changes (ω value) of 3.4 indicates NA gene to be under strong selection pressure. Further, seven putative N-glycosylation sites were observed in the NA protein. The mapping of specific aa changes, their mutational and functional analysis need to be carried out to ascertain their role in pathogenecity of the virus.

Buffalopox virus (BPXV), a close variant of vaccinia virus (VACV) has emerged as a zoonotic pathogen. The host tropism of poxviruses is governed by host-range genes. Among the host-range genes: E3L, K3L, and C7L are essential for virus replication by preventing interferon resistance, whereas B5R is essential for spread of the virus and evasion from the host’s immune response as in VACV. We report sequence analysis of host-range genes: E3L, K3L, C7L, and membrane protein gene (B5R) of BPXVs from buffalo, cattle, and human from recent outbreaks in India—their phylogenetic relationship with reference strain (BP4) and other Orthopoxviruses. BPXVs revealed a sequence homology with VACVs including zoonotic Brazilian VACV-like viruses. The aa sequences of E3L and K3L genes were 100 % similar in buffalo, cattle, and human isolates. However, four significant point mutations (I11K; N12K and S36F in C7L gene and D249G in B5R gene) were observed specific to buffalo isolate only. This signifies that different strains of BPXV were circulated during the outbreak. The mutations in C7L and B5R could play an important role in adaptation of BPXV in human and cattle which needs further functional studies. The strain of BPXV isolated from buffalo may not be adopted in human and cow. Various point mutations were observed in the host-range genes of reference strain (BPXV-BP4) which may be due to several passages of virus in cell culture. The phylogeny constructed based on concatenated gene sequences revealed that BPXVs are not as closely related to vaccine strain (Lister and Lister-derived strain—LC16m8), as hypothesized earlier, rather they are more closely related to reference strain (BPXV-BP4) and other vaccinia and vaccinia-like viruses such as Passatempo and Aracatuba viruses. The availability of information regarding host tropism determinants would allow us to understand molecular mechanism of species tropism of poxviruses which would be useful in unveiling new strategies to control zoonotic poxviral infections.

This study reports the first conclusive evidence of zoonotic camelpox virus (CMLV) infection in humans associated with outbreaks in dromedarian camels ( Camelus dromedaries ) in northwest region of India during 2009. CMLV infection is usually restricted to camels and causes localised skin lesions but occasionally leads to generalised form of disease. However, the present outbreak involved camel handlers and attendants with clinical manifestations such as papules, vesicles, ulceration and finally scabs over fingers and hands. In camels, the pock-like lesions were distributed over the hairless parts of the body. On the basis of clinical and epidemiological features coupled with serological tests and molecular characterization of the causative agent, CMLV zoonosis was confirmed in three human cases. Clinical samples such as skin scabs/swabs and blood collected from affected animals and humans were analysed initially, for the presence of CMLV-specific antigen and antibodies by counter immunoelectrophoresis (CIE); serum neutralization test (SNT); plaque-reduction neutralization test (PRNT) and indirect immunoperoxidase test which was later confirmed by amplification of CMLV-specific ankyrin repeat protein (C18L) gene. Virus isolation was successful only from samples collected from camels. Further, sequence analyses based on three full-length envelope protein genes (A27L, H3L and D8L) revealed 95.2–99.8% and 93.1–99.3% homology with other Orthopoxviruses at nucleotide and amino acid levels, respectively. Phylogram of the three genes revealed a close relationship of CMLV with Variola virus (VARV). Considering the emerging and re-emerging nature of the virus, its genetic relatedness to VARV, zoonotic potential and productivity losses in camels; the control measures are imperative in curtailing economic and public health impact of the disease. This is the first instance of laboratory confirmed camelpox zoonosis in India.

An outbreak of equine influenza (EI) was reported in India in June, 2008 after a gap of two decades. The outbreak started from Jammu and Kashmir (Katra), northern state of India and spread to the other parts of the country affecting equines in 11 states. The virus (H3N8) was isolated from nasal swabs obtained from clinical cases in various locations in the country including Katra (Jammu and Kashmir), Mysore (Karnataka) and Ahmedabad (Gujarat) using embryonated chicken eggs. The virus isolates were identified as H3N8 by haemagglutination inhibition (HI) test titration with standard serum and by sequencing of full-length haemagglutinin (HA) gene and partial sequence of neuraminidase (NA) gene. Paired serum samples ( n = 271) showing more than fourfold rise in antibody titres tested from 11 states confirmed equine influenza. Serum samples ( n = 2517) of equines from 13 states of the country screened by HI test revealed 687 (26.85%) samples positive for antibodies to EI (H3N8). Phylogenetic analysis of the haemagglutinin (HA) gene confirmed the virus to be closely related to Clade 2 of the Florida sublineage in American lineage. Comparison of deduced amino acid sequence of HA gene with EIV isolates from various lineages showed substitutions in the antigenic regions C and D. HA1 gene sequence had highest amino acid identity to A/eq/Gansu/7/08 and A/eq/Hubei/6/08 isolates from China and Inner-Mongolia isolate, while the complete HA gene sequence was closest to A/eq/A/eq/Newmarket/5/03, A/eq/Bari/05 and A/eq/Kentucky/05/02 isolates. Recent outbreaks of Mongolia, China and India by clade 2 EI viruses imply their predominance in Asia in addition to Europe.