T. Riyesh

Zoonotic cases of camelpox infection in India

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.

Sequence and phylogenetic analysis of host-range (E3L, K3L, and C7L) and structural protein (B5R) genes of buffalopox virus isolates from buffalo, cattle, and human in India

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 100xa0% 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.

Emetine inhibits replication of RNA and DNA viruses without generating drug-resistant virus variants

Abstract 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. HighlightsAntiviral activity of emetine was extended against PPRV, NDV, BPXV and BHV‐1.Protective efficacy of emetine was evaluated in ovo against BPXV and NDV.Emetine treatment results in reduced synthesis of viral genome in infected cells.Emetine can directly inhibit specific viral polymerases, though it may have some other targets as well.Emetine‐resistant viral mutants are unlikely to occur.

Complexities in Isolation and Purification of Multiple Viruses from Mixed Viral Infections: Viral Interference, Persistence and Exclusion

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.

Draft Genome Sequence of Pasteurella multocida subsp. multocida B:2 Strain VTCCBAA264 Isolated from Bubalus bubalis in North India

ABSTRACT 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.

Advances in peste des petits ruminants vaccines

n Abstractn n Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants that leads to high morbidity and mortality thereby results in devastating economic consequences to the livestock industry. PPR is currently endemic across most parts of Asia and Africa, the two regions with the highest concentration of poor people in the world. Sheep and goats in particularly contribute significantly towards the upliftment of livelihood of the poor and marginal farmers in these regions. In this context, PPR directly affecting the viability of sheep and goat husbandry has emerged as a major hurdle in the development of these regions. The control of PPR in these regions could significantly contribute to poverty alleviation, therefore, the Office International des Epizooties (OIE) and Food and Agricultural Organization (FAO) have targeted the control and eradication of PPR by 2030 a priority. In order to achieve this goal, a potent, safe and efficacious live-attenuated PPR vaccine with long-lasting immunity is available for immunoprophylaxis. However, the live-attenuated PPR vaccine is thermolabile and needs maintenance of an effective cold chain to deliver into the field. In addition, the infected animals cannot be differentiated from vaccinated animals. To overcome these limitations, some recombinant vaccines have been developed. This review comprehensively describes about the latest developments in PPR vaccines.n n

Genetic characterization of equine herpesvirus 1 isolates from abortion outbreaks in India.

Equine herpesvirus 1 (EHV1) is a common pathogen of horses that causes upper respiratory tract disease, abortion, neonatal death and neurological disease. The neurological form of disease is called equine herpesvirus myeloencephalopathy (EHM). During the past decade, the incidence of EHM has been on the rise in Europe, North America, Australia and Asia. Some EHV1 isolates causing EHM exhibit a single-nucleotide polymorphism (SNP) in the DNA polymerase gene (ORF30) at position 2254 (A2254 to G2254). Further, based on polymorphism in the ORF68, EHV1 isolates have been classified into different groups. The aim of the present study was to estimate the genetic diversity of EHV1 and to determine the prevalence of the neuropathogenic genotype of EHV1 in India. Out of 133 clinical specimens from abortion cases in northern India, 56 were positive for EHV1 infection. Analysis of the A/G SNP by real-time PCR and sequence analysis revealed that 54 of 56 samples (96.43xa0%) were of the non-neuropathogenic genotype (A2254), while two (3.57xa0%) had the neuropathogenic marker (G2254). Sequence analysis of the polymorphic region of ORF68 of EHV1 isolates (nxa0=xa09) from India indicated that the Delhi/1998, Tohana-2/2013, Hisar-2/2014 and Hisar-15/1990 isolates belonged to group 4, while the Jind/1996, Rajasthan/1998, Delhi-3/2007 and Tohana-5/1996 isolates clustered within group 5. One isolate (Hisar-7/1990) exhibited SNPs at positions C710 and C713, forming a separate group. Here, we report for the first time the detection of neuropathogenic genotypes of EHV1 in India and show that Indian EHV1 isolates cluster within groups 4 and 5.

Isolation and characterization of a bacteriophage with broad host range, displaying potential in preventing bovine diarrhoea

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 47.3xa0% of calf diarrhoeal isolates of Escherichia coli, in vitro. The isolated phage resembled the characteristics of Myoviridae family and showed ~97xa0% 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.

Isolation and genetic characterization of swinepox virus from pigs in India.

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.

Genetic characterization and phylogenetic analysis of host-range genes of Camelpox virus isolates from India

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 94.5–100xa0% identity at both nucleotide (nt) and amino acid (aa) levels. All genes showed identity (59.3–98.4xa0%) with cowpox virus (CPXV) while three genes—crmB, ckbp and b5r showed similarity (92–96.5xa0%) 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.