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1.
Sci Rep ; 14(1): 6773, 2024 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-38514747

RESUMO

Haemorrhagic septicaemia (HS) is an economically important disease affecting cattle and buffaloes and the livelihoods of small-holder farmers that depend upon them. The disease is caused by Gram-negative bacterium, Pasteurella multocida, and is considered to be endemic in many states of India with more than 25,000 outbreaks in the past three decades. Currently, there is no national policy for control of HS in India. In this study, we analysed thirty year (1987-2016) monthly data on HS outbreaks using different statistical and mathematical methods to identify spatial variability and temporal patterns (seasonality, periodicity). There was zonal variation in the trend and seasonality of HS outbreaks. Overall, South zone reported maximum proportion of the outbreaks (70.2%), followed by East zone (7.2%), Central zone (6.4%), North zone (5.6%), West zone (5.5%) and North-East zone (4.9%). Annual state level analysis indicated that the reporting of HS outbreaks started at different years independently and there was no apparent transmission between the states. The results of the current study are useful for the policy makers to design national control programme on HS in India and implement state specific strategies. Further, our study and strategies could aid in implementation of similar approaches in HS endemic tropical countries around the world.


Assuntos
Doenças dos Bovinos , Septicemia Hemorrágica , Pasteurella multocida , Animais , Bovinos , Septicemia Hemorrágica/epidemiologia , Septicemia Hemorrágica/veterinária , Septicemia Hemorrágica/microbiologia , Búfalos/microbiologia , Surtos de Doenças , Índia/epidemiologia , Doenças dos Bovinos/microbiologia
2.
Genome ; 67(1): 13-23, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37639729

RESUMO

Pasteurella multocida causes acute/chronic pasteurellosis in porcine, resulting in considerable economic losses globally. The draft genomes of two Indian strains NIVEDIPm17 (serogroup D) and NIVEDIPm36 (serogroup A) were sequenced. A total of 2182-2284 coding sequences (CDSs) were predicted along with 5-6 rRNA and 45-46 tRNA genes in the genomes. Multilocus sequence analysis and LPS genotyping showed the presence of ST50: genotype 07 and ST74: genotype 06 in NIVEDIPm17 and NIVEDIPm36, respectively. Pangenome analysis of 61 strains showed the presence of 1653 core genes, 167 soft core genes, 750 shell genes, and 1820 cloud genes. Analysis of virulence-associated genes in 61 genomes indicated the presence of nanB, exbB, exbD, ptfA, ompA, ompH, fur, plpB, fimA, sodA, sodC, tonB, and omp87 in all strains. The 61 genomes contained genes encoding tetracycline (54%), streptomycin (48%), sulphonamide (28%), tigecycline (25%), chloramphenicol (21%), amikacin (7%), cephalosporin (5%), and trimethoprim (5%) resistance. Multilocus sequence type revealed that ST50 was the most common (34%), followed by ST74 (26%), ST13 (24%), ST287 (5%), ST09 (5%), ST122 (3%), and ST07 (2%). Single-nucleotide polymorphism and core genome-based phylogenetic analysis clustered the strains into three major clusters. In conclusion, we described the various virulence factors, mobile genetic elements, and antimicrobial resistance genes in the pangenome of P. multocida of porcine origin, besides the rare presence of LPS genotype 7 in serogroup D.


Assuntos
Infecções por Pasteurella , Pasteurella multocida , Animais , Suínos , Pasteurella multocida/genética , Filogenia , Lipopolissacarídeos , Infecções por Pasteurella/veterinária , Fatores de Virulência/genética
3.
One Health ; 17: 100609, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37583365

RESUMO

Crimean Congo Hemorrhagic Fever (CCHF), is an emerging zoonosis globally and in India. The present study focused on identifying the risk factors for occurrence of CCHF in the Indian state of Gujarat and development of risk map for India. The past CCHF outbreaks in India were collated for the analyses. Influence of land use change and climatic factors in determining the occurrence of CCHF in Gujarat was assessed using Bayesian spatial models. Change in maximum temperature in affected districts was analysed to identify the significant change points over 110 years. Risk map was developed for Gujarat using Bayesian Additive Regression Trees (BART) model with remotely sensed environmental variables and host (livestock and human) factors. We found the change in land use patterns and maximum temperature in affected districts to be contributing to the occurrence of CCHF in Gujarat. Spatial risk map developed using CCHF occurrence data for Gujarat identified density of buffalo, minimum land surface temperature and elevation as risk determinants. Further, spatial risk map for the occurrence of CCHF in India was developed using selected variables. Overall, we found that combination of factors such as change in land-use patterns, maximum temperature, buffalo density, day time minimum land surface temperature and elevation led to the emergence and further spread of the disease in India. Mitigation measures for CCHF in India could be designed considering disease epidemiology and initiation of surveillance strategies based on the risk map developed in this study.

4.
Acta Trop ; 240: 106848, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36773849

RESUMO

Anthrax is an economically important livestock disease affecting subsistence farmers and it is of zoonotic importance. Anthrax is endemic in many states of India including Karnataka. Identification of spatial risk factors for occurrence of anthrax and development of predictive risk maps are required for planning adequate vaccination in high-risk areas as well as targeted surveillance activities in animals, humans and environment. In this study, village level anthrax outbreak locations from Karnataka (1997-2016) were geo-referenced and predictive risk map was developed using temporally Fourier Processed remotely sensed variables. A non-linear discriminant analysis approach was used to develop the risk map for Karnataka. Elevation was identified as top predictor variable in the 10 variables selected. The predicted risk map showed good accuracy and validation statistics when evaluated using different metrics (Kappa, sensitivity, specificity, AUC). The predicted risk map also showed good correspondence with past outbreaks. Further, we used Bayesian Penalised spline method to estimate species response curves for top 10 variables selected. The validated risk map can be used in planning vaccination strategy and surveillance in high-risk areas.


Assuntos
Antraz , Animais , Humanos , Antraz/epidemiologia , Teorema de Bayes , Índia/epidemiologia , Surtos de Doenças , Fatores de Risco , Gado
5.
Arch Microbiol ; 204(6): 328, 2022 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-35576020

RESUMO

Black quarter (BQ) is an infectious disease affecting cattle and small ruminants worldwide caused by Gram-positive anaerobic bacterium Clostridium chauvoei. In this study, a draft genome sequence of C. chauvoei NIVEDIBQ1 strain isolated from clinical case of black quarter was analyzed. Sequence analysis indicated that genome had 2653 predicted coding DNA sequences, harbored numerous genes, mobile genetic elements for pathogenesis, and virulence factors. Computational analysis revealed that strain contained 30 virulence-associated genes. An intact genomic region highly similar to the Clostridium phage was present in the genome. Presence of CRISPR systems and the transposon components likely contribute to the genome plasticity. Strain encode diverse spectrum of degradative carbohydrate-active enzymes (CAZymes). Comparative SNP analysis revealed that the genomes of the C. chauvoei strains analyzed were highly conserved. Phylogenetic analysis of strains and available genome (n = 21) based on whole-genome multi-locus sequence typing (wgMLST) and core orthologous genes showed the clustering of strains into two different clusters suggesting geographical links.


Assuntos
Clostridium chauvoei , Animais , Composição de Bases , Bovinos , Clostridium chauvoei/genética , Genoma Bacteriano , Tipagem de Sequências Multilocus , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
6.
Gene ; 826: 146452, 2022 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-35339640

RESUMO

Pasteurella multocida, a Gram-negative bacterium with ubiquitous nature, is known to affect wide range of host species worldwide with varied clinical manifestations including haemorrhagic septicaemia (HS) in bovines. Although, HS causing P. multocida strains were identified and characterized by conventional tools and PCR assays, diverse strains are indistinguishable by these tools in the face of disease outbreaks. In this study, draft genomes of three virulent P. multocida serotype B:2 strains (NIVEDIPm32, NIVEDIPm34 and NIVEDIPm35) were analyzed following whole genome sequencing, assembly, annotation and compared them with existing global genomes (n = 43) of bovine origin in the database. Three draft genomes of NIVEDIPm strains consisted of 40-52 contigs with GC content of ∼40.4%. The genome size and predicted genes content was ∼2.3 Mb and 2181-2189, respectively. Besides, the presence of various mobile genetic elements, antimicrobial resistance genes and biofilm related genes suggested their vital roles in virulence; further, adaptation to the host immune system as well as host pathogen interaction. Multi locus sequence analysis based on RIRDC scheme showed the presence of ST122 in all the three strains. wgMLST based phylogenic analysis suggested that HS causing Indian virulent field strains differed geographically and showed diversity from existing HS vaccine strain P52. The phylogenetic tree revealed that North Indian strains share high similarity with strains of Pakistan than South Indian Strain. Notably, a high divergence of SNPs between the HS causing circulating virulent strains of India and current HS vaccine strain P52 suggested an imminent need for relook in to HS vaccination strategy for livestock in India.


Assuntos
Septicemia Hemorrágica , Infecções por Pasteurella , Pasteurella multocida , Animais , Bovinos , Hibridização Genômica Comparativa , Septicemia Hemorrágica/genética , Septicemia Hemorrágica/microbiologia , Septicemia Hemorrágica/veterinária , Infecções por Pasteurella/microbiologia , Infecções por Pasteurella/veterinária , Filogenia , Sorogrupo
7.
Vet Parasitol ; 289: 109338, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33359970

RESUMO

Canine babesiosis, a tick-borne haemoprotozoan disease of dogs, is of significance globally due to its rapid spread. A precise confirmatory diagnosis is required to curtail the rapid spread of infection. Our study described the evaluation of recombinant BgSA3 protein based indirect ELISA for sero-diagnosis and sero-surveillance of Babesia gibsoni infection in dogs. A partial BgSA3 gene segment (1921 bp) of B. gibsoni, encoding for recombinant truncated BgSA3 (75 kDa) protein devoid of predicted signal peptide (23 aa) at N-terminus and transmembrane region (20 aa) at C-terminus, was expressed in E. coli using a pET28a(+) vector. The rBgSA3 protein purified under native conditions using Ni-NTA superflow cartridge was confirmed by SDS-PAGE and Western blotting using sera from dogs infected/uninfected with B. gibsoni, and erythrocyte lysate/ plasma from infected/uninfected dogs. The rBgSA3 protein was specific only to B. gibsoni antibodies but did not react with uninfected sera. Further, rBgSA3 protein was evaluated for sero-diagnosis/sero-surveillance using Indirect-ELISA format. There was no cross reactivity to B. vogeli, E. canis, H. canis and D. repens infected dogs serum samples. The diagnostic sensitivity and specificity of rBgSA3 based I-ELISA was found to be 86.4 and 93.1 % respectively, in comparison with cytb based PCR assay. Additionally, rBgSA3-ELISA evaluated using survey serum samples (n = 287), detected 11.85 % samples as positive. In conclusion, B. gibsoni infection, an emerging disease is prevalent in the present study area and the standardized rBgSA3 protein based indirect-ELISA was found to be a specific and sensitive test for large scale sero-diagnosis and sero-surveillance of B. gibsoni infection in dogs.


Assuntos
Babesia/isolamento & purificação , Babesiose/parasitologia , Doenças do Cão/parasitologia , Ensaio de Imunoadsorção Enzimática/veterinária , Proteínas de Protozoários/sangue , Animais , Antígenos de Protozoários , Babesiose/sangue , Babesiose/diagnóstico , Doenças do Cão/diagnóstico , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Masculino , Vigilância da População , Estudos Soroepidemiológicos
8.
Biofouling ; 36(8): 938-950, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-33059484

RESUMO

Biofilm production, hitherto an uncharacterized feature among circulating Pasteurella multocida strains, was studied along with the antibiotic susceptibility pattern. On the basis of biofilm formation ability, all the strains were categorized into four groups under six different culture conditions: strong biofilm-forming (22%), moderate (19%), weak (51%), and non-adherent (7%). Strains from serogroups A and B formed significant biofilms in at least one culture condition whereas strains from serogroup D were unable to form biofilms. All strains were found to be susceptible to tetracycline. In addition, the correlation between diverse factors (host, capsule type, clinical condition and the tadD gene) as well as antimicrobial susceptibility in biofilm production were analyzed by Joint distribution models, and showed that enrofloxacin and azithromycin resistant strains were positively correlated with strong biofilm production.


Assuntos
Biofilmes , Pasteurella multocida , Antibacterianos/farmacologia , Anti-Infecciosos , Testes de Sensibilidade Microbiana
9.
Infect Genet Evol ; 85: 104564, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32979548

RESUMO

Virulence associated and/or housekeeping/repetitive genes either in single or multiple copies are being extensively targeted for bacterial pathogen detection and differentiation in epidemiological studies. In the present study, isolation of Pasteurella multocida from different animals and their genetic profiling based on the capsular types, virulence and repetitive elements (ERIC/REP) were carried out. A total 345 clinical samples from apparently healthy and diseased (pneumonic, septicaemia) animals (sheep, goat, pig, cattle, buffalo and rabbits) from different geographical regions of Karnataka, Uttar Pradesh, Mizoram and Assam states of India were screened. A total of 32% of the samples were found positive, of which 41 P. multocida isolates recovered. Virulence profiling of isolates indicated that omp87, ompA, ptfA, sodA, sodC, nanB, fur and exbB were present in 100% of isolates. Whereas, prevalence of other genes were; nanH (90%), ompH (71%), pfhA (63%), plpB (80%), hsf-1 (12%), hsf-2 (37%), pmHAS (78%), toxA (73%), hgbA (37%), hgbB (81%), tbpA (78%) and fimA (98%), among isolates. There was no influence of host or place on prevalence of virulence genes when assessed by fitting a Hierarchial Bayesian ordinal regression model. There was correlation (positive and negative) between broad groups of virulence genes. Both repetitive gene profiles (ERIC and REP) generated multiple amplicons (~200 to ~4000 bp). Cluster analysis with ERIC profiles revealed 5 clusters and 3 non- typable isolates with higher discriminatory power (D = 0.7991) than the REP-PCR profiles (D = 00.734) which revealed 4 clusters and 6 non- typable isolates. The results showed that a considerable level of genetic diversity exists among circulating P. multocida isolates despite belonging to the same geographical origin. The genetic diversity or clustering based on either virulence or repetitive elements among isolates could be largely driven by multiple factors acting together which lead to manifestations of particular disease symptoms.


Assuntos
Doenças dos Animais/microbiologia , Genes Bacterianos , Variação Genética , Infecções por Pasteurella/veterinária , Pasteurella multocida/genética , Sequências Repetitivas de Ácido Nucleico , Fatores de Virulência/genética , Animais , Teorema de Bayes , Interações Hospedeiro-Patógeno , Pasteurella multocida/classificação , Filogenia , Filogeografia , Virulência/genética
10.
Infect Genet Evol ; 85: 104472, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32711078

RESUMO

Sheeppox and goatpox are highly contagious viral diseases of small ruminants causing severe economic losses to the livestock farmers. The disease is enzootic in Asia including India, Middle East and African countries. In the present study, a total of 28 isolates from twenty five sheeppox and goatpox disease outbreaks were phylogenetically analyzed based on P32 gene/protein along with homology modeling and docking using heparan sulfate and UDP-glucose. Three distinct lineage-specific clusters as per their host origin were recorded. Multiple sequence analysis of P32 gene revealed that genetically similar sheeppox virus (SPPV) and goatpox virus (GTPV) strains are circulating in India. Phylogenetically, Lumpy skin disease (LSDV) and SPPV had a closer genetic relationship than GTPV. Comparative sequence alignment indicated conservation of various motifs such as glycosaminoglycan (GAG), chemokine like motif (CX3C) and Asp-Glu-any other residue-Asp (D/ExD), as well as viral specific signature residues in SPPV and GTPV isolates. Structurally, P32 protein of SPPV and GTPV with mixed α helices and ß sheets resembled with crystal structure of homologue vaccinia virus H3L protein. Docking studies in P32 protein of SPPV and GTPV revealed conserved binding pattern with heparan sulfate which is involved in the virus attachment and varied glycosyltransferase fold with UDP-glucose. These findings may help in development of suitable vaccines/diagnostics and therapeutics against capripoxviruses.


Assuntos
Capripoxvirus/classificação , Capripoxvirus/genética , Doenças das Cabras/virologia , Infecções por Poxviridae/genética , Doenças dos Ovinos/virologia , Proteínas do Envelope Viral/genética , Animais , Cabras/virologia , Índia , Filogenia , Mapeamento de Interação de Proteínas , Análise de Sequência de DNA , Ovinos/virologia
11.
Protein Expr Purif ; 155: 15-20, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30217599

RESUMO

Strategic design and suitable purification techniques are of paramount importance in the production of recombinant proteins, if intended for use in a diagnostic assay. However, there is no single protocol that can be universally adopted for obtaining proteins in requisite quality and quantity across various platforms. In this study, we have targeted proteins of bluetongue virus (BTV), which is the causative agent of an arthropod-borne infectious disease in ruminants. Traditionally, serological diagnosis of the disease has rested upon either virus neutralization test or on an ELISA test that employed a recombinant structural (VP1, VP7) protein. Among the non-structural (NS) proteins of BTV, NS1 and NS3, are preferred candidate antigens in development of immuno-diagnostics as these provide the option for identifying recent/ongoing infection. However, the difficulty in production/purification of recombinant full length NS proteins of BTV in sufficient quantity and quality in various expression systems, due to inherent structural complexities, have restricted their wider applicability as immunodiagnostic reagents. To circumvent the difficulties associated with production/purification, we developed a novel NS1 and NS3 fusion gene (∼1302 bp) encoding for NS1 N-terminus (1M to G252 aa) and NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains along with intervening variable central domain (118A to A182 aa) of bluetongue virus 23. This construct was cloned, over-expressed and efficiently purified by single step affinity chromatography under unique denaturing/renaturing condition. The purified fusion protein was found suitable for detection of antibodies against BTV in an indirect ELISA (iELISA).


Assuntos
Vírus Bluetongue/genética , Proteínas não Estruturais Virais/genética , Animais , Bluetongue/virologia , Vírus Bluetongue/química , Clonagem Molecular/métodos , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Ovinos , Proteínas não Estruturais Virais/química
12.
J Virol Methods ; 261: 112-120, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30149033

RESUMO

Orf or contagious ecthyma, is a highly contagious transboundary disease of sheep and goats. For sero-diagnosis of orf, recombinant antigen based assays are considered as alternatives to conventional approaches such as serum neutralization test (SNT) and counter-immuno-electrophoresis (CIE). A major envelope protein of orf virus (ORFV), F1L, is highly immunogenic and is a candidate for use in these assays. In this study, the F1L gene of the ORFV-59/05 strain encoding a recombinant mature F1L protein (1M-D302 aa) with a C- terminal truncation, was produced as a fusion protein (∼50 kDa) in Escherichia coli. The immunogenic potential of purified rF1L was confirmed by detecting specific anti-F1L antibody responses in sera collected from immunized rabbits and guinea pigs using ELISA and SNT. An indirect-ELISA based on rF1L was developed and optimized. In comparison to SNT by ROC analysis in the detection of ORFV specific antibodies, this new assay exhibited a diagnostic specificity of 94.04% and 92.53% with sheep and goat sera, respectively, while the sensitivity was 89.22% and 94.25%, for sheep and goat sera. No cross reactivity was noted with sera collected from small ruminants infected with other transboundary diseases (goatpox, sheeppox, peste des petits ruminants, foot-and-mouth disease and bluetongue). Furthermore, the rF1L-ELISA applied to screen the vaccinated/challenged goat sera resulted in better detection (30%) than by SNT (28%) in spite of lower levels of antibodies which could be due to predominant cell mediated immune response in vaccinated animals. This study highlighted the potential utility of rF1L protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of sero-diagnostic assay for surveillance of ORFV infection in sheep and goats.


Assuntos
Anticorpos Antivirais/sangue , Ectima Contagioso/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Doenças das Cabras/diagnóstico , Vírus do Orf/imunologia , Doenças dos Ovinos/diagnóstico , Proteínas Virais/imunologia , Animais , Cabras , Cobaias , Curva ROC , Coelhos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e Especificidade , Ovinos , Proteínas Virais/genética
13.
Gene ; 663: 72-82, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29655893

RESUMO

Orf virus (ORFV), a member of the genus Parapoxvirus in the family Poxviridae, is the cause of orf, a highly contagious zoonotic viral disease that affects mainly sheep and goats. In the present study, the sequence and phylogenetic analysis of Indian ORFV isolates (n = 15) from natural outbreaks in sheep and goats belonging to different geographical regions were analysed on the basis of F1L gene along with homology modelling of F1L protein. Multiple sequence alignments revealed highly conserved C-terminus and heterogeneity of N-terminus region of F1L among all orf viruses studied. Further, a comparative sequence alignment indicated conservation of various motifs such as glycosaminoglycan (GAG), Asp/Glu-any residue-Asp (D/ExD) and a Cx3C chemokine like motif among all poxviruses and unique motifs (proline rich region [PRR] and Lys-Gly-Asp [KGD]), in parapoxviruses including ORFV isolates irrespective of geography and host species. Phylogenetically, two major clusters were noticed which included Indian orf isolates along with foreign isolates. Structurally, ORFV F1L resembled the topology as exhibited by its homologue vaccinia virus H3 protein with mixed ß/α folds and ligand binding specificity in docking models. We noted that despite differences in host cell specificity and pathogencity, poxvirus proteins especially ORFV F1L protein and its homologues presumed to share similarities as they are highly conserved irrespective of species and countries of origin. Further, the study also indicated the possibilities of differentiation of ORFV strains based on N-terminal heterogeneity despite highly conserved C-terminal region with conserved motifs.


Assuntos
Vírus do Orf/genética , Análise de Sequência de DNA/métodos , Proteínas Virais/química , Proteínas Virais/genética , Animais , Sequência Conservada , Cabras/microbiologia , Modelos Moleculares , Simulação de Acoplamento Molecular , Vírus do Orf/isolamento & purificação , Filogenia , Domínios Proteicos , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Ovinos/microbiologia
14.
Antiviral Res ; 141: 174-178, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28259752

RESUMO

Buffalopox virus (BPXV) and other vaccinia-like viruses (VLVs) are causing an emerging/re-emerging zoonosis affecting buffaloes, cattle and humans in India and other countries. A27L and H3L are immuno-dominant major envelope proteins of intracellular mature virion (IMV) of orthopoxviruses (OPVs) and are highly conserved with an ability to elicit neutralizing antibodies. In the present study, two recombinant proteins namely; rA27L (21S to E110; ∼30 kDa) and rH3L(1M to I280; ∼50 kDa) of BPXV-Vij/96 produced from Escherichia coli were used in vaccine formulation. A combined recombinant subunit vaccine comprising rA27L and rH3L antigens (10 µg of each) was used for active immunization of adult mice (20µg/dose/mice) with or without adjuvant (FCA/FIA) by intramuscular route. Immune responses revealed a gradual increase in antigen specific serum IgG as well as neutralizing antibody titers measured by using indirect-ELISA and serum neutralization test (SNT) respectively, which were higher as compared to that elicited by individual antigens. Suckling mice passively administered with combined anti-A27L and anti-H3L sera showed a complete (100%) pre-exposure protection upon challenge with virulent BPXV. Conclusively, this study highlights the potential utility of rA27L and rH3L proteins as safer candidate prophylactic antigens in combined recombinant subunit vaccine for buffalopox as well as passive protective efficacy of combined sera in employing better pre-exposure protection against virulent BPXV.


Assuntos
Imunização Passiva , Imunogenicidade da Vacina , Infecções por Poxviridae/prevenção & controle , Vírus Vaccinia/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Animais Lactentes , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Soros Imunes/administração & dosagem , Imunoglobulina G/sangue , Infecções por Poxviridae/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Vacinação/métodos , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/imunologia , Vírus Vaccinia/química , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem
15.
Arch Virol ; 162(4): 953-962, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27995337

RESUMO

Orf, or contagious ecthyma, a highly contagious transboundary disease of sheep and goats, is caused by a double-stranded DNA virus (ORFV) belonging to the genus Parapoxvirus of the family Poxviridae. The ORFV genome encodes the major envelope proteins B2L and F1L, which have been found to be highly immunogenic and have multiple functional characteristics. In order to investigate the functional properties of the B2L protein, in this study, the B2L gene of ORFV strain 59/05, encoding recombinant mature B2L (aa 1M-D334), was produced as a fusion protein in Escherichia coli. The functional characteristics of purified rB2L fusion protein (~60 kDa) were evaluated in vivo and in vitro, showing that this protein had lipase and immunomodulatory activities. Immunization trials involving laboratory animals (mice, rabbits and guinea pigs) using either constant or graded doses of rB2L fusion protein with or without adjuvants (FCA, alum) as well as co-administration with candidate rErns-Ag protein of classical swine fever virus (CSFV) indicated that the rB2L protein is immunogenic and has immunomodulatory properties. This study shows the potential utility of the rB2L protein as a safe and novel adjuvant in veterinary vaccine formulations.


Assuntos
Ectima Contagioso/virologia , Vírus do Orf/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Ectima Contagioso/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Cobaias , Imunização , Lipase/administração & dosagem , Lipase/genética , Lipase/imunologia , Masculino , Camundongos , Vírus do Orf/genética , Coelhos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Recombinação Genética , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
16.
Biologicals ; 44(5): 352-9, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27448505

RESUMO

Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization.


Assuntos
Sequência de Aminoácidos , Vírus Bluetongue , Deleção de Sequência , Proteínas não Estruturais Virais , Animais , Bluetongue/diagnóstico , Bluetongue/imunologia , Vírus Bluetongue/química , Vírus Bluetongue/genética , Vírus Bluetongue/imunologia , Escherichia coli , Expressão Gênica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Ruminantes/imunologia , Ruminantes/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
17.
Antiviral Res ; 126: 108-16, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26723250

RESUMO

Buffalopox virus, a zoonotic Indian vaccinia-like virus, is responsible for contagious disease affecting mainly buffaloes, cattle and humans. H3L gene, encoding for an immunodominant major envelope protein of intracellular mature virion of orthopoxviruses, is highly conserved and found to elicit neutralizing antibodies. Therefore in the present study, the immunogenicity and protective efficacy of the recombinant H3L protein of buffalopox virus in laboratory animal models has been evaluated. A partial H3L gene encoding for the C-terminal truncated ectodomain of H3L protein (1M to I280) of BPXV-Vij/96 strain was cloned, over-expressed and purified as histidine-tagged fusion protein (50 kDa) from Escherichia coli using Ni-NTA affinity chromatography. The purified rH3L protein was further used for active immunization of guinea pig (250 µg/dose) and adult mice (10 µg and 50 µg/dose) with or without adjuvants (alum, Freund's Complete Adjuvant and CpG). Subsequently, a gradual increase in antigen specific serum IgG as well as neutralizing antibody titres measured by using indirect-ELISA and serum neutralization test respectively, was noted in both guinea pigs and mouse models. Suckling mice immunized passively with anti-H3L serum showed 80% pre-exposure prophylaxis upon challenge with virulent buffalopox virus strain. An indirect-ELISA based on rH3L protein showed no cross-reactivity with hyperimmune sera against sheeppox virus (SPPV), goatpox virus (GTPV), orf virus (ORFV), foot- and- mouth disease virus (FMDV), peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) during the course of study. The study highlights the potential utility of rH3L protein as a safer prophylactic and diagnostic reagent for buffalopox.


Assuntos
Formação de Anticorpos/imunologia , Vírus Bluetongue/imunologia , Proteínas de Transporte/imunologia , Proteínas Recombinantes , Vírus Vaccinia/imunologia , Vaccinia/virologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Sequência de Bases , Vírus Bluetongue/genética , Capripoxvirus/imunologia , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Feminino , Vírus da Febre Aftosa/imunologia , Cobaias , Imunoglobulina G/sangue , Masculino , Camundongos , Modelos Animais , Vírus do Orf/imunologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Infecções por Poxviridae/diagnóstico , Infecções por Poxviridae/prevenção & controle , Profilaxia Pré-Exposição , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinação , Vaccinia/imunologia , Vaccinia/prevenção & controle , Vírus Vaccinia/genética , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação
18.
Vaccine ; 33(41): 5396-5405, 2015 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-26319070

RESUMO

Buffalopox virus (BPXV), an Indian variant of vaccinia virus (VACV), is a zoonotic agent and affects buffaloes, cattle and humans. A27L is one of the conserved major immuno-dominant envelope proteins of orthopox viruses (OPVs) involved in viral entry/maturation and elicits neutralizing antibodies. In this study, the A27L gene of BPXV-Vij/96 strain encoding recombinant mature A27L (21S to E110) and C-terminal truncated A27L-LZD (21S to N84aa) proteins were cloned and over-expressed in Escherichia coli as fusion proteins. Structurally, A27L of BPXV was similar to that of VACV and found to contain four regions including a potential coiled-coil motif (CCM) in the centre (43 to 84aa). Oligomerization of recombinant A27L fusion protein (∼30 kDa) leads to the formation of dimer/trimers/tetramers under non-reducing conditions. Further, the purified rA27L protein was used for active immunization of rabbit (250 µg/rabbit) and adult mice (10 µg and 50 µg/mice) with or without adjuvants (FCA, alum and CpG). Immune response measured by using indirect-ELISA and SNT revealed a gradual increase in antigen specific serum IgG as well as neutralization antibody titers. Upon challenge with virulent BPXV strain, a protection of 60% was observed in suckling mice passively administered with anti-rA27L sera. No cross-reactivity of rA27L protein with hyperimmune sera against ORFV, GTPV, SPPV, PPRV, FMDV and BTV was noticed in indirect-ELISA. The study indicated that the rA27L protein is a safe and potential prophylactic as well as diagnostic antigen for buffalopox.


Assuntos
Proteínas Recombinantes , Vírus Vaccinia/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Sequência Conservada , Soros Imunes/imunologia , Imunização , Camundongos , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Terciária de Proteína , Coelhos , Sensibilidade e Especificidade , Vaccinia/diagnóstico , Vaccinia/imunologia , Vaccinia/patologia , Vírus Vaccinia/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação
19.
Virus Genes ; 51(2): 244-51, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26318174

RESUMO

Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.


Assuntos
Vírus Bluetongue/fisiologia , Multimerização Proteica , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Análise de Sequência de DNA , Proteínas não Estruturais Virais/química
20.
Virus Genes ; 51(1): 33-8, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25971425

RESUMO

Recent developments in molecular biology shed light on cross-species transmission of SPPV and GTPV. The present study was planned to characterize the capripoxviruses which were circulating in the field condition among sheep and goats using RPO30 gene-based viral lineage (SPPV/GTPV) differentiating PCR and sequencing of RPO30 and GPCR genes from clinical samples. Out of 58 scabs (35 sheep and 23 goats) screened, 27 sheep and 18 goat scabs were found positive for capripox virus infections. With the exception of one sheep and one goat scabs, all the positive samples yielded amplicon size according to host origin, i.e. SPPV in sheep and GTPV in goats. In the above two exceptional cases, goat scab and sheep scab yielded amplicon size as that of SPPV and GTPV, respectively. Further, sequencing and phylogenetic analyses of complete ORFs of RPO30 and GPCR genes from six sheep and three goat scabs revealed that with the exception of above two samples, all had host-specific signatures and clustered according to their host origin. In case of cross-species infecting samples, sheep scab possessed GTPV-like signatures and goat scab possessed SPPV-like signatures. Our study identifies the circulation of cross-infecting SPPV and GTPV in the field and warrants the development of single-strain vaccine which can protect the animals from both sheeppox and goatpox diseases.


Assuntos
Capripoxvirus/classificação , Capripoxvirus/isolamento & purificação , DNA Viral/química , DNA Viral/genética , Doenças das Cabras/virologia , Infecções por Poxviridae/veterinária , Doenças dos Ovinos/virologia , Animais , Capripoxvirus/genética , Transmissão de Doença Infecciosa , Doenças das Cabras/transmissão , Cabras , Índia , Dados de Sequência Molecular , Infecções por Poxviridae/transmissão , Infecções por Poxviridae/virologia , Ruminantes , Análise de Sequência de DNA , Ovinos , Doenças dos Ovinos/transmissão
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