Investigating the Influence of ANTXR2 Gene Mutations on Protective Antigen Binding for Heightened Anthrax Resistance.

Bacillus anthracis is the bacterium responsible for causing the zoonotic disease called anthrax. The disease presents itself in different forms like gastrointestinal, inhalation, and cutaneous. Bacterial spores are tremendously adaptable, can persist for extended periods and occasionally endanger human health. The Anthrax Toxin Receptor-2 (ANTXR2) gene acts as membrane receptor and facilitates the entry of the anthrax toxin into host cells. Additionally, mutations in the ANTXR2 gene have been linked to various autoimmune diseases, including Hyaline Fibromatosis Syndrome (HFS), Ankylosing Spondylitis (AS), Juvenile Hyaline Fibromatosis (JHF), and Infantile Systemic Hyalinosis (ISH). This study delves into the genetic landscape of ANTXR2, aiming to comprehend its associations with diverse disorders, elucidate the impacts of its mutations, and pinpoint minimal non-pathogenic mutations capable of reducing the binding affinity of the ANTXR2 gene with the protective antigen. Recognizing the pivotal role of single-nucleotide polymorphisms (SNPs) in shaping genetic diversity, we conducted computational analyses to discern highly deleterious and tolerated non-synonymous SNPs (nsSNPs) in the ANTXR2 gene. The Mutpred2 server determined that the Arg465Trp alteration in the ANTXR2 gene leads to altered DNA binding (p = 0.22) with a probability of a deleterious mutation of 0.808; notably, among the identified deleterious SNPs, rs368288611 (Arg465Trp) stands out due to its significant impact on altering the DNA-binding ability of ANTXR2. We propose these SNPs as potential candidates for hypertension linked to the ANTXR2 gene, which is implicated in blood pressure regulation. Noteworthy among the tolerated substitutions is rs200536829 (Ala33Ser), recognized as less pathogenic; this highlights its potential as a valuable biomarker, potentially reducing side effects on the host while also reducing binding with the protective antigen protein. Investigating these SNPs holds the potential to correlate with several autoimmune disorders and mitigate the impact of anthrax disease in humans.

Development of recombinant capsid protein-based single serum dilution ELISA for sero-detection of porcine circovirus type 2 infection in pigs.

Porcine Circovirus 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) of swine and is one of the reasons for severe economic loss in swine industry. In India, there is a considerable prevalence rate of PCV2 infection in pig population, PCV2d being the most prominent genotype. Proper sero-diagnosis and sero-surveillance of the disease is formulated as an effective control measure. In this study, a recombinant capsid protein-based single serum dilution indirect ELISA was developed for determination of antibody titre of the infected pigs. The capsid protein (Cap) of PCV2d was produced in Saccharomyces cerevisiae cells and the capsid protein was purified by affinity chromatography. This recombinant protein was used as a coating antigen to develop a cost effective, highly sensitive and specific single serum dilution ELISA. The in-house developed ELISA was optimized to be used in a 1:200 single serum dilution. The developed ELISA along with a commercial ELISA kit were compared with a sensitive immuno-peroxidase assay (IPMA) by receiver-operating characteristics (ROC) test. Our results showed that the developed single serum dilution ELISA had a higher sensitivity and specificity in comparison to the commercial ELISA. The area under the ROC curve (AUC) also confirmed that the developed ELISA had a better overall diagnostic performance in comparison to the commercial ELISA kit.

Application of machine learning models for risk estimation and risk prediction of classical swine fever in Assam, India.

The present study is aimed to develop an early warning system of Classical swine fever (CSF) disease by applying machine learning models and to study the climate-disease relationship with respect to the spatial occurrence and outbreaks of the disease in the north-eastern state of Assam, India. The disease incidence data from the year 2005 to 2021 was used. The linear discriminant analysis (LDA) revealed that significant environmental and remote sensing risk factors like air temperature, enhanced vegetation index, land surface temperature, potential evaporation rate and wind speed were significantly contributing to CSF incidences in Assam. Furthermore, the climate-based disease modelling was applied to relevant ecological and environmental risk factors determined using LDA and risk maps were generated. The western and eastern regions of the state were predicted to be at high risk of CSF with presence of significant hotspots. For the districts that are significantly clustered, the Basic reproduction number (R0) was calculated after the predicted results were superimposed onto the risk maps. The R0 value ranged from 1.04 to 2.07, implying that the eastern and western regions of Assam are more susceptible to CSF. Machine learning models were implemented using R statistical software version 3.1.3. The random forest, classification tree analysis and gradient boosting machine were found to be the best-fitted models for the study group. The models' performance was measured using the Receiving Operating Characteristic (ROC) curve, Cohen's Kappa, True Skill Statistics, Area Under ROC Curve, ACCURACY, ERROR RATE, F1 SCORE, and Logistic Loss. As a part of the suggested study, these models will help us to understand the disease transmission dynamics, risk factors and spatio-temporal pattern of spread and evaluate the efficacy of control measures to battle the economic losses caused by CSF outbreaks. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00847-6.

Prevalence of bovine tuberculosis and analysis of risk factors among the dairy farms in and around Guwahati metropolitan city, India.

Bovine tuberculosis (bTB), a neglected zoonotic disease caused by Mycobacterium bovis is being reported worldwide. The present work was carried out from December 2020 to November 2021 to assess the prevalence and risk factors of bTB in peri-urban and urban dairy farms of Guwahati, Assam, India. A questionnaire was used to collect data on knowledge about bTB on 36 farms, and ten animals per farm were screened by single intradermal comparative cervical tuberculin test (SICCT) to determine the prevalence of bTB, giving a total of 360 animals. The demographic data of the farmers revealed that 61.1% respondents were illiterate, 66.7% had no awareness about bovine tuberculosis and 41.7% consumed unpasteurised milk and milk products. SICCT showed that 38 cattle from 18 of the farms were positive reactors for bTB, yielding an overall animal level prevalence of 10.55% (95% confidence interval (CI = 7.58-14.2%) and a 50% herd prevalence (95% CI 32.9-67.1%). Animals 5 years and above were found to be more likely to be positive for bTB (17.18%). The study highlighted the widespread prevalence of bovine tuberculosis in peri-urban and urban dairy farms of Guwahati which gives a picture also about other major cities of India. Hence, it is of utmost importance to undertake a comprehensive epidemiological study in such cities for effective control and prevention of bTB in a one health approach.

Evaluation of an in-house IgM/IgG lateral flow assay for serodiagnosis of human brucellosis.

This study aimed to evaluate the diagnostic efficacy of an in-house lateral flow assay (LFA) for the detection of IgM/IgG anti-Brucella antibodies for rapid serodiagnosis of human brucellosis. Three groups of sera samples including 476 from high-risk individuals, 27 from culture-confirmed patients, and 43 from healthy blood donors were used for evaluation of LFA. In comparison with iELISA, the sensitivity, specificity, and accuracy of LFA were >95%, >99%, and 99% respectively. Considering the very good agreement, accuracy, simplicity, and rapidity, LFAs might be useful as a point of care test for the diagnosis of human brucellosis in resource-limited laboratories.

Transmission of African swine fever virus to the wild boars of Northeast India.

BACKGROUND: India recorded the first outbreak of African swine fever (ASF) in North-eastern region (NER) in the year 2020. AIM: The current study was undertaken to investigate the transmission of African swine fever virus (ASFV) in the wild boars of Northeast India, particularly of Assam. MATERIAL AND METHODS: ASF suspected mortal tissue remains and blood samples of wild boars collected from different locations of Assam were screened for molecular detection of swine viruses which includes Classical swine fever virus, Porcine Circovirus 2, Porcine reproductive and respiratory syndrome virus and ASFV. RESULTS: One sample each from Manas and Nameri National Parks was detected positive for ASFV. Besides this, one of the samples was positive for CSFV and one of the ASFV positive samples was also positive for PCV2. Several striking gross and microscopic alterations were noticed in different organs of ASFV infected animals. Sequencing and phylogenetic analysis of B646L gene confirmed the presence of ASFV genotype-II in wild boars. Circulation of similar genotype in domestic pigs of NER in the contemporary period as well as locations near to the aforementioned national parks indicates the transmission of ASFV from domestic to wild boars. CLINICAL RELEVANCE: The detection of ASFV in the wild boars of Assam is alarming as it is an impending threat to pig population and other endangered species (particularly Pygmy hog), making it increasingly daunting to control the disease. CONCLUSION: Chances are high for ASFV to become endemic in Assam region if stringent measures are not taken at proper time.

Computational analysis of immunogenic epitopes in the p30 and p54 proteins of African swine fever virus.

African swine fever (ASF) is a highly infectious viral disease of pigs, which causes acute fatal haemorrhage and is a severe concern to the global pork industry. The present study followed computational approaches to identify B- and T-cell epitopes for the p30 and p54 proteins of the African swine fever virus (ASFV) by interacting with the swine leukocyte antigen (SLA) alleles. The amino acid sequences of p30 and p54 were analysed for variability and relative solvent accessibility, and their three-dimensional structures were predicted and validated. Molecular dynamics simulation was performed to study the structural and dynamic properties of the protein. Six and five linear B-cell epitopes have been predicted for p30 and p54, respectively. Four and three discontinuous B-cell epitopes have been predicted for p30 and p54, respectively. Further, the top five T-cell epitopes for SLA-1, 2, and 3 have been listed for both proteins. These results can help us to understand the immunodominant regions in the p30 and p54 proteins of ASFV and potentially assist in designing peptide-based diagnostics and vaccines. Also, the identified T-cell epitopes may be considered for peptide-based vaccine design against ASFV.Communicated by Ramaswamy H. Sarma.

Molecular characterization of porcine circovirus 2 circulating in Assam and Arunachal Pradesh of India.

PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVADs) which affect pigs worldwide. Currently, there is a worldwide genotype prevalence switch from PCV2b to PCV2d, which has led to increased virulence of the circulating virus strains leading to vaccine failures and selection pressure. In the present study, the PCV2 genotypes circulating in north eastern region (NER) of India particularly the states of Assam and Arunachal Pradesh was characterized by isolation, sequencing and phylogenetic analysis of cap gene. The phylogenetic analysis revealed that the PCV2 isolates circulating in pigs of Assam and Arunachal Pradesh were mostly of PCV2d genotype. Hence, it can be concluded that PCV2d genotype is the most dominating genotype in NER and priority should be given to this genotype for development of future vaccine candidate against PCV2 in India.

Thermostability and Immunogenicity of Genotype II Avian Orthoavulavirus (AOaV-1) Isolates from Duck (Anas platyrhynchos) and Parrot (Eclectusroratus).

Newcastle disease (ND) is a highly contagious viral disease of poultry causing significant economic losses worldwide. Vaccination is considered the most reliable approach to curb the economic menace that is ND, but the thermolabile nature of Newcastle disease virus (NDV) vaccination poses a significant threat to its protective efficacy. This study aimed to profile the thermostability of NDV isolates from duck (As/Km/19/44) and parrot (As/WB/19/91) and evaluate their immunogenic potential in chicks. Fusion protein cleavage site (FPCS) and phylogenetic analysis demonstrated the lentogenic nature of both the isolates/strains and classified them as class II genotype II NDV. The characterized NDV isolates were adapted in specific-pathogen-free (SPF) chicks by serially passaging. Biological pathogenicity assessment of chicken-adapted As/Km/19/44 (PSD44C) and As/WB/19/91 (PSP91C) revealed both the isolates to be avirulent with a mean death time (MDT) of more than 90 h and an intracerebral pathogenicity index (ICPI) ranging from 0.2 to 0.4. Both of the NDV isolates displayed varied thermostability profiles. PSD44C was the most thermostable strain as compared to PSP91C and the commercially available LaSota vaccine strain. The immunogenicity of PSD44C and LaSota was significantly higher than PSP91C. Based on these results, it is concluded that NDV isolate PSD44C is more thermostable and immunogenic when administered intraocularly without any adverse effects. Therefore, PSD44C is suitable for further research and vaccine development.

Draft Genome Sequence Analysis of the Genotype II African Swine Fever Virus from India.

African swine fever virus (ASFV) entered the northeastern (NE) part of India early in 2020, causing huge economic loss to the piggery sector. Here, we are presenting a brief report on the draft genome sequence of an ASFV strain ABTCVSCK_ASF007 from Assam state of NE India belonging to genotype II.

Prevalence of Japanese encephalitis (JE) virus in mosquitoes and animals of the Asian continent: A systematic review and meta-analysis.

BACKGROUND: Japanese encephalitis (JE) is a viral zoonotic disease that has been found in several countries of Asia and is responsible for high mortality and morbidity of men and animals in rural and sub-urban endemic areas due to the virus re-circulation among diverse hosts and vectors. The present study estimates the prevalence of the JE virus in the vector and animal population of the Asian continent using a systematic review and meta-analysis. METHODS: The Cochran collaborators' Preferred Reporting Items for Systematic Reviews and Meta-Analysis [PRISMA] guidelines were used for systematic review and meta-analysis. The heterogeneity was observed in meta-regression analysis due to several factors including region, species, and different diagnostic assays used in various studies. Thus we did sensitivity and subgroup analysis. RESULTS: The prevalence of the JE virus was calculated using a total sample size of 47,391. Subgroup analysis revealed the JE virus prevalence of 39% in the Southeast Asia region, followed by East Asia with 35% and South Asia with 15% prevalence. Hence, the overall pooled prevalence of the JE virus was 26% in the Asian continent. CONCLUSIONS: The highest proportion of infection was found in pigs amongst all animals, reinforcing the fact that they can be used as sentinels to predict outbreaks in humans. The findings of this study will enable researchers and policymakers in better understanding the disease's spatial and temporal distribution, as well as in creating and implementing location-specific JE prevention and control measures.

Complete genome analysis of African swine fever virus isolated from domestic pigs during the first ASF outbreaks in India.

African swine fever (ASF), considered as the most dreadful swine disease due to its very high mortality, emerged in India in 2020. The complete genome analysis of ASF viruses isolated during the first outbreaks in India showed a few unique non-synonymous mutations in MGF 369-11L, MGF 505-4R, K205R and B263R genes. Frame shifts in the protein coding sequences were observed in DP60R, ASFV-G_ACD 00190, MGF 110-10-L-MGF110-14L fusion, MGF 360-14L and I267L genes of Indian ASF viruses as compared to ASFV/Georgia/2007. Complete genome based phylogenetic analysis of p72-genotype-II viruses showed the clustering of Indian isolates with ASFV/Wuhan/2019 in a separate clade. Phylogenetic analysis of concatenated sequences of 14 open reading frames (ORF) having single nucleotide polymorphisms (SNP) showed distinct grouping of Indian ASFVs with other Asian ASFVs. This is the first complete genome characterization of ASF viruses isolated from domestic pigs in India. The results indicate that number of Tandem Repeat Sequence (TRS) in the intergenic region between I73R and I329L genes, and the 14 ORFs with SNP reported in this study could be the genetic determinants to differentiate the closely related p72-genotype II viruses circulating in Asia.

Whole-Genome Sequence of a Porcine Circovirus Type 2 Strain Detected in Assam, India.

Porcine circovirus-associated disease caused by porcine circovirus type 2 (PCV2) is a vital threat to the global pig industry. In this study, we have characterized the complete genome sequence of a PCV2 isolate, namely, Assam-01, belonging to the genotype PCV2d.

First complete genome characterization of duck plague virus from India.

In this study, we report the complete genome sequencing of the Duck plague virus from India for the first time. The sequencing was done on the MinION nanopore sequencer from Oxford Nanopore Technologies. The closest relative is the European strain 2085v, with 99.98 and 99.8% identity at the amino acid and nucleotide level respectively. Moreover, 72 out of 77 ORFs are completely conserved between the 2 strains. The high similarity with the European strain over the only three other pathogenic strains reported from China points to the circulation of European strain in India. The fly pathways of migratory birds and co-habitation with native species being a probable reason. More complete genome data from diverse sampling locations are needed to characterize the genomic features, develop diagnostics, vaccines, and understand the evolution of the virus.

Gut microbiome architecture of wild greater one-horned rhinoceros:a vulnerable species from Kaziranga National Park, India.

Rhinoceros unicornis, also known as the greater one-horned rhinoceros (GoHR), is a vulnerable wildlife species found in the Indian subcontinent with an estimated global population of 3582, of which an estimated 2995 resides in India. The Kaziranga National Park of Assam is the home to ~80.56% of the GoH population in India. Recent advances in genetics and microbial studies underscored the importance of gut microbial symbiosis as a crucial factor for host metabolic health and environmental interaction, particularly for higher mammals. Alteration of the normal microbiome can also be an indicator of chronic disease and infection. Freshly voided dung samples from nine dung heaps of free ranging or wild GoH rhinoceros were collected from Kaziranga National Park for mapping the gut microbial architecture through 16S-metagenomic approach. In our sample, the GoH gut harbours 168.8±12.55 (SE) bacteria-specific OTUs belonging to 21 phyla of which the gram-negative Proteobacteria is the most abundant phyla. Other abundant phylas found in the GoH gut are Firmicutes and Bacteroidetes. Although the GoH rhinoceros gut can utilize fibrous plant by microbial fermentation, the aerobic, nonfermenting Acinetobacter (20.7%), Stenotrophomonas (17.8%) and Brevundimonas (9.1%) constitute about 50% of all identified genus. Functional prediction of the GoH microbiome reveals that>50% of the bacteria present are involved in metabolism followed by cellular processes and information processing. A significant proportion (>1%) are associated with different diseases. In summary, our study characterized bacterial communities of nine wild GoH to identify some unique features and its implication in disease and survival of GoH.

Evidence of Transmission of Goatpox between Domestic Goats and Wild Himalayan Goral (Naemorhedus goral) in Arunachal Pradesh, India.

Examination of carcasses of Himalayan goral (Naemorhedus goral) revealed nodular, pox-like eruptions in the skin. Similar disease was also seen in domestic goats (Capra aegagrus hircus) in the same area. Goatpox virus was identified as the etiology of the disease in both cases, with probable transmission between the species.

KOBUVIRUS DETECTION IN THE CRITICALLY ENDANGERED PYGMY HOG (PORCULA SALVANIA), INDIA.

Pygmy hogs (Porcula salvania) are the smallest and rarest wild suid. It is categorized as a Critically Endangered species as per the Red List of the International Union for Conservation of Nature. This study reports the first detection of a single-stranded RNA virus species, Aichivirus C, belonging to the genus Kobuvirus (KobV) and the family Picornaviridae, in pygmy hogs. KobV species are identified as a cause of acute gastroenteritis among children in India. As of now, there exists no report on the detection of KobV in animals from India. We used a detection assay based on reverse transcription-polymerase chain reaction for KobV screening in pygmy hogs from a conservation center in India. The 3D polymerase gene-based molecular analysis revealed KobV presence in the Indian wild suid, pygmy hogs. Of the 15 samples tested, three were found positive for picornaviruses and were negative for rotavirus A, rotavirus C, astrovirus, picobirnavirus and caliciviruses. Nucleotide-based sequence analysis of the partial 3D polymerase gene revealed close identity with porcine KobV from the Czech Republic (JX232619, 90.6%-91.6%) and Hungary (NC_011829, 89.8%-91.6%), wherein one of the current study strains clustered with the Czech Republic JX232619 strain in the phylogenetic tree. Further investigation of the role of KobV in health and disease of pygmy hogs is warranted.

Investigation of congenital tremor associated with Classical swine fever virus genotype 2.2 in an organized pig farm in north-eastern India.

The present study describes an outbreak of Classical swine fever (CSF) in an organized pig farm followed by an episode of CSF virus (CSFV) associated congenital tremors in piglets. The outbreak was recorded in a newly procured herd of Hampshire pigs housed adjacent to the existing pigs of the farm. The recorded CSF outbreak caused a mortality of 100% in the newly procured and 54.28% in the existing herd. As the disease subsides, the clinically recovered boars were served naturally with Tamworth gilts. Though, the sows farrowed on usual gestation period, litters born to each sow showed congenital tremors and eventually died within 24 h of birth. Necropsy analysis of affected piglets was indicative of CSFV infection and was further confirmed using RT-PCR signifying a transplacental infection. The CSFV strains from the initial outbreak and post outbreak episode of congenital tremors were successfully isolated in PK-15 cells and detected in indirect FAT and RT-PCR. Phylogenetic analysis based on E2 gene and 5'NTR of CSFV grouped the isolates within the genotype 2.2 and revealed close resemblance with previously reported Indian isolates of CSFV genotype 2.2 origin. To the best of our knowledge, this is the first report of CSFV induced congenital form reported from India under natural conditions.

First complete genome characterization of swinepox virus directly from a clinical sample indicates divergence of a Eurasian-lineage virus.

In this study, we report the complete genome sequence of swinepox virus from a clinical sample from a naturally occurring infection in India. The sequencing was done on a Nanopore MinION sequencer from Oxford Nanopore Technologies. Two new annotations were added to the genome. Three of the genes were found to have frameshifts, which might be of importance in relation to infection. When compared to the only other reported whole genome sequence of swinepox virus, which was obtained from an isolate from America in 1999, our sequence is only 98.19% identical at the nucleotide level. The average amino acid sequence identity of the viral proteins, based on the common 149 annotations, is also 98.19%, demonstrating that these viruses are distinctly divergent. Owing to the fact that swinepox virus infects only swine, it could not have entered America until the introduction of swine in the 16th century from Europe. The swinepox viruses in both continents have continued to evolve independently. The sequence divergence identified here indicates a Eurasian-lineage virus that is geographically distinct from the American-lineage swinepox virus.

Sero-epidemiology of porcine parvovirus, circovirus, and classical swine fever virus infections in India.

Reproductive problems in swine caused by porcine viruses pose a serious threat to the pig industry in developing countries like India. For evaluating the true extent of porcine infections, a total of 1308 representative sera samples were collected from 92 different pig farms covering 8 North-Eastern states and Punjab state of Northern India during a period of 2 years (2011-2013). Sera samples were tested for the presence of antibodies against porcine parvovirus (PPV), porcine circovirus-2 (PCV-2), and classical swine fever virus (CSFV) using commercial enzyme-linked immunosorbent assay (ELISA) kits. In the North-Eastern states, the seroprevalence of CSFV in non-vaccinated animals was 6.30% and that of PCV2 and PPV was 6.28% and 1.24%, respectively. In Punjab, the seroprevalence of CSFV in non-vaccinated animals was 44.44% and seroprevalence of PCV-2 and PPV was 34.07% and 39.10%, respectively. Detection of antibodies against more than one virus revealed that 4.66% animals had co-infection with PCV-2 and PPV, 1.75% with CSF and PPV, 1.98% with CSF and PCV-2, and 1.75% with all the three viruses. The receiver operator characteristics (ROC) curve analysis depicted that piglet mortality, parvovirus, and CSFV were the most important parameters with an AUC value of 0.997, 0.897, and 0.973, respectively. Incidence of single or co-infection with different viruses showed that the occurrence of single infection was significantly more prevalent than co-infection. This study provides useful information to set up future epidemiologic, flock management, and public animal health policies for the prevention and control of PCV-2, PPV, and CSF in India.