Low molecular weight proteins, known as chemokines, facilitate the migration and localization of immune cells to the site of infection and injury. One of the first chemokines identified, CXCL8 functions as a key neutrophil activator, recruiting neutrophils to sites of inflammation. Several viral infections, including zoonotic coronaviruses and poxviruses, have been reported to induce the expression of CXCL8. Dromedary camels are known to harbor several potentially zoonotic pathogens, but critical immune molecules such as chemokines remain unidentified. We report here the identification of CXCL8 from the dromedary camel - the first chemokine identified from camelids. The complete dromedary CXCL8 cDNA sequence as well as the corresponding gene sequence from dromedary and two New World camelids - alpaca and llama were cloned. CXCL8 mRNA expression was relatively higher in PBMC, spleen, lung, intestine, and liver. Poly(I:C) and lipopolysaccharide stimulated CXCL8 expression in vitro, while interferon treatment inhibited it. In vitro infection with potentially zoonotic camelpox virus induced the expression of CXCL8 in camel kidney cells. Toxicological studies on camelids have been limited, and no biomarkers have been identified. Hence, we also evaluated CXCL8 mRNA expression as a potential biomarker to assess heavy metal toxicity in camel kidney cells in vitro. CXCL8 expression was increased after in vitro exposure to heavy metal compounds of cobalt and cadmium, suggesting potential utility as a biomarker for renal toxicity in camels. The results of our study demonstrate that camel CXCL8 plays a significant role in immunomodulatory and induced toxicity responses in dromedary camels.

Viral infections activate pattern recognition receptors in the host, triggering an innate immune response that involves the production of interferons, which, in turn, stimulates the expression of antiviral effector genes. Viperin is one of the most highly induced interferon-stimulated genes and displays broad antiviral activity, especially against tick-borne viruses. Of late, camelid-borne zoonotic viruses have been on the rise in the Arabian Peninsula, but research into camelid antiviral effector genes has been limited. This is the first report of an interferon-responsive gene from the mammalian suborder Tylopoda to which modern camels belong. From camel kidney cells treated with dsRNA mimetic, we cloned viperin cDNA encoding 361 amino acid protein. Sequence analysis of camel viperin reveals high levels of amino acid conservation, particularly within the RSAD domain. Compared to kidney, the relative mRNA expression of viperin was higher in blood, lung, spleen, lymph nodes, and intestines. The in-vitro expression of viperin was induced by poly(I:C) and interferon treatment in camel kidney cell lines. Viperin expression was subdued in camel kidney cells infected with the camelpox virus during the early stages of infection, suggesting possible suppression by the virus. Overexpression of camel viperin through transient transfection significantly enhanced the resistance of cultured camel kidney cell lines to infection with camelpox virus. Research into the role of viperin in host immunity against emerging viral pathogens of camels will provide insight into novel mechanisms of antiviral activity of the protein, viral immune evasion strategies, and enable the development of better antivirals.

The last two decades have seen the emergence of three highly pathogenic coronaviruses with zoonotic origins, which prompted immediate attention to the underlying cause and prevention of future outbreaks. Intensification of camel husbandry in the Middle East has resulted in increased human-camel interactions, which has led to the spread of potentially zoonotic viruses with human spillover risks like MERS-coronavirus, camelpox virus, etc. Type-I interferons function as the first line of defense against invading viruses and are pivotal for limiting viral replication and immune-mediated pathologies. Seven novel dromedary camel interferon delta genes were identified and cloned. Functional characterization of this novel class of IFNs from the mammalian suborder tylopoda is reported for the first time. The camel interferon-delta proteins resemble the reported mammalian counterparts in sequence similarity, conservation of cysteines, and phylogenetic proximity. Prokaryotically expressed recombinant camel interferon-δ1 induced IFN-stimulated gene expression and also exerted antiviral action against camelpox virus, an endemic zoonotic virus. The pre-treatment of camel kidney cells with recombinant camel IFN-δ1 increased cell survival and reduced camelpox virus in a dose-dependent manner. The identification of novel IFNs from species with zoonotic spillover risk such as camels, and evaluating their antiviral effects in-vitro will play a key role in improving immunotherapies against viruses and expanding the arsenal to combat emerging zoonotic pathogens.

The COVID-19 pandemic is a wake-up call on the zoonotic viral spillover events and the need to be prepared for future outbreaks. Zoonotic RNA viruses like the Middle East respiratory syndrome coronavirus (MERS-CoV) are potential pathogens that could trigger the next pandemic. Dromedary camels are the only known animal source of MERS-CoV zoonotic infections, but little is known about the molecular antiviral response in this species. IFN-β and other type-I interferons provide the first line of defense against invading pathogens in the host immune response. We identified the IFNB gene of the dromedary camel and all extant members of the family Camelidae. Camelid IFN-β is unique with an even number of cysteines in the mature protein compared to other eutherian mammals with an odd number of cysteines. The viral mimetic poly(I:C) strongly induced IFN-β expression in camel kidney cells. Induction of IFN-β expression upon infection with camelpox virus was late and subdued when compared to poly(I:C) treatment. Prokaryotically expressed recombinant dromedary IFN-β induced expression of IFN-responsive genes in camel kidney cells. Further, recombinant IFN-β conferred antiviral resistance to camel kidney cells against the cytopathic effects of the camelpox virus, an endemic zoonotic pathogen. IFN-β from this unique group of mammals will offer insights into antiviral immune mechanisms and aid in the development of specific antivirals against pathogens that have the potential to be the next zoonotic pandemic.

Investigations into the molecular immune response of dromedary camel, a key livestock species of the arid, have been limited due to the lack of species-specific reagents. Here we describe for the first time, the identification and characterization of type I IFNs of dromedary camel, which are the most important cytokines in the innate host immune response against viruses. We cloned camel IFN-α coding sequences and identified a total of eleven subtypes. The canonical IFN-α subtype designated as IFN-α1 contained a 555-bp Open Reading Frame encoding a protein of 184 amino acids. Recombinant IFN-α1 protein was produced in E. coli and purified from inclusion bodies. Recombinant camel IFN-α1 induced the mRNA expression of interferon-stimulated genes (ISGs) in camel kidney cells. The purified protein also showed potent in-vitro antiviral activity against Camelpox Virus in kidney cells. The identified camel IFN-α protein and the subtypes will facilitate a better understanding of the host immune response to viral infections in camel and the development of potential antiviral biologicals for zoonotic diseases for which camel act as a reservoir.

Accurate diagnosis at an early stage of infection is essential for the successful management of any contagious disease. The coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus is a pandemic that has affected 214 countries affecting more than 37.4 million people causing 1.07 million deaths as of the second week of October 2020. The primary diagnosis of the infection is done either by the molecular technique of RT-qPCR by detecting portions of the RNA of the viral genome or through immunodiagnostic tests by detecting the viral proteins or the antibodies produced by the host. As the demand for the test increased rapidly many naive manufacturers entered the market with novel kits and more and more laboratories also entered the diagnostic arena making the test result more error-prone. There are serious debates globally and regionally on the sensitivity and specificity of these tests and about the overall accuracy and reliability of the tests for decision making on control strategies. The significance of the test is also complexed by the presence of asymptomatic carriers, re-occurrence of infection in cured patients as well as by the varied incubation periods of the infection and shifting of the viral location in the host tissues. In this paper, we review the techniques available for SARS-CoV-2 diagnosis and probable factors that can reduce the sensitivity and specificity of the different test methods currently in vogue. We also provide a checklist of factors to be considered to avoid fallacious practices to reduce false positive and false negative results by the clinical laboratories.

Interleukin-26 (IL-26) is a member of the IL-10 family of cytokines. Though conserved across vertebrates, the IL-26 gene is functionally inactivated in a few mammals like rat, mouse and horse. We report here the identification, isolation and cloning of the cDNA of IL-26 from the dromedary camel. The camel cDNA contains a 516 bp open reading frame encoding a 171 amino acid precursor protein, including a 21 amino acid signal peptide. Sequence analysis revealed high similarity with other mammalian IL-26 homologs and the conservation of IL-10 cytokine family domain structure including key amino acid residues. We also report the identification and cloning of four novel transcript variants produced by alternative splicing at the Exon 3-Exon 4 regions of the gene. Three of the alternative splice variants had premature termination codons and are predicted to code for truncated proteins. The transcript variant 4 (Tv4) having an insertion of an extra 120 bp nucleotides in the ORF was predicted to encode a full length protein product with 40 extra amino acid residues. The mRNA transcripts of all the variants were identified in lymph node, where as fewer variants were observed in other tissues like blood, liver and kidney. The expression of Tv2 and Tv3 were found to be up regulated in mitogen induced camel peripheral blood mononuclear cells. IL-26-Tv2 expression was also induced in camel fibroblast cells infected with Camel pox virus in-vitro. The identification of the transcript variants of IL-26 from the dromedary camel is the first report of alternative splicing for IL-26 in a species in which the gene has not been inactivated.

The mechanism by which type I interferon-mediated antiviral response is mounted by hosts against invading pathogen is an intriguing one. Of late, an endoplasmic reticulum transmembrane protein encoded by a gene called stimulator of interferon genes (STING) is implicated in the innate signalling pathways and has been identified and cloned in few mammalian species including human, mouse and pig. In this article, we report the identification of STING from three different species of a highly conserved family of mammals - the camelids. cDNAs encoding the STING of Old World camels - dromedary camel (Camelus dromedarius) and bactrian camel (Camelus bactrianus) and a New World camel - llama (Llama glama) were amplified using conserved primers and RACE. The complete STING cDNA of dromedary camel is 2171bp long with a 706-bp 5 untranslated regions (UTR), an 1137-bp open reading frame (ORF) and a 328-bp 3 UTR. Sequence and phylogenetic analysis of the ORF of STING from these three camelids indicate high level of similarity among camelids and conservation of critical amino acid residues across different species. Quantitative real-time PCR analysis revealed high levels of STING mRNA expression in blood, spleen, lymph node and lung. The identification of camelid STING will help in better understanding of the role of this molecule in the innate immunity of the camelids and other mammals.

Duck virus enteritis (DVE) also known as duck plague, is a viral infection of ducks caused by duck enteritis virus (DEV). The control of the disease is mainly done by vaccination with a chicken embryo-adapted live virus that is known to be poorly immunogenic and affords partial protection. Further, the risk of harboring other infectious agents in the embryo particularly the deadly and zoonotic avian influenza virus is also high. In this paper, we report propagation of a chicken embryo-adapted vaccine strain of duck enteritis virus in duck embryo fibroblast (DEF) cell line. Thirty serial passages were done in DEF cell that made the vaccine virus further attenuated which was tested in ducks. The growth behaviors of the virus in DEF cells were studied and at 30th passage level the virus titre was found to be 10(6.8) TCID(50)/ml. Ducks were immunized with this virus and challenged after 21 days with high dose of virulent DEV. All the immunized ducks withstood challenge with no clinical symptoms in any of the ducks while all the control ducks died. DEF cell which is free from other infectious agents appears to be a good system for cultivation of duck enteritis virus vaccine strain.

In the present study cytogenetic profiling and genetic characterization using microsatellite genotyping was carried out on Assamese swamp buffalo population. Microsatellite data analysis for 23 markers indicated substantial genetic variation existing in the buffalo population. Sufficient allelic diversity was observed with 133 alleles present across different loci. The genetic diversity analysis of Assamese buffaloes displayed moderate to high level of within population variability in terms of mean number of alleles per locus (5.78) and heterozygosity estimates, viz. mean observed (Ho = 0.618) and expected heterozygosity (He = 0.665). The Assamese buffalo population showed low heterozygote deficiency (F-IS=0.029). Mean polymorphic information content (PIC) across the 23 loci was 0.611, which indicated suitability of the marker panel for diversity analysis in these buffaloes. Bottleneck analysis indicated no recent genetic bottleneck in the Assamese buffalo population. Further, cytogenetic screening of 46 Assamese buffaloes indicated riverine (2n=50) or hybrid (2n=49) status. None of the animals had a typical pure swamp karyotype (2n=48). Thus, majority of the investigated animals were characterized as riverine type, which is in contrast to the general classification of Assamese buffaloes as swamp type derived from their morphological and behavioral similarity to swamp buffaloes.

India is the home of best much buffalo breeds in the world. Majority of them are riverine type found in almost the entire country extending from north to south and west. However buffalo populations in the eastern and north-eastern region are yet to be precisely classified, though these have been generally considered to be swamp type based on their phenotypic resemblance to swamp type buffaloes. Manipuri buffaloes, essentially reared for draught and meat purposes have been characterized phenotypically and cytogenetically in this study. Morphometric data collected from 56 adult buffaloes from hilly and plain areas revealed differences in their body measurements. Cytogenetic analysis of 40 randomly selected animals (7 males and 33 females) from both the locations revealed diploid chromosome count of 48, twenty-three pairs of autosomes and a pair of sex chromosomes, typical of swamp type buffaloes. Most distinctive karyotypic feature of swamp buffalo observed in all the animals was the size of the fourth pair of metacentric chromosome. Comparison of mitochondrial D-loop sequence of these buffaloes with representative riverine and swamp type buffaloes further corroborated this conclusion. This report, thus, is the first confirmed documentation of existence of pure swamp type buffaloes in Manipur state.

Signaling Lymphocyte Activation Molecule-SLAM (CD150) molecule has been reported as a putative receptor for most morbilliviruses for their respective host species. In this study, we determined the complete nucleotide sequence of the gene coding for the morbillivirus receptor-SLAM from the four species, namely, goat (Capra hircus), sheep (Ovis aries), Indian cattle (Bos indicus), and buffalo (Bubalus bubalis). The nucleotide (nt) open reading frame sequence of SLAM gene in all the four species studied was 1017 nucleotides in length encoding a polypeptide of 339 amino acids (aa), similar to Bos taurus, but different from canine, human, marmoset, and mouse SLAM, which were 1029, 1008, 1011, and 1032 nts, respectively, in length, and coding for 343, 336, 337, and 344 aa, respectively. Sequence analysis revealed 96.3–98.5% and 92.9–96.8% identities among the four species at the nt and aa level, respectively. Sequence diversity at aa level between various species revealed that the critical functional region of SLAM protein among different species is relatively conserved, thereby facilitating this molecule to act as a receptor for morbillivirus. Phylogenetic relationship based on the aa sequences of SLAM protein revealed that caprine, ovine, cattle, and buffalo fall under a defined cluster but caprine SLAM is more closely related to ovine, followed by bovine.

Objective: Mutations in connexin 26 gene (GJB2) are the most common cause of hearing loss in different populations. The aim of our study was to determine the prevalence of GJB2 mutations in the population of Kerala, India.Methods: This study was conducted on the genomic DNA of 86 affected subjects and their relatives from 59 families of Kerala, India. Mutation detection was done by sequencing and PCR-RFLP.Results: 36% of the probands had mutations in the GJB2 gene. We found that 45% (15/33) of the families that had a family history of deafness had mutations in GJB2 gene. Two different mutations were identified. W24X mutation was detected in 32.5% of the affected patients. Analysis of control samples revealed a carrier frequency of 0.0357 for this mutation. The estimation of haplotype frequency revealed that there was a significant association between the W24X mutation and the haplotype in this region with respect to the markers, D13S143 and D13S175 suggesting a founder effect for this mutation in this population. A novel mutation, R32L was detected in 3.5% of the affected patients. Structural prediction revealed that this mutation alters the helical structure of the first transmembrane domain of GJB2 protein resulting in defective gap junctions.Conclusion: Mutations in connexin26 is responsible for 36% of non-syndromic sensorineural deafness in the population of Kerala, India. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

The role of A1 and A2 beta casein milk variants and human health is a matter of concern for scientific investigations. The status of A1/A2 beta casein variants in Bos taurus cattle breeds from different countries have shown presence of A1 variant in European cattle, which has been linked to range of illness. However, no data on beta casein A1/A2 frequency is available on diverse Indian cattle (Bos indicus) breeds. Also no report is available on river buffalo breeds which contribute more than 55% of total milk produced in the country. In this study we report the frequency of beta casein variants among 618 animals of 15 zebu cattle breeds and 231 buffaloes of 8 river buffalo breeds. The beta casein A1/A2 frequency data indicated the predominance of A2 variant (0.987) in zebu cattle breeds while the river buffalo indicated only A2 milk type. The results point towards the origin of A2 variant in Bos indicus cattle. This is the first report of A1/A2 milk variant from majority of Indian zebu cattle and riverine buffaso breeds.

Purification of bluetongue virus (BTV) group-specific VP7 protein, expressed in prokaryotic system as histidine-tagged fusion protein is described in the present study. The major antigenic portion of VP7 gene of BTV 23 was amplified from the extracted RNA by reverse transcription polymerase chain reaction and cloned. The recombinant expression construct (pET-VP7) was identified by the polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion truncated VP7 protein with a molecular mass of 36 kDa was determined by Western blot analysis using anti-His antibody. The expressed VP7 was purified to near homogeneity by chromatography on nickel-agarose column as judged by sodium dodesyl sulfate-polyacrylamide gel electrophoresis analysis. The purified VP7 protein was recognized by antibody to BTV in Western blot analysis. The capability of the recombinant VP7 protein to differentiate hyperimmune serum of rabbit to BTV from normal rabbit serum was evident in the enzyme-linked immunosorbent assay (ELISA). The purified VP7 reacted well with the 24 BTV serotype-specific sera obtained from OIE Reference Laboratory on bluetongue. Our results indicated that the expressed VP7 protein could be used as antigen for development of antibody-capture ELISA for detection BTV group-specific antibodies. This recombinant protein may also be used as antigen in competitive ELISA format.

The livestock husbandry scenario during 2004-06 in some selected villages of Nainital district (Uttarakhand) was assessd from a total of 300 respondents. The information about the existing housing, breeding, milking, feeding and health care practices of livestock was documented. Most of the farmers in the study area kept their animals in kachha house having poor lighting and drainage facility. Breeding of animals is mainly through natural service with available bulls and the artificial insemination (AI) service was at the primitive level (21 %). The milking practice involved mostly stripping method and the farmers were very much (80%) aware of clean milk production and they sale their milk to dairy co-operatives. The livestocks were mostly dependent on the locally available feeding resources like oak leaves and unclassified grasses in the village grazing land/forest area for feeding of their livestock round the year. A very few farmers were aware of vaccination programme against important livestock diseases (HS, FMD, PPR, etc.). The major constraints in livestock development in the study area were - (i) deficiency of quality feed and folder, (ii) poor conception rate (from AI), (iii) prolonged age at first calving (4-6 years), (iv) unhygienic housing/resting place of animals, (v) poor disease management system, (vi) high morbidity and mortality in livestock, and (vii) little knowledge about vaccination against animal diseases. There is a need to introduce scientific animal health package and practices for adoption amongst small and marginal farmers. The scenario can be improved by providing quality feed and fodder to animals, sound health and management practices and suitable animal breeding practices, which are sustainable in the hill regions.

A trial using avermectins (ivermectin and doramectin) was conducted in crossbred cattle maintained at high altitude (> 7500 feet above MSL). Animals positive for gastrointestinal nematode infection (n=24) based on fecal egg counts were randomly divided into groups A, Band C of 8 animals each. Ivermectin and doramectin injection were given in all animals of group A and B, respectively, through subcutaneous route at the recommended dose while the animals of group C were kept as untreated control. Efficacy ofthe drugs was calculated on 14th day post treatment by fecal egg count reduction test (FECRT). Both ivermectin and doramectin were 100% effective against commonly occurring GI nematodes of cattle.

Introduction: Recently there has been an apparent increase of outbreaks due to foot-and-mouth disease (FMD) Asia 1. Spread has occurred into regions where this serotype has never or rarely been recorded People’s Republic of China (including Hong Kong), Russian Federation and Mongolia. To determine the relationships between FMD virus isolates from the various outbreaks, a phylogenetic study using complete VP1 gene sequences was undertaken. Materials and Methods: RNA extracted from either clinical samples or cell culture grown virus was subjected to RT-PCR using foot-and-mouth disease virus (FMDV)-specific primers targeting regions either side of the VP1 gene. The complete sequence of the VP1 gene was directly determined using standard automated sequencing techniques. Results: The VP1 region of more than 140 FMD Asia 1 viruses were amplified by RT-PCR and directly sequenced. Phylogenetic trees were generated by the Neighbor-joining method using the MEGA 3.1 software package. Recent virus isolates fell into six genetic groups. Discussion: FMDV serotype Asia 1 was recently responsible for multiple disease outbreaks throughout much of eastern Asia. Normally this serotype occurs in Southern and Southeast Asia and has been regularly reported in Afghanistan, India, Iran, Malaysia, Nepal, Pakistan and Thailand. However, at the end of 2004 through to 2006, in addition to some of these countries, outbreaks of Asia 1 were reported in several provinces or autonomous regions of the People’s Republic of China, Mongolia, Myanmar, several regions of eastern Russia, Tajikistan and Vietnam. Phylogenetic analysis of complete VP1 gene sequences from FMDV isolates responsible for these outbreaks showed that they were caused by viruses that belonged to six different lineages within the Asia 1 serotype. It appears from this study that some of these viruses have spread rapidly over large distances and are genetically very closely related to isolates collected 25 years ago in India.

A total of 366 faecal samples of goats (75 in summer, 173 in monsoon and 118 in winter) were collected in the year 2004–2005 and faecal eggs were estimated by modified McMaster egg counting technique. Data on nematode infection at experimental goat farm revealed 96.00, 87.28 and 98.30% incidence during summer, monsoon and winter, respectively. Eggs per gram (EPG) of GI nematodes in goats was higher during monsoon (1717) followed by summer (838) and winter (775). Tapeworm infection (Moniezia sp.) was found almost similar (about 10%) in all three seasons.