Complete genome sequencing and evolutionary analysis of dengue virus serotype 1 isolates from an outbreak in Kerala, South India.

In this study, dengue virus (DENV) isolates from a localized, small-scale, non-seasonal dengue outbreak were genetically characterized. The outbreak occurred during the pre-monsoon months (April-May) in a medical college campus in Kerala, South India in 2009 affecting 76 people. Analysis of 39 viral RNA positive serum samples by a serotype specific reverse-transcription polymerase chain reaction identified dengue virus serotype 1 (DENV1) as the causative strain. Formation of a distinct genetic clade was revealed in the initial phylogenetic analysis using nucleotide sequences of a partial (303 bp) Capsid-Pre-membrane protein (C-PrM) coding region of 37 outbreak strains. The sequences of these strains clustered with that of the Genotype III DENV-1 strains from India, and 32 among them formed a single major sub-clade. Whole-genome sequencing (10,693 bp) of two strains (RGCB585/2009 and RGCB592/2009) selected from this major sub-clade, and subsequent phylogenetic analysis using the full-length coding region sequence showed that the sequences grouped with that of the isolates from Thailand (1980), Comoros (1993), Singapore (1993), and Brunei (2005) among the Indo-Pacific isolates. The sequences of the two strains had a nucleotide identity of 97-98 % and an amino acid identity of 98-99 % with these closely related strains. Maximum amino acid similarity was shown with the Singapore 8114/93 isolate (99.6 %). Four mutations-L46M in the capsid, D278N in the NS1, L123I, and L879S in the NS5 protein coding regions-were seen as signature substitutions uniformly in RGCB585/2009 and RGCB592/2009; in another isolate from Kerala (RGCB419/2008) and in the Brunei isolate (DS06-210505). These four isolates also had in common a 21-nucleotide deletion in the hyper-variable region of the 3'-non-translated region. This first report on the complete genome characterization of DENV-1 isolates from India reveals a dengue outbreak caused by a genetically different viral strain. The results point to the possibility of exotic introduction of these circulating viral strains in the region.

Correction: Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0059350.].

Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction.

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. RESULTS: Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. CONCLUSION: Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

Genetic characterization of 2006-2008 isolates of Chikungunya virus from Kerala, South India, by whole genome sequence analysis.

Chikungunya virus (CHIKV), a positive-stranded alphavirus, causes epidemic febrile infections characterized by severe and prolonged arthralgia. In the present study, six CHIKV isolates (2006 RGCB03, RGCB05; 2007 RGCB80, RGCB120; 2008 RGCB355, RGCB356) from three consecutive Chikungunya outbreaks in Kerala, South India, were analyzed for genetic variations by sequencing the 11798 bp whole genome of the virus. A total of 37 novel mutations were identified and they were predominant in the 2007 and 2008 isolates among the six isolates studied. The previously identified E1 A226V critical mutation, which enhances mosquito adaptability, was present in the 2007 and 2008 samples. An important observation was the presence of two coding region substitutions, leading to nsP2 L539S and E2 K252Q change. These were identified in three isolates (2007 RGCB80 and RGCB120; 2008 RGCB355) by full-genome analysis, and also in 13 of the 31 additional samples (42%), obtained from various parts of the state, by sequencing the corresponding genomic regions. These mutations showed 100% co-occurrence in all these samples. In phylogenetic analysis, formation of a new genetic clade by these isolates within the East, Central and South African (ECSA) genotypes was observed. Homology modeling followed by mapping revealed that at least 20 of the identified mutations fall into functionally significant domains of the viral proteins and are predicted to affect protein structure. Eighteen of the identified mutations in structural proteins, including the E2 K252Q change, are predicted to disrupt T-cell epitope immunogenicity. Our study reveals that CHIK virus with novel genetic changes were present in the severe Chikungunya outbreaks in 2007 and 2008 in South India.

Genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in Ernakulam, Kerala, South India.

Dengue fever, a mosquito-borne viral infection, causes significant morbidity and has become endemic in the Indian subcontinent. Virus strains currently circulating in many parts of the country are not well studied at the molecular level. In the present study, genetic characterization of virus strains from a dengue outbreak that occurred in and around a tertiary care hospital in Ernakulam, Kerala in the year 2008 has been reported. By reverse transcription polymerase chain reaction (RT-PCR), 37 out of 75 (49.3%) clinically suspected cases were positive for dengue viral RNA. Among these, 21 (56.8%) samples showed concurrent infection with multiple serotypes of the virus. Majority of the combined infections were caused by dengue serotype 2 and 3. Co-infections with type 1 and 2 in two patients, and type 1, 2 and 3 in one patient were also observed. The core-pre-Membrane (CprM) junction nucleotide sequencing and phylogenetic analysis revealed that the type 1 strains were related to the viral strains reported from Delhi-2001 and Gwalior-2002 dengue outbreaks, while the type 2 strains were related to the strains from Gwalior-2001 epidemic. Sequences of type 3 strains did not show clear relation to any of the previous Indian isolates, and in the phylogenetic analysis, they formed a distinct lineage within the Indian type 3 strains. This study indicates hyperendemicity of dengue in the region with the presence of multiple serotypes and high rates of co-infection, and local genomic evolution of the viral strains involved in this outbreak.

Molecular characterization and expression of interferon-gamma of Asian elephant (Elephas maximus).

Tuberculosis (TB) caused by Mycobacterial organisms has emerged as one of the major diseases in captive elephants. In vitro Interferon-gamma (IFN-gamma) assay is being used as an ancillary test for early detection of TB in domestic and captive wild animals. In the present study, basic sequence information and immunological cross-reactivity of this major cytokine of Asian elephants were explored. At predicted amino acid level, IFN-gamma of Asian elephant showed maximum identity to that of horse (73%). Other IFN-gamma amino acid sequences that showed high level identity were that of giant panda (72%), dog (71%), nine-banded armadillo (69%), cattle (63%) and human (62%). IFN-gamma promoter sequences of Asian elephant, human, cattle and mouse showed high level conservation of the putative transcription factor binding sites, TATA box and transcriptional start site. The functionally important human IFN-gamma promoter elements, such as AP-2IRE-BE, YY1-gammaIFN-BED, ATFCS and AP-1gammaINF binding sites, were absolutely conserved in the corresponding elephant sequence. There was only a single nucleotide variation in the other two important elements, NFAT-gammaINF and IFN-gammaPE, indicating the highly conserved regulation of IFN-gamma expression across different species. Phylogenetic analysis based on IFN-gamma protein sequences revealed a closer relation of Asian elephants and nine-banded armadillo. This shows a closer evolution of these members of Afrotheria and Xenarthra, respectively; and supports the previous reports based on mitochondrial DNA studies. In Western blot analysis, IFN-gamma of Asian elephant expressed in Escherichia coli was detected using an anti-bovine IFN-gamma monoclonal antibody, indicating immunological cross-reactivity.

Cloning and biological characterization of buffalo (Bubalus bubalis) interferon-gamma.

Interferon-gamma, a major immunomodulatory cytokine, of Indian water buffalo (Bubalus bubalis) was characterized at molecular level. Complementary DNA and essential promoter region were cloned and sequenced, and functional recombinant protein was expressed in bacterial system. The cDNA has 97.8% nucleotide identity with 11-nucleotide and four-amino acid variations, and the essential promoter region has 98.4% identity with five-nucleotide variations and a four-nucleotide deletion in comparison with the corresponding bovine sequences. All the major promoter elements such as NF IL-2 like motif, cyclosporin sensitive binding element and GATA motif are strictly conserved. Recombinant buffalo-IFN-gamma expressed in bacterial system reacted with an anti-bovine-IFN-gamma monoclonal antibody in Western blot and showed antiviral activity against buffalo pox virus in cultured Madin-Darby bovine kidney (MDBK) cells by inhibiting virus induced cytopathic effect. The study shows high level sequence similarity of IFN-gamma among ruminants. In view of the immunomodulatory and antiviral activities of IFN-gamma, this molecule will be useful in better understanding of the immune system of water buffaloes.

Buffalo (Bubalus bubalis) interleukin-12: analysis of expression profiles and functional cross-reactivity with bovine system.

Interleukin-12, a heterodimeric pro-inflammatory cytokine, from water buffaloes (Bubalus bubalis) was analyzed for its for its tissue specific expression and functionality. Concanavalin A stimulated splenocytes displayed an up-regulation of the IL-12 p40 subunit 8-24h post-stimulation, whereas the p35 subunit did not show any quantitative variation at different time intervals. Basal level expressions of both the subunits were observed by RT-PCR in spleen. In addition p40 transcripts could be detected in liver and p35 in brain and muscle tissues as well in very low levels. Functional recombinant buffalo IL-12 was expressed in HEK 293T cells as a heterodimer using foot-and-mouth disease virus 2A polypeptide as a linker. Culture supernatants from transfected cells contained a hetero-dimeric p70 subunit as revealed in western blot of the proteins separated by native polyacrylamide gel electrophoresis (PAGE) using a monoclonal antibody against bovine IL-12 p40. IL-12 containing culture supernatant induced production of nitric oxide in cultured splenocytes of both buffalo and bovine origin. Our study reveals that buffalo IL-12, which shares a high-level sequence identity with bovine IL-12, also has functional cross-reactivity with the bovine immune cells.

Molecular characterization and prokaryotic expression of buffalo (Bubalus bubalis) Interleukin-6.

The study describes the characterization of Interleukin-6 cDNA and essential promoter sequences of the Indian Water Buffalo (Bubalus bubalis) and expression of the recombinant IL-6 in Escherichia coli. Buffalo IL-6 shows very high nucleotide level identity of the cDNA (98.7%) and promoter (98%) sequences with the corresponding cattle sequences. All the major regulatory elements of IL-6 promoter like AP-1, Multiple Response Element, NF-IL6, ETS binding domain and NF-kappaB binding sites show absolute conservation. Basal level IL-6 mRNA is detected in organs like liver, lung and spleen. Concanavalin A stimulated splenocytes produced maximum IL-6 mRNA at 8h poststimulation. Recombinant IL-6 production in JM109 (DE3) and BL21 (DE3) pLysS bacterial system is substantially enhanced by supplementation of rare codon tRNAs through co-transformation with a second plasmid. BL21 (DE3) pLysS strain is a more efficient producer of the IL-6 as it expressed two-fold more protein than by JM109 (DE3) cells. The study shows high-level conservation of IL-6 regulatory and coding sequences between cattle and buffalo, and indicates the use of a common reagent for studying the effects of this cytokine in these species.

Inhibition of Anatid Herpes Virus-1 replication by small interfering RNAs in cell culture system.

RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. It is projected as a potential tool to inhibit viral replication. In the present paper, we demonstrate the suppression of replication of an avian herpes virus (Anatid Herpes Virus-1, AHV-1) by siRNA mediated gene silencing in avian cells. The UL-6 gene of AHV-1 that codes for a protein involved in viral packaging was targeted. Both cocktail and unique siRNAs were attempted to evaluate the inhibitory potential of AHV-1 replication in duck embryo fibroblast (DEF) cell line. DEF cells were chemically transfected with different siRNAs in separate experiments followed by viral infection. The observed reduction in virus replication was evaluated by cytopathic effect, viral titration and quantitative real time PCR (QRT-PCR). Among the three siRNA targets used the unique siRNA UL-B sequence was found to be more potent in antiviral activity than the cocktail and UL6-A-siRNA sequences.

Molecular cloning and expression profile analysis of interleukin-10 and interleukin-18 cDNA of Indian water buffalo (Bubalus bubalis).

The cDNAs encoding the interleukin-10 and interleukin-18 of Indian water buffalo (Bubalus bubalis) were cloned and sequenced. A 537 bp IL-10 cDNA fragment and a 623 bp IL-18 cDNA fragment were amplified by reverse transcriptase polymerase chain reaction (RT-PCR) from concanavalin A stimulated splenocytes. Sequence analysis of these cytokines revealed high level conservation at nucleic acid and protein level. Both these cytokines also showed strict conservation in the predicted secondary structure and critical amino acid residues compared to the ruminant homologues. Basal level expression of both IL-10 and IL-18 was observed in liver, lung and spleen. The expression level of IL-10 was not affected by mitogenic stimulation, whereas IL-18 was up regulated upon stimulation. The availability of these cytokine molecules will aid in the study of their role in the immunology and pathogenesis of infections in water buffalo.

Cloning and sequencing of truncated gIV glycoprotein gene of an Indian isolate of bovine herpesvirus 1.

Bovine herpesvirus 1 (BHV-1) has been reported from the Indian subcontinent in late 70's. In order to identify the origin of an Indian isolate of BHV-1 (IBR/H 167 VS) and its molecular relationship to known strains of BHV-1, a 680 bp region of the glycoprotein gene gIV was amplified by polymerase chain reaction (PCR), cloned and sequenced. Comparison of this sequences with the corresponding one of an European strain of BHV-1 (Cooper) revealed more than 99% nucleotide (nt) homology. We conclude that the Indian isolate under study does not differ from the Cooper strain regarding the gIV gene.

Mycobacterium phlei as an oral immunomodulator with Newcastle disease vaccine.

Experiments were conducted in chickens to understand the effects of oral immunomodulation. Heat inactivated M phlei, a commensal Mycobacterium and a non-specific immunomodulator, was administered orally prior to live Newcastle disease F (ND F) strain vaccination. In experimental birds it lead to an enhanced cell mediated Immune response (CMI) against the vaccine. There was a reduction in the Haemagglutination inhibiting (HI) antibodies. However, it did not affect the protection against a virulent challenge, as the protection percentage was more or less same in vaccinated birds irrespective of the M.phlei administration. M. phlei administration could not enhance the immune response to inactivated ND F vaccine administered orally. The results indicate that M. phlei favours a CMI response to orally administered live ND F vaccine. It may be of potential use in enhancing CMI against vaccines and a cheaper alternative to costlier recombinant cytokines.

Functional characterization of the CC chemokine RANTES from Pekin duck (Anas platyrhynchos).

RANTES (Regulated upon Activation, Normal T-cell Expressed and Secreted) is a key pro-inflammatory cytokine that belongs to the CC-group of chemokines. The present study was carried out to functionally characterize the previously identified RANTES homologue in domestic duck (GenBank Accession No. AY641435). Recombinant duck RANTES was expressed in Escherichia coli-based and HEK293T cell-based systems. A tRNA supplementation strategy was required to express the protein in E. coli due to the presence of rare codons. In biological assays using HEK293T cell-expressed protein, RANTES was found to mediate chemotaxis of DT-40 chicken B cells and primary duck splenocytes at a concentration of 0.505µg/ml (0.6µM). Immunostaining of the migrated splenocytes using anti-duck CD4 and CD8 monoclonal antibodies and subsequent flow cytometric analysis showed enhanced chemotaxis of CD8+ cells. The recombinant RANTES exhibited in vitro antiviral activity by inhibiting infection of chicken embryo fibroblast cells with duck enteritis virus (DEV) at the same concentration. The effect could be neutralized by rabbit anti-duck RANTES polyclonal serum. The mechanism seems to be direct on viral particles as evidenced by the need for co-incubation of RANTES with DEV prior to the infection for antiviral activity, and also by the enhanced binding of DEV to E. coli expressed purified RANTES on ELISA-based assays. Our results show that the duck RANTES has overlapping biological properties with its mammalian orthologue, and also has possible functional cross-reactivity with chicken immune cells indicated by the chemotaxis of DT-40 cells.

Molecular evidences for the chemosensitizing efficacy of liposomal curcumin in paclitaxel chemotherapy in mouse models of cervical cancer.

The microtubule-targeting antineoplastic agent, paclitaxel, is highly efficacious against a wide spectrum of human cancers. However, dose-limiting toxicity and development of drug resistance limit its clinical application. Development of novel strategies that overcome chemoresistance and sensitize cancer cells to paclitaxel can enhance the therapeutic effect of this drug. We have previously shown that curcumin, a natural polyphenol, enhances paclitaxel-induced cytotoxicity in vitro through downregulation of nuclear factor (NF)-κB and Akt pathways. This study was undertaken to determine whether this synergism exists in vivo and to elucidate the underlying molecular mechanisms. Mouse cervical multistage squamous cell carcinoma model using 3-methylcholanthrene (3-MC) and a xenograft model of human cervical cancer in nonobese diabetic severe combined immunodeficient (NOD-SCID) mice using HeLa cells were used to evaluate the synergism. We observed that the combined treatment of curcumin and paclitaxel induced a synergestic reduction in the tumor incidence as well as tumor volume of animals compared with the individual treatments of paclitaxel or curcumin, although curcumin alone could not induce any significant effect at the concentration used. The results suggest that a suboptimal concentration of curcumin augments the antitumor action of paclitaxel by downregulating the activation and downstream signaling of antiapoptotic factors and survival signals such as NF-κB, Akt and mitogen-activated protein kinases that have significant roles in proliferation, survival, angiogenesis and metastasis. This study revealed for the first time that 3-MC-induced tumorigenesis in mice is associated with a strong constitutive activation of NF-κB activity. Furthermore, we also observed that pre-exposure of carcinoma cells isolated from 3-MC-induced tumors to curcumin potentiates paclitaxel-induced apoptosis. Overall, the findings of this preclinical study provide a strong rationale for the validation of this combination through clinical trials. As curcumin could effectively downregulate all these survival signals induced by paclitaxel, we suggest it as a potent chemosensitizer to improve the therapeutic index of paclitaxel.

Discovery of Anas platyrhynchos avian beta-defensin 2 (Apl_AvBD2) with antibacterial and chemotactic functions.

The cationic, cysteine-rich peptides called beta-defensins play a major role in the innate immune response. Here, we describe the identification and characterization of the duck beta-defensin-2 homologue, Anas platyrhynchos avian beta-defensin 2 (Apl_AvBD2). The 195 base pair open reading frame (ORF) of Apl_AvBD2 has 83% identity with Gga_AvBD2 (chicken) and 85% identity with Mga_AvBD2 (turkey) at nucleotide level. The gene corresponding to the coding region is comprised of three exons and two introns in both Apl_AvBD2 and Gga_AvBD2. The predicted secondary structure of Apl_AvBD2 has the classical "beta-defensin core motif" formed by the beta-sheet rich structure. Apart from mild expression in tissues like kidney, lung, brain, bursa of Fabricious and ovary, Apl_AvBD2 mRNA show a very high level constitutive expression in bone marrow and spleen, indicating that it is a myeloid defensin. Purified recombinant Apl_AvBD2 demonstrated in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria, with a minimum bactericidal concentration (MBC) of 3.7 microM against Micrococcus luteus NCIM 2871 and Escherichia coli NCIM 2685, and of 2.2 microM against Reimerella anatipestifer. The immunomodulatory potential of Apl_AvBD2 was shown by chemotaxis of DT-40 chicken B-lymphocytes. The widespread tissue distribution and the potent bactericidal and chemotactic activity make Apl_AvBD2 an important molecule in the innate immune response in ducks. It may play a vital role in the immune response of these birds against bacterial and viral pathogens.

Immunomodulation by duck defensin, Apl_AvBD2: in vitro dendritic cell immunoreceptor (DCIR) mRNA suppression, and B- and T-lymphocyte chemotaxis.

The present study analyzed the immunomodulatory potential of a newly identified duck beta-defensin, Apl_AvBD2. Recombinant Apl_AvBD2 expressed in HEK293T cells induced a concentration dependent in vitro migration of duck splenocytes, and spleen B- and T-lymphocytes, which was specifically inhibited by anti-Apl_AvBD2 polyclonal antibodies. Among the transcripts of 13 immunologically important genes analyzed in cultured splenocytes for the early immunomodulatory effect of Apl_AvBD2, dendritic cell immunoreceptor (DCIR) mRNA was found to be significantly down-regulated. However, there were no major changes in the expression levels of transcripts for cell surface proteins (MHC I, MHC II 2 beta-chain, TCR-beta, TLR-7, DCAR, CD44, and CD58) and cytokines (IL-2, IFN-gamma, RANTES, MIP-1beta-like and MCP-1 like chemokines). Our results reveal chemotactic and immunomodulatory properties of Apl_AvBD2, two important functions that would help in employing this protein as a molecular adjuvant with avian vaccines.

Duck (Anas platyrhynchos), Japanese quail (Coturnix coturnix japonica) and other avian interleukin-2 reveals significant conservation of gene organization, promoter elements and functional residues.

We compared the gene, promoter and cDNA sequences, and the predicted protein structure of duck and quail interleukin-2 (IL-2), a major immunomodulatory cytokine, with the known sequences of other avian and human IL-2. Analysis of the gene organization showed significant similarity with the overall organization of mammalian IL-2 genes, with four exons and three introns and a very short 5' untranslated regions. The second intron was the biggest in all the IL-2 sequences. The third intron was of similar size in chicken and quail, whereas in duck it was found to be slightly longer. Promoter sequence analysis of the IL-2 gene revealed remarkable conservation of the functionally important residues. The transcription factor binding sites such as those for AP-1, NF-AT, CD 28 RE and OCT, the TATA box and the predicted transcription start site with respect to chicken IL-2 sequence showed total conservation in duck, quail and turkey IL-2 promoters. Comparative analysis of the avian IL-2 cDNAs such as those of chicken, turkey, duck, quail, goose and Muscovy duck, revealed significant conservation of the nucleotide and predicted amino acid sequences. They showed nucleotide identity levels varying from 75% to 85%, amino acid level identity from 58% to 72% and amino acid similarity from 71% to 80% with each other. In the predicted protein secondary structure, the four essential alpha-helices and the hydrophobic amino acids in the heptad repeats forming the core structure of IL-2 molecules were conserved in all the avian and the human IL-2. The present study reveals high-level conservation of the gene; cDNA structure and regulatory elements of avian IL-2. This indicates highly conserved functions and probable functional cross-reactivity of this major immunomodulatory cytokine among birds.

Interleukin-12 subunits p35 and p40 of Indian water buffalo (Bubalus bubalis) maintain high sequence homology with those of other ruminants.

The immune system of Indian water buffalo (Bubalus bubalis), one of the major dairy animals of the tropics, has received little attention. cDNAs encoding the two subunits of the heterodimeric interleukin (IL)-12 of Indian water buffalo were isolated from concanavalin A-stimulated lymphocytes. The 710-bp p35 and 1012-bp p40 subunits were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), cloned, sequenced and compared with other ruminant sequences. The IL-12 p35 subunit cDNA had nine nucleotide variations and shared 98.1% amino acid identity with the cattle IL-12 p35. The IL-12 p40 cDNA had 13 nucleotide variations and had 97.5% amino acid identity with the cattle IL-12 p40. Both the subunits showed strict conservation in the predicted secondary structure and critical amino acid residues compared with other ruminant IL-12 molecules. Buffalo IL-12 p40 recombinant protein expressed in Escherichia coli cross-reacted with cattle anti-IL-12 p40 monoclonal antibody. Our study indicates a high level of conservation of this key cytokine among ruminants.