The present study was aimed to investigate the role of cannibalism in transmission of H5N1 avian influenza virus to house crows (Corvus splendens). Four crows were intranasally inoculated with 108.0 EID50 (A/crow/India/01CA249/2021) H5N1 highly pathogenic avian influenza (HPAI) virus and were observed for 14 days for any overt signs of illness. Two of the infected crows showed signs of wing paralysis, incoordination, and torticollis. For cannibalism experiment, two crows showing clinical signs were euthanized on 14th day post-infection (dpi) and were kept in the isolator and four naïve healthy crows were introduced along with the euthanized crows. The viscera from the infected carcasses were eaten by all the four crows. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. All four crows showed clinical signs viz., dullness, reluctance to move with ruffled feathers on 6th day post cannibalism along with neurological signs including incoordination and paralysis of the wings. All the crows gradually recovered after showing clinical signs and were euthanized on 21st day of observation period. Virus excretion was observed from 3rd to 11th day post cannibalism through both oropharyngeal and cloacal routes with maximum shedding through oropharyngeal route. The virus was isolated from lungs and trachea of one the infected crows at 21st day after euthanasia. All the four crows seroconverted against H5N1 virus infection at 14th day post cannibalism. Our study confirms the transmission of H5N1 virus in crows through cannibalism and highlights how H5N1 virus might circulate in a crow colony once they become infected.

We investigated the experimental infection of two highly pathogenic avian influenza H5N1 viruses isolated from crow (A/crow/Assam/142119/2008) and chicken (A/chicken/Sikkim/151466/2009) in house crows (Corvus splendens). Both viruses caused infection in crows, where four out of six and three out of six crows succumbed to H5N1 infection within 11 days post challenge by crow and chicken viruses, respectively. The major clinical signs in crows were wing paralysis, circling and torticollis. The virus shedding detected from swabs was not persistent in both crow nor chicken viruses. Both viruses were isolated more frequently from oral swabs than from cloacal swabs. Both virus strains were isolated from brain, lungs, heart, liver, pancreas, spleen, large intestines of crows that succumbed to H5N1 infection. The surviving birds seroconverted in response to H5N1 virus infection. Microscopically, both viruses caused coagulative necrosis in pancreas and kidneys. Brain showed gliosis and neuronal degeneration. This experimental study highlights that crows could be infected with H5N1 viruses from different hosts with minor differences in pathogenicity. Therefore, it is imperative to carry out surveillance of highly pathogenic avian influenza H5N1 virus in synanthropic birds along with biosecurity measures to mitigate the H5N1 spread in poultry population. Keywords: chicken virus; crow virus; highly pathogenic avian influenza; house crows.

Porcine Reproductive and Respiratory Syndrome (PRRS), caused by PRRS virus (PRRSV), is one of the most important devastating diseases of pigs, characterized by reproductive failure in sows, and respiratory disease with heavy mortality in piglets. PRRS virus has been reported to elevate the levels of proinflammatory cytokines in the serum of infected pigs. High Mobility Group Box-1 (HMGB-1) protein is a cellular biomolecule belonging to the Danger Associated Molecular Patterns (DAMP) family, which stimulates immune cells to release pro inflammatory cytokines upon release out of cells. The role of HMGB-1 in the pathogenesis of PRRSV remains largely unknown. In the present study, HMGB-1 levels in serum samples collected from six-week-old piglets infected intra-nasally with 2 x 10(5.75) TCID50/mL of Indian PRRSV (Ind-297221/2013) was estimated by ELISA up to 21 days post infection (dpi). Pro-inflammatory cytokine mRNA (IL-1 beta, IL-6 and TNF- alpha) expression in PBL was estimated by SYBR green based real time PCR. Mean HMGB-1 concentration in serum was found to be significantly elevated in PRRSV infected piglets on 6 dpi as compared to uninfected control piglets. At mRNA level, significant increase in expression of HMGB-1 was observed from 4 to 5 dpi and from 11 to 13 dpi. IL-1 beta and IL-6 mRNA were significantly upregulated between 4 and 6 dpi. Significant increase in TNF-alpha gene expression was seen only on 7 and 9 dpi. Higher levels of pro-inflammatory cytokines and HMGB-1 could be correlated with fever which was observed within 7 dpi in all the infected piglets and additionally around 13 dpi in the animal that died on 17 dpi. Thus, elevated HMGB-1 level in PRRSV infected piglets could be correlated with concurrent increase in pro-inflammatory cytokine (IL-6) mRNA. In-vitro studies were conducted in PRRSV infected Porcine Pulmonary Alveolar Macrophages (PAM) to ascertain HMGB-1 role in PRRS pathogenesis. The results of both in-vivo and in-vitro studies showed that HMGB-1 plays an important role in mediating the pro inflammatory cytokine responses in PRRS pathogenesis.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important transboundary viral disease of pigs confronting the swine industry worldwide. This study was aimed to assess the pathogenic potential of PRRS virus belonging to genotype 2 that emerged in India in 2013. Nine 6-week-old piglets were inoculated intranasally with 2 x 10(5.75) TCID50/ml of PRRSV (Ind-297221/2013). Three piglets were kept as uninfected controls. Blood and nasal swabs were collected daily up to 7 days post-infection (dpi) and on alternate days subsequently. Piglets were necropsied for tissue sample collection either on death or after euthanasia on 7, 14 or 21 dpi (one uninfected control and three PRRSV-infected piglets per interval). The virus caused high fever, typical blue ear, weight loss, respiratory distress, diarrhoea and leucopenia between 2 and 8 dpi. Two infected piglets died (on 3 and 17 dpi) during the course of study. The presence of virus in serum and nasal secretion was observed up to 19 and 17 dpi, respectively, with the maximum load between 4 and 7 dpi. Seroconversion started 6 dpi and the mean PRRSV antibody titre reached up to 640 by 21 dpi. Virus load was highest in tonsils at all the intervals, whereas in spleen and lymph nodes load was higher in later intervals. Major microscopic lesions in PRRSV-infected piglets included moderate to severe interstitial pneumonia, lymphoid depletion in tonsils and lymph nodes (cystic), thymic atrophy, reactive hyperplasia followed by lymphoid depletion in spleen. PRRSV antigen was consistently demonstrated by immunoperoxidase test in the lungs, spleen, tonsils and lymph nodes. Antigen distribution was more widespread on 7 and 14 dpi than on 21 dpi. The findings establish that the Indian PRRSV is highly pathogenic to piglets.

A total of 160 day-old broiler chicks were divided randomly into four groups with two replicates of 20 birds each. The birds were fed with basal diet, basal diet with CFO, basal diet with aflatoxin, basal diet with CFO and AF. Serum globulin level was significantly reduced in birds fed with AF only on 14th day of treatment. Microscopically, mild degree of congestion, histiocytosis and cellular sparcity of the periarteriolar lymphoid component were observed in the spleen whereas lymphoid cell depletion in thymus and bursal follicles were prominent. Additionally, thymus revealed moderate degree of congestion and haemorrhages in widened medulla, histiocytosis and degeneration of Hassall's corpuscles. In CFO and AF fed birds, the extent and degree of lesions were lesser with compact arrangement of lymphoid cell with widening of cortex was observed mainly during later days of experiment. There was widening of lymphoid component of caecal tonsils up to mucosal layer in CFO fed birds.

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.

The study was designed to characterize and compare chicken bone marrow and peripheral blood monocyte derived dendritic cells (chBM-DC and chMoDC) and to evaluate inflammatory cytokine and chemokine alterations in response upon LPS stimulation. Typical morphology was observed in DCs from 48h of culture using recombinant chicken GM-CSF and IL-4. Maturation of DCs with LPS (1μg/ml) showed significant up regulation of mRNA of surface markers (CD40, CD80, CD83, CD86, MHC-II and DC-LAMP (CD208)), pro-inflammatory cytokines (IL-1β, IL-6, TNF-α (LITAF)), iNOS, chemokine CXCli2 and TLRs4 and 15. Basal level of TLR1 mRNA expression was higher followed by TLR15 in both DCs irrespective of their origin. Expression of iNOS and CXCLi2 mRNA in mature DCs of both origins were higher than other surface molecules and cytokines studied. Hence, its level of expression can also be used as an additional maturation marker for LPS induced chicken dendritic cell maturation along with CD83 and CD40. LPS matured DCs of both origins upregulated IL-12 and IFN-γ. Based on CD40 and CD83 mRNA expression, it was observed that LPS induced the maturation in both DCs, but chMoDCs responded better in expression of surface markers and inflammatory mediator genes.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging zoonotic disease in India, first reported from Gujarat, in December 2010. In the present study, CCHFV genome was detected in Hyalomma ticks infesting cattle collected in early 2011, from Undani village, of Mehsana district of Gujarat, India under post human CCHF outbreak surveillance activity. The full-length ‘S’ segment of CCHFV was amplified using one-step RT-PCR, from total RNA isolated from these ticks. The 1673 bp amplicon was further cloned in to pGEM®-T Easy vector and confirmed by restriction digestion. This full length ‘S’ segment was sequenced and sequence was submitted to NCBI GenBank database under Accession No JX051650.