Induction profiles of mRNA of toll like receptors and cytokines in chickens pre-exposed to low pathogenic avian influenza H9N2 virus followed by challenge with highly pathogenic avian influenza H5N1 virus.

Herein, the induction of TLRs and cytokines in chickens pre-exposed to low pathogenic avian influenza H9N2 virus followed by challenge with highly pathogenic avian influenza (HPAI) H5N1 virus was studied. Four groups (1-4) of chickens inoculated with 106 EID50 of H9N2 virus were challenged with 106 EID50 of H5N1 virus on days 1, 3, 7 and 14 post H9N2 inoculation, respectively. In groups (1-4) TLRs and cytokines induction was studied in chicken PBMCs on day 3 post H5N1 challenge. In H5N1 control group TLRs (1, 2, 5 and 7) cytokines (IFNα, IFNß, IFNγ, IL1ß, IL2, IL4, IL8 and TGF ß3) were down regulated. In group 1 down regulation of cytokines and TLRs was similar to H5N1 control birds. Down regulation of TLRs and cytokines in H5N1 control and group 1 resulted death of all the chickens. In group 2, up-regulation of TLRs (3, 7 and 15) and induction of TNFα, IFNα, IFNß, IFNγ aided virus clearance leading to survival of all the chickens. In group 3 significant up-regulation of TLRs (3, 4 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL4, IL6, IL8, IL10 and TGF ß3) was detected. In group 4 significant up-regulation of TLRs (2, 3, 7 and 15) and significant induction of cytokines (IFNγ, TNFα, IL1ß, IL2, IL6, IL8 and IL10) was detected. In groups 3 and 4 simultaneous and significant induction of pro-inflammatory, antiviral and anti-inflammatory cytokine resulted cytokine dysregulation leading to death of (2/6) and (3/6) chickens respectively. Hence, the study revealed TLRs and cytokines role in modulating the H5N1 infection outcome in chickens pre-exposed to H9N2 virus.

Partial heterologous protection by low pathogenic H9N2 virus against natural H9N2-PB1 gene reassortant highly pathogenic H5N1 virus in chickens.

Low pathogenic avian influenza H9N2 and highly pathogenic avian influenza H5N1 viruses continue to co-circulate in chickens. Prior infection with low pathogenic avian influenza can modulate the outcome of H5N1 infection. In India, low pathogenic H9N2 and highly pathogenic H5N1 avian influenza viruses are co-circulating in poultry. Herein, by using chickens with prior infection of A/chicken/India/04TI05/2012 (H9N2) virus we explored the outcome of infection with H5N1 virus A/turkey/India/10CA03/2012 natural PB1 gene reassortant from H9N2. Four groups (E1-E4) of SPF chickens (n = 6) prior inoculated with 10(6) EID50 of H9N2 virus were challenged with 10(6) EID50 of H5N1 natural reassortant (PB1-H9N2) virus at days 1 (group E1); 3 (group E2); 7 (group E3) and 14 (group E4) post H9N2 inoculation. The survival percentage in groups E1-E4 was 0, 100, 66.6 and 50%, respectively. Virus shedding periods for groups E1-E4 were 3, 4, 7 and 9 days, respectively post H5N1 challenge. Birds of group E1 and E2 were shedding both H9N2 and H5N1 viruses and mean viral RNA copy number was higher in oropharyngeal swabs than cloacal swabs. In group, E3 and E4 birds excreted only H5N1 virus and mean viral RNA copy number was higher in most cloacal swabs than oral swabs. These results indicate that prior infection with H9N2 virus could protect from lethal challenge of reassortant H5N1 virus as early as with three days prior H9N2 inoculation and protection decreased in groups E3 and E4 as time elapsed. However, prior infection with H9N2 did not prevent infection with H5N1 virus and birds continue to excrete virus in oropharyngeal and cloacal swabs. Amino acid substitution K368E was found in HA gene of excreted H5N1 virus of group E3. Hence, concurrent infection can also cause emergence of viruses with mutations leading to virus evolution. The results of this study are important for the surveillance and epidemiological data analysis where both H9N2 and H5N1 viruses are co-circulating.

Recombinant nucleocapsid protein based single serum dilution ELISA for the detection of antibodies to infectious bronchitis virus in poultry.

Avian infectious bronchitis is ubiquitous and highly contagious disease of poultry, with profound effect on commercial poultry production. For effective control of infectious bronchitis virus (IBV), quick and specific diagnosis is of utmost importance. In this study, the virus was isolated from clinical samples from India and the full length nucleocapsid (N) gene was amplified, cloned and expressed in a prokaryotic system. The purified recombinant N protein based single serum dilution enzyme linked immunosorbent assay (ELISA) was developed for IBV to measure specific antibody in the sera of chickens. A total of 310 chicken sera samples were tested using the commercial IDEXX kit along with the assay developed. A linear correlation was obtained between predicted antibody titres at a single working dilution of 1:100 and the corresponding serum titres observed as determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The developed equation was then used to extrapolate predicated ELISA antibody titer from corrected absorbance readings of the single working dilution. The assay proved to be specific (95.8%) and sensitive (96.8%) when compared to the commercial IDEXX ELISA test.

Genotype characterization of commonly used Newcastle disease virus vaccine strains of India.

Newcastle disease is an avian pathogen causing severe economic losses to the Indian poultry industry due to recurring outbreaks in vaccinated and unvaccinated flocks. India being an endemic country, advocates vaccination against the virus using lentogenic and mesogenic strains. Two virus strains which are commonly used for vaccination are strain F (a lentogenic virus) and strain R2B (a mesogenic virus). Strain F is given to 0-7 days old chicks and R2B is given to older birds which are around 6-8 weeks old. To understand the genetic makeup of these two strains, a complete genome study and phylogenetic analysis of the F, HN genes of these vaccine strains were carried out. Both the viral strains had a genome length of 15,186 nucleotides and consisted of six genes with conserved complimentary 3' leader and 5' trailer regions. The fusion protein cleavage site of strain F is GGRQGRL and strain R2B is RRQKRF. Although both the viral strains had different virulence attributes, the length of the HN protein was similar with 577 amino acids. Phylogenetic analysis of F, HN and complete genome sequences grouped these two strains in genotype II category which are considered as early genotypes and corroborated with their years of isolation.

Egg Drop Syndrome-76 (EDS-76) in Japanese quails (Coturnix coturnix japonica): an experimental study revealing pathology, effect on egg production/quality and immune responses.

Egg Drop Syndrome-76 (EDS-76) is a recognized disease of chickens and Japanese Quails, which is of high economic importance due to its drastic negative effects on egg production in laying birds. The aim of the present study was to better understand the EDS-76 viral disease process in Japanese quails (Coturnix coturnix japonica), since very limited studies have been conducted in this species of birds. For this purpose, an experimental study was conducted with infection of EDS-76 virus in laying Japanese quails to reveal pathology, effect on egg production/quality and immune responses of this virus in these birds. By 7, 9 and 13-15 Days Post Infection (DPI), drop as well as aberrant egg production and lower mean egg quality were observed compared to control birds. Significant histopathological changes were observed in genitalia and spleen. Haemagglutination Inhibition (HI) and Enzyme Linked Immunosorbent Assay (ELISA) titres rose rapidly by 2nd week when it became maximum; thereafter declined and maintained at low levels up to 10 week post infection. The mean total protein values in infected quail gradually increased to 4.10±0.05/100 mL without any change in mean albumen value at 12 DPI. In conclusion, the course of the EDS-76 is significant not only in chickens but also in quails even though it occurs occasionally in quails. Explorative pathological, blood biochemical and immunological studies are suggested during EDS-76 viral disease course in quails. This would aid in formulating effective disease prevention and control measures for this economically important disease of poultry.

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis.

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.

Formation of subviral particles of the capsid protein VP2 of infectious bursal disease virus and its application in serological diagnosis.

Infectious bursal disease virus (IBDV) is an immunosuppressive disease of young chicken characterized by severe depletion of B-lymphocytes in the bursa of Fabricius. To provide antigen for diagnostic tests, its major structural protein VP2 was expressed in the yeast Saccharomyces cerevisiae. Electron microscopy of purified VP2 protein demonstrated that when expressed from yeast cells VP2 protein forms subviral particles (SVPs) of approximately 20nm in diameter. A recombinant VP2 antigen-based single serum dilution enzyme linked immunosorbent assay (ELISA) using the SVPs detected IBDV specific antibodies in chickens. A linear relationship was found between the predicted antibody titres at a single working dilution of 1:1000 and the corresponding observed serum titres when determined by the standard serial dilution method. Regression analysis was used to construct a standard curve from which an equation was derived which confirmed their correlation. The equation was then used to convert the corrected absorbance readings of the single working dilution directly into the predicted ELISA antibody titres. The assay proved to be sensitive, specific and accurate as compared to the serum neutralization test and agar gel immunodiffusion test. The recombinant VP2 antigen is a suitable alternative to whole viral antigen.