Molecular characterization of Indian races of Fusarium oxysporum f. sp. lentis (Fol) based on secreted in Xylem (SIX) effector genes and development of a SIX11 gene-based molecular marker for specific detection of Fol.

Fusarium wilt of lentil caused by Fusarium oxysporum f. sp. lentis (Fol) is a destructive pathogen limiting lentil production in India. In the present study, Secreted in Xylem (SIX) effectors genes were explored in Indian races of Fol and also a diagnostic tool for reliable detection of the disease was developed. Four SIX effectors genes, SIX11, SIX13, SIX6 and SIX2 were identified in 12 isolates of Fol belonging to seven races. SIX11 was present in all the races while SIX 13 was absent in race 6 and SIX6 was present only in race 4. The phylogenetic analysis revealed the conserved nature of the SIX genes within the forma specialis and showed sequence homology with F. oxysporum f. sp. pisi. The presence of three effectors, SIX11, SIX13 and SIX6 in race 4 correlates with high disease incidence in lentil germplasms. The in-silico characterization revealed the presence of signal peptide and localization of the effectors. Further SIX11 effector gene present in all the isolates was used to develop Fol-specific molecular marker for accurate detection. The marker developed could differentiate F. oxysporum f. sp. lycopersici, F. solani, F. oxysporum, Rhizoctonia solani and Sclerotium rolfsii and had a detection limit of 0.01ng µL- 1. The effector-based marker detection helps in the unambiguous detection of the pathogen under field conditions.

Expression profiling and characterization of key RGA involved in lentil Fusarium wilt Race 5 resistance.

Fusarium wilt is a major threat to lentil production in India and worldwide. The presence of evolving virulent races has imposed the necessity of reliable management practices including breeding for resistance using unexplored germplasms. The magnitude of resistance by the plant is determined by rapid recognition of the pathogen and induction of defence genes. Resistance gene analogues have been key factors involved in the recognition and induction of defence response. In the present study, the expression of key RGA previously cloned was determined in three resistant accessions (L65, L83 and L90) and a susceptible accession (L27). The expression was assessed via qPCR at 24, 48 and 72 hpi against virulent race5 (CG-5). All the RGAs differentially transcribed in resistant and susceptible accession showed temporal variation. RGA Lc2, Lc8, Ln1 and Lo6 produced cDNA signals during early infection (24 hpi) predicting its involvement in recognition. LoRGA6 showed significant upregulation in L65 and L83 while downregulating in L27 and the full length of LoRGA6 loci was isolated by 5' and 3' RACE PCR. In-silico characterization revealed LoRGA6 loci code for 912 amino acids long polypeptide with a TIR motif at the N terminal and eight LRR motifs at the C terminal. The tertiary structure revealed a concave pocket-like structure at the LRR domain potentially involved in pathogen effectors interaction. The loci have ADP binding domain and ATPase activity. This has further paved the path for functional analysis of the loci by VIGS to understand the molecular mechanism of resistance.

Unraveling Microbial Volatile Elicitors Using a Transparent Methodology for Induction of Systemic Resistance and Regulation of Antioxidant Genes at Expression Levels in Chili against Bacterial Wilt Disease.

Microbial volatiles benefit the agricultural ecological system by promoting plant growth and systemic resistance against diseases without harming the environment. To explore the plant growth-promoting efficiency of VOCs produced by Pseudomonas fluorescens PDS1 and Bacillus subtilis KA9 in terms of chili plant growth and its biocontrol efficiency against Ralstonia solanacearum, experiments were conducted both in vitro and in vivo. A closure assembly was designed using a half-inverted plastic bottle to demonstrate plant-microbial interactions via volatile compounds. The most common volatile organic compounds were identified and reported; they promoted plant development and induced systemic resistance (ISR) against wilt pathogen R. solanacearum. The PDS1 and KA9 VOCs significantly increased defensive enzyme activity and overexpressed the antioxidant genes PAL, POD, SOD, WRKYa, PAL1, DEF-1, CAT-2, WRKY40, HSFC1, LOX2, and NPR1 related to plant defense. The overall gene expression was greater in root tissue as compared to leaf tissue in chili plant. Our findings shed light on the relationship among rhizobacteria, pathogen, and host plants, resulting in plant growth promotion, disease suppression, systemic resistance-inducing potential, and antioxidant response with related gene expression in the leaf and root tissue of chili.

Screening and Biocontrol Potential of Rhizobacteria Native to Gangetic Plains and Hilly Regions to Induce Systemic Resistance and Promote Plant Growth in Chilli against Bacterial Wilt Disease.

Plant growth-promoting rhizobacteria (PGPR) is a microbial population found in the rhizosphere of plants that can stimulate plant development and restrict the growth of plant diseases directly or indirectly. In this study, 90 rhizospheric soil samples from five agro climatic zones of chilli (Capsicum annuum L.) were collected and rhizobacteria were isolated, screened and characterized at morphological, biochemical and molecular levels. In total, 38% of rhizobacteria exhibited the antagonistic capacity to suppress Ralstonia solanacearum growth and showed PGPR activities such as indole acetic acid production by 67.64% from total screened rhizobacteria isolates, phosphorus solubilization by 79.41%, ammonia by 67.75%, HCN by 58.82% and siderophore by 55.88%. We performed a principal component analysis depicting correlation and significance among plant growth-promoting activities, growth parameters of chilli and rhizobacterial strains. Plant inoculation studies indicated a significant increase in growth parameters and PDS1 strain showed maximum 71.11% biocontrol efficiency against wilt disease. The best five rhizobacterial isolates demonstrating both plant growth-promotion traits and biocontrol potential were characterized and identified as PDS1-Pseudomonas fluorescens (MN368159), BDS1-Bacillus subtilis (MN395039), UK4-Bacillus cereus (MT491099), UK2-Bacillus amyloliquefaciens (MT491100) and KA9-Bacillus subtilis (MT491101). These rhizobacteria have the potential natural elicitors to be used as biopesticides and biofertilizers to improve crop health while warding off soil-borne pathogens. The chilli cv. Pusa Jwala treated with Bacillus subtilis KA9 and Pseudomonas fluorescens PDS1 showed enhancement in the defensive enzymes PO, PPO, SOD and PAL activities in chilli leaf and root tissues, which collectively contributed to induced resistance in chilli plants against Ralstonia solanacearum. The induction of these defense enzymes was found higher in leave tissues (PO-4.87-fold, PP0-9.30-fold, SOD-9.49-fold and PAL-1.04-fold, respectively) in comparison to roots tissue at 48 h after pathogen inoculation. The findings support the view that plant growth-promoting rhizobacteria boost defense-related enzymes and limit pathogen growth in chilli plants, respectively, hence managing the chilli bacterial wilt.

Experimental infection and pathology of two highly pathogenic avian influenza H5N1 viruses isolated from crow and chicken in house crows (Corvus splendens).

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.

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep.

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15 days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5 × 10(5) TCID50 of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5'-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.

Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA.

The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.

Pathology of a H5N1, highly pathogenic avian influenza virus, in two Indian native chicken breeds and a synthetic broiler line.

In this study, susceptibility to H5N1 virus infection was studied in two Indian native chicken breeds viz. Kadaknath and Aseel (Peela) and an Indian synthetic broiler strain (Synthetic dam line (SDL-IC). Fifty birds from each genetic group were infected intra-nasally with 1000 EID50 of a highly pathogenic avian influenza virus (HPAIV) strain A/chicken/Navapur/India/7972/ 06 (H5N1) and observed for a period of 10 days. Significant differences in severity of clinical signs, gross lesions and time for onset of symptoms were observed. The overall severity of clinical signs and gross lesions was less in SDL-IC broilers as compared to the other two genetic groups. The mortality percentages were 100, 98 and 92% with Mean Death Time (MDT) of 3.12, 5.92 and 6.96 days, respectively for the two native breeds Kadaknath and Aseel (Peela), the and SDL-IC broiler strain. Comparison of histological lesions revealed differences in disease progression among the genetic groups. Vascular lesions such as disseminated intravascular coagulopathy (DIC) were predominant on 3 days post infection (dpi) in Kadaknath, and on 5 and 6 dpi in Aseel (Peela) and SDL-IC broiler. The mean log2 HA titres of the re-isolated virus from various organs of H5N1 AIV infected birds of the three genetic groups ranged from 2.32 (lung, trachea and bursa) to 5.04 (spleen) in Kadaknath; 2.32 (lung) to 6.68 (brain) in Aseel (Peela); and 2.06 (liver) to 7.01 (lungs and kidney) in SDL-IC broiler. These results suggest that the susceptibility to H5N1 highly pathogenic avian influenza virus infection differed among the three breeds; Kadaknath being highest followed by Aseel (Peela) and synthetic SDL-IC broiler. This is possibly the first report on the differences in the susceptibility of the India native breeds to H5N1 virus infection and its severity.

Survivability of Highly Pathogenic Avian Influenza H5N1 Virus in Poultry Faeces at Different Temperatures.

Highly pathogenic avian Influenza (HPAI) is an important zoonotic disease and is becoming a great threat to poultry industry. India has experienced continual outbreaks of H5N1 HPAI virus since February, 2006 especially in Eastern India. Survivability in poultry faeces is an important determinant in evaluating the persistence of the virus in the poultry sheds and their vicinity. In this paper, survivability of Indian H5N1 HPAI virus in dry and wet poultry faeces at 42, 37, 24 and 4 °C, respectively is reported. The effect of different temperatures was determined by linear regression model and defined in terms of linear equation. The virus survived up to 18 h at 42 °C, 24 h at 37 °C, 5 days at 24 °C and 8 weeks at 4 °C in dry and wet faeces, respectively. The coefficients of determination (R(2)) values for dry and wet faeces revealed that the difference in viral persistence in dry and wet faeces at all temperatures was not very marked. Results of the present study indicated that H5N1 HPAI virus may remain viable for extended periods of time in faeces at low temperatures and may act as a long term source of influenza virus in the environment.

Genetic variety of bovine viral diarrhea virus 1 strains isolated from sheep and goats in India.

UNLABELLED: Antigenic and genetic typing of pestiviruses isolated from Indian sheep and goats was carried out. Testing of 1777 sheep and 1026 goat blood samples collected between 2004 and 2008 resulted in isolation of twelve pestiviruses, seven from sheep and five from goats. All of them were antigenically typed as bovine viral diarrhea virus 1 (BVDV-1). Both the partial 5ʹ-UTR and entire non-structural autoprotease (Npro) gene of the pestiviruses were amplified by RT-PCR and sequenced. The phylogenetic analysis confirmed all twelve sheep and goat pestiviruses as BVDV-1 and they were further classified into two subtypes, BVDV-1b (seven) and BVDV-1c (five). This is for the first time that BVDV-1c was detected in sheep and goats. However, no association between the subtype and geographic area of origin was observed. Although closely related, BVDV-1b and BVDV-1c isolates of sheep and goats were placed in a different clade than previously reported Indian BVDV-1b/BVDV-1c isolates. This study confirmed widespread prevalence of BVDV-1 in Indian sheep and goats that has significance in the epidemiology of bovine viral diarrhea. KEYWORDS: bovine viral diarrhea virus; BVDV-1; goat; Npro; genetic typing; sheep; 5ʹ-UTR.

Bioefficacy of novel cyanobacteria-amended formulations in suppressing damping off disease in tomato seedlings.

Biological control of plant pathogens is receiving increasing relevance, as compared to chemical methods, as they are eco-friendly, economical and indirectly improve plant quality and yield attributes. An investigation was undertaken to evaluate the potential of antagonistic cyanobacteria (Anabaena variabilis RPAN59 and A. oscillarioides RPAN69) fortified formulations for suppressing damping off disease in tomato seedlings challenged by the inoculation of a fungal consortium (Pythium debaryanum, Fusarium oxysporum lycopersici, Fusarium moniliforme and Rhizoctonia solani). Treatment with A. variabilis amended formulations recorded significantly higher plant growth parameters, than other treatments, including biological control (Trichoderma formulation) and chemical control (Thiram-Carbendazim). The A. variabilis amended compost-vermiculite and compost formulations exhibited 10-15 % lower disease severity and 40-50 % higher values than chemical and biological control treatments in terms of fresh weight and height of the plants. In future, in depth analyses regarding the mechanism involved in biocontrol by cyanobacteria and evaluation of these formulations under field conditions are proposed to be undertaken.

Antiviral activity of crude extracts of Eugenia jambolana Lam. against highly pathogenic avian influenza (H5N1) virus.

Crude extracts of leaves and bark of E. jambolana were tested for antiviral activity against highly pathogenic avian influenza virus (H5N1) by CPE reduction assay in three different layouts to elucidate virucidal, post-exposure and preexposure antiviral activity of the extracts. The cold and hot aqueous extracts of bark and hot aqueous extract of leaves of E. jambolana showed significant virucidal activity (100% inhibition) which was further confirmed in virus yield reduction assay (-98 to 99% reduction) and by egg based in ovo assay. The selective index (CC50/EC50) of hot aqueous extract (248) and cold aqueous extract (43.5) of bark of E. jambolana showed their antiviral potential against H5N1 virus. The significant virucidal activity of leaves and bark of E. jambolana merits further investigation as it may provide alternative antiviral agent for managing avian influenza infections in poultry farms and potential avian-human transmission.

Isolation and characterization of influenza A virus (subtype H5N1) that caused the first highly pathogenic avian influenza outbreak in chicken in Bhutan.

We characterized Influenza A/H5N1 virus that caused the first outbreak of highly pathogenic avian influenza (HPAI) in chickens in Bhutan in 2010. The virus was highly virulent to chicken, killing them within two days of the experimental inoculation with an intravenous pathogenicity index (IVPI) of 2.88. For genetic and phylogenetic analyses, complete genome sequencing of 4 viral isolates was carried out. The isolates revealed multiple basic amino acids at their hemagglutinin (HA) cleavage site, similar to other "Qinghai-like" H5N1 isolates. The receptor-binding site of HA molecule contained avian-like amino acids ((222)Q and (224)G). The isolates also contained amino acid residue K at position 627 of the PB2 protein, and other markers in NS 1 and PB1 proteins, highlighting the risk to mammals. However, the isolates were sensitive to influenza drugs presently available in the market. The sequence analysis indicated that the Bhutan viruses shared 99.1-100% nucleotide homology in all the eight genes among themselves and 2010 chicken isolate from Bangladesh (A/chicken/Bangladesh/1151-11/2010) indicating common progenitor virus. The phylogenetic analysis indicated that the Bhutan isolates belonged to sub-clade 2.2.3 (EMA 3) and shared common progenitor virus with the 2010 Bangladesh virus. Based on the evidence of phylogeny and molecular markers, it could be concluded that the outbreaks in Bhutan and Bangladesh in 2010 were due to independent introductions of the virus probably through migratory birds.

Comparison of a nucleoprotein gene based RT-PCR with real time RT-PCR for diagnosis of avian influenza in clinical specimens.

A nucleoprotein (NP) gene based reverse transcription polymerase chain reaction (npRT-PCR) assay was developed in our laboratory which could detect 35.09% of the experimental samples negative for virus isolation in first passage but positive by third passage. Reducing the reaction volume to 12.5 µl did not alter the test sensitivity and the results did not vary when duplicate samples were run in a different thermal cycler. The positive and negative agreements of this test in clinical specimens were compared with a matrix gene based real time RT-PCR with virus isolation as standard. A total of 516 clinical specimens including tissues, swabs and feces submitted from various States of India as part of active surveillance for avian influenza were tested by npRT-PCR, RRT-PCR and virus isolation in 9-11 day old embryonated specific pathogen free chicken eggs. The positive and negative agreements of npRT-PCR with virus isolation were found to be 0.909±0.022 and 0.980±0.004 respectively and that of RRT-PCR with virus isolation were 0.902±0.023 and 0.977±0.005 respectively. Since the positive and negative agreements of both npRT-PCR and RRT-PCR tests were similar, we suggest that this test can be used by peripheral veterinary laboratories that do not have real time PCR facility for active surveillance of AIV.

Small interfering RNAs targeting viral structural envelope protein genes and the 5ʹ-UTR inhibit replication of bovine viral diarrhea virus in MDBK cells.

Bovine viral diarrhea viruses (BVDVs) are important pathogens of cattle that occur worldwide, and for which no antiviral therapy is available. In the present study, the inhibitory effect of small interfering (si) RNAs on bovine viral diarrhea virus 1 (BVDV-1) replication in cultured bovine cells was explored. Four synthetic siRNAs were designed to target structural envelope region genes (Erns, E1, and E2) and one cocktail of siRNA was generated to target the 5ʹ-UTR of the BVDV-1 genome. The inhibitory effects of siRNAs were assessed by determination of infectious viral titer, viral antigen and viral RNA. The siRNA cocktail and three of the synthetic siRNAs produced moderate anti-BVDV-1 effect in vitro as shown by 25%-40% reduction in BVDV-1 antigen production, 7.9-19.9-fold reduction in viral titer and 21-48-fold reduction in BVDV-1 RNA copy number. Our findings suggest that siRNA cocktail targeted at the 5ʹ-UTR is a stronger inhibitor of BVDV-1 replication and the targets for siRNA inhibition can be extended to BVDV-1 structural envelope protein genes.

Emergence of amantadine-resistant avian influenza H5N1 virus in India.

This study reports the genetic characterization of highly pathogenic avian influenza (HPAI) virus (subtype H5N1) isolated from poultry in West Bengal, India. We analyzed all the eight genome segments of two viruses isolated from chickens in January 2010 to understand their genetic relationship with other Indian H5N1 isolates and possible connection between different outbreaks. The hemagglutinin (HA) gene of the viruses showed multiple basic amino acids at the cleavage site, a marker for high virulence in chickens. Of greatest concern was that the viruses displayed amino acid substitution from serine-to-asparagine at position 31 of M2 ion channel protein suggesting emergence of amantadine-resistant mutants not previously reported in HPAI H5N1 outbreaks in India. Amino acid lysine at position 627 of the PB2 protein highlights the risk the viruses possess to mammals. In the phylogenetic trees, the viruses clustered within the lineage of avian isolates from India (2008-2009) and avian and human isolates from Bangladesh (2007-2009) in all the genes. Both these viruses were most closely related to the viruses from 2008 in West Bengal within the subclade 2.2.3 of H5N1 viruses.

Phylogenetic evidence of multiple introduction of H5N1 virus in Malda district of West Bengal, India in 2008.

Outbreaks of H5N1 avian influenza virus were reported in 15 districts of West Bengal State in India in early 2008 and subsequent re-occurrence in 5 districts in December, 2008 to May, 2009. We have sequenced complete genome of 12 viruses isolated from early 2008 outbreak and from recurrent outbreak and determined the phylogenetic relationship between the viruses isolated from the two outbreaks. One of the virus isolated in early 2008 from Malda district (A/chicken/West Bengal/81760/2008) clustered with Korean and Russian isolates of 2006 in European-Middle Eastern-African (EMA) 3 sub-lineage of sub-clade 2.2, whereas other viruses showed close genetic relationship with 2007-2009 isolates of Bangladesh. Nucleotide sequence analysis revealed that the PB1-F2 protein expression might be completely abolished due to mutated start codon ((95)ATG(97)→(95)ACG(97)) in this isolate but in all other isolates it was completely expressed. Hence, we conclude that there were two separate introductions of H5N1 viruses in Malda district and this H5N1 virus was not epidemiologically dominant as the viruses isolated subsequently from the same district and region did not share close relationship with this virus. The failure of this virus to spread to adjoining areas suggests that the culling and disposal operations initiated by Government of India were effective.

Influence of dose of inocula on outcome of clinical disease in highly pathogenic avian influenza (H5N1) infections--an experimental study.

Twelve-week-old Vanaraja (an Indian native dual purpose breed) chickens were inoculated intranasally with different doses (100, 1000, and 10,000 mean embryo infective dose [EID50]) of H5N1 virus, and the clinical disease and pathologic changes were compared. Although the overall severity of clinical signs was more severe in the 100 EID50 group, the progression of the clinical disease was slower with delayed onset of mortality when compared with the other two groups. The mean death time of the 100 EID50 group (4.57 days) differed significantly from that of the 10,000 EID50 group (3.60 days) and from that of the 1000 EID50 group (3.33 days). Similarly, overall severity of gross lesions was expressed more in the 100 EID50 group. The histopathologic lesions were of a more hemorrhagic and necrotic nature in the 100 EID50 group, histopathologic lesions were of an inflammatory/proliferative nature in the 1000 EID50 group, and a tendency for intravascular coagulopathy was observed in the 10,000 EID50 group. These differences may be assigned to the influence of dose in the outcome of disease.

Production and characterization of monoclonal antibodies against NS1 protein of H5N1 avian influenza virus.

A panel of 11 murine monoclonal antibodies (MAbs) was generated against NS1 (non-structural) protein of highly pathogenic avian influenza H5N1 virus using whole viral antigen as immunogen and as antigen in the preliminary screening of clones followed by a more specific screening through recombinant NS1 protein-based ELISA. Isotyping showed two clones 10E2 (IgG2b) and 5H8 (IgG3) to be IgG type and 6B9 and 4C4 to be IgA type, while the rest could not be isotyped due to weak reactivity with NS1 antigen. The subclone 10E2E7G3 was selected for subsequent work, which showed comparatively higher OD in NS1-based ELISA. The reactivity of 10E2E7G3 with H5N1 virus as well as recombinant NS1 protein was confirmed in Western blot analysis. The anti-NS1 MAbs produced in the present work may be valuable in developing an immunodiagnostic assay as a DIVA test for differentiating infected from vaccinated birds in AIV.

Isolation and molecular characterization of a H5N1 virus isolated from a Jungle crow (Corvus macrohynchos) in India.

In 2008, India experienced widespread outbreaks of H5N1 virus in West Bengal, Tripura, and Assam. The virus was detected in Kamrup district of Assam in November 2008 and subsequently spread to eight more districts. Two Jungle or Large billed crows (Corvus macrohynchos) were found dead in a hospital campus at about 8 km from the foci of initial detection of the virus in the same district. One of the crows was positive for H5N1 avian influenza virus by virus isolation, real time RT-PCR, and RT-PCR tests. Full length sequencing of all the eight segments of the virus was carried out. The phylogenetic analysis indicated that all the eight genes grouped with clade 2.2 viruses and were closely related to the human isolate of Bangladesh and avian isolates from India, Bangladesh, Kuwait, Germany, and Saudi Arabia. The molecular analysis indicated avian receptor (alpha 2,3 sialic acid) specificity, susceptibility to oseltamivir and amantadine group of antivirals and lower pathogenicity to mice.