Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

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Not AvailableGenetic and antigenic analysis of H5N1 viruses, isolated in India during a period from year 2006 to 2010, was carried out for selection of the potential H5-HA (haemagglutinin) gene donor virus for developing a reverse genetics based DIVA marker H5 vaccine for poultry in India. Out of the 47 H5N1 viruses (clade 2.2), 14 representative viruses were selected on the basis of amino acid sequence analysis of HA1 gene for further antigenic characterization. Using antigenic cartography, an antigenic map was constructed based on the data of cross-HI (haemagglutinin inhibition) titration of 14 sera versus 14 viruses to visualize the relatedness among the antigens and antigenic coverage of the sera. Sera against five H5N1 viruses (A/crow/Assam/142119/2008, A/chicken/West Bengal/100879/2008, A/chicken/West Bengal/155505/2009, A/chicken/West Bengal/80995/2008 and A/chicken/West Bengal/81760/2008) exhibited maximum (100 %) antigenic coverage, hence, were selected as the potential HA donor viruses. However, the virus strain A/chicken/West Bengal/80995/2008 matched completely with the consensus amino acid sequence of the 47 viruses, therefore, was considered the best HA donor candidate out of the five showing 100 % antigenic coverage. The present study demonstrates a stepwise methodology for logical selection of vaccine strain or HA gene donor strain for developing H5 vaccines using genetic and antigenic data.Not Availabl

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Not AvailableIn a cross-sectional study, prevalence of ovine herpesvirus 2 (family: Herpesviridae, subfamily: Gammaherpesvirinae, genus Macavirus and species: Ovine herpesvirus 2) infection was estimated in sheep population of Karnataka state in India. Based on the three stage cluster sampling method, whole blood samples (356) of sheep were collected from 11 sheep-dense districts of the state. The samples were tested for presence of OvHV-2 genome by recommended hemi-nested polymerase chain reaction (PCR) test. The true prevalence of OvHV-2 infection in sheep population of Karnataka was 24.44 %. Of the 11 district surveyed, highest true prevalence of 42.42 % (CI 25.56-59.29) was found in Raichur followed by Tumkur (39.02 %, CI 24.09-53.96). Inverse distance weighted interpolation of prevalence indicated that OvHV-2 prevalence within a given district is not uniform and there are areas of varied prevalence. The nucleotide sequence of the 422 bp DNA fragment, amplified in PCR, matched 99 % with OvHV-2 reference sequence and other sequences reported from India. Grouping of OvHV-2 sequences obtained from Karnataka with those from Andhra Pradesh, Tamil Nadu and Jammu and Kashmir in the neighbour joining tree indicated a close relationship among the OvHV-2s circulating in India. This is the first study in the country where systematic screening of sheep population of a state for the presence of OvHV-2 infection has been carried out, which indicated a widespread prevalence calling for an urgent need for policy measures to prevent economic losses due to the disease in susceptible cattle and buffalo species.Not Availabl

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Not AvailableSingle chain fragment variable (ScFv) antibodies specific to the nucleoprotein (NP) of avian influenza virus (AIV) were developed using a phage display system. The variable heavy (VH) and the variable light (VL) chain gene fragments were derived from spleen cells of Balb/c mouse immunized with a recombinant NP (rNP) antigen (∼63 kDa) of H5N1 influenza virus. The VH and the VL DNA fragments were assembled through a flexible linker DNA to generate ScFv DNA that was cloned subsequently in a phagemid to express ScFv protein in Escherichia coli cells. The specific reactivity of the ScFv with the rNP antigen and viral antigen (H5N1) was confirmed by Western blot and ELISA. A competitive inhibition ELISA (CI-ELISA) was developed using the rNP and the anti-NP ScFv for detection of type-specific antibodies to AIV in chicken sera. The ScFv based CI-ELISA was compared with hemagglutination inhibition (HI) test and agar gel immunodiffusion (AGID) test over 850 sera. Sensitivity of the CI-ELISA was 100% with HI and AGID and specificity was 98.7% with HI and 100% with AGID. Copyright © 2014 Elsevier B.V. All rights reserved.Not Availabl

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Not AvailableIn a cross-sectional study, prevalence of ovine herpesvirus 2 (family: Herpesviridae, subfamily: Gammaherpesvirinae, genus Macavirus and species: Ovine herpesvirus 2) infection was estimated in sheep population of Karnataka state in India. Based on the three stage cluster sampling method, whole blood samples (356) of sheep were collected from 11 sheep-dense districts of the state. The samples were tested for presence of OvHV-2 genome by recommended hemi-nested polymerase chain reaction (PCR) test. The true prevalence of OvHV-2 infection in sheep population of Karnataka was 24.44 %. Of the 11 district surveyed, highest true prevalence of 42.42 % (CI 25.56-59.29) was found in Raichur followed by Tumkur (39.02 %, CI 24.09-53.96). Inverse distance weighted interpolation of prevalence indicated that OvHV-2 prevalence within a given district is not uniform and there are areas of varied prevalence. The nucleotide sequence of the 422 bp DNA fragment, amplified in PCR, matched 99 % with OvHV-2 reference sequence and other sequences reported from India. Grouping of OvHV-2 sequences obtained from Karnataka with those from Andhra Pradesh, Tamil Nadu and Jammu and Kashmir in the neighbour joining tree indicated a close relationship among the OvHV-2s circulating in India. This is the first study in the country where systematic screening of sheep population of a state for the presence of OvHV-2 infection has been carried out, which indicated a widespread prevalence calling for an urgent need for policy measures to prevent economic losses due to the disease in susceptible cattle and buffalo species.Not Availabl

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Not AvailableAn inactivated vaccine was developed using the rgH5N2 virus (6 + 2 reassortant) generated by plasmid based reverse genetics system (RGS) with WSN/33/H1N1 as backbone virus. Following mutation of the basic amino acid cleavage site RRRKKR*GLF to IETR*GLF, the H5-HA (haemagglutinin) gene of the selected donor H5N1 virus (A/chicken/West Bengal/80995/2008) of antigenic clade 2.2 was used along with the N2-NA gene from H9N2 field isolate (A/chicken/Uttar Pradesh/2543/2004) for generation of the rgH5N2 virus. A single dose (0.5 ml/bird) of the inactivated rgH5N2 vaccine protected 100% of the vaccinated chickens (n = 10) on 28(th) dpv (early challenge) and 90% of the vaccinated chickens (n = 10) on 200(th) dpv (late challenge) against high dose challenge with HPAI virus (10(9) EID50/bird). Challenge virus shedding via oropharynx and cloaca of the vaccinated chickens was detectable by realtime RT-PCR during 1-5 dpc and 1-9 days dpc in the early and the late challenge, respectively. The protective level of antibodies (mean HI titre > 128) was maintained without booster vaccination for 200 days. The present study provides the experimental evidence about the extent of protection provided by a reverse genetics based vaccine for clade 2.2 H5N1 viruses against challenge with high dose of field virus at two different time points (28 dpv and 200 dpv). The challenge study is uniquely different from the previous similar experiments on account of 1000 times higher dose of challenge and protection at 200 dpv. The protection and virus shedding data of the study may be useful for countries planning to use H5 vaccine in poultry especially against the clade 2.2 H5N1 viruses.Not Availabl

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Not AvailableIn the present study, 27 field materials from different sources were tested in SYBR Green I dye based Real Time PCR optimized using WHO recommended conventional PCR primers targeting Bacillus anthracis protective antigen (PA) gene of pX01 plasmid. The assay had detection limit of 0.000002 pg in terms of target DNA quantity and up to 4 copies of pX01 plasmid per reaction, in terms of copy number. Melt curve analysis of PCR products of field samples showed deviation up to 3.2°C in melting temperature from standard anthrax strain suggestive of possible mutation/ mutations in PA gene. Targeted region of PA (596 bp) encompasses domain 2, involved in its binding with lethal factor (LF) and/or edema factor (EF) and mutation in this region may alter the efficacy of PA to traffic toxins inside the cells and results in differed level of virulence of pathogen. The test is useful not only for detection but may be suggestive of mutation also. Evaluation of a protective antigen gene based SYBR Green I real time PCR for detection of Bacillus anthracis in field samples (PDF Download Available). Available from: https://www.researchgate.net/publication/265013123_Evaluation_of_a_protective_antigen_gene_based_SYBR_Green_I_real_time_PCR_for_detection_of_Bacillus_anthracis_in_field_samples [accessed Jun 3, 2017].Not Availabl

Advances in Aquaculture Vaccines Against Fish Pathogens: Global Status and Current Trends.

In recent years, aquaculture has attained a major economic revolution, however, infectious diseases of bacterial, viral, mycotic and parasitic origin are the most significant restrictive agents in the improvement of intensified aquaculture, which has become a fast blooming seafood industry. For environment-friendly aquaculture and human health concerns owing to the rise in incidences of antimicrobial resistant microbes and food safety hazards, the immunoprophylaxis or vaccination strategies are highly effective and economical in protecting the health of fish and aquaculture animals from various infectious agents. Advancements in science have paved newer avenues in both basic and applied research areas for developing and designing novel and effective vaccines, as well as improving existing vaccines for rendering protection from various types of infectious diseases. Current advances in vaccines and vaccinology offer valuable opportunities to discover new vaccine candidates to combat fish pathogens, including mycotic and parasitic agents, for which vaccines are still lacking. This review focuses on the current knowledge, recent advances and future perspectives of vaccines and vaccination in the aquaculture industry, from traditional inactivated and attenuated vaccines to new generation vaccines comprising of recombinant, subunit, vectored, genetically engineered, DNA and peptide vaccines, reverse vaccinology and plant-based edible vaccines, and nanovaccines.Maryam Dadar and Kuldeep Dhama would like to thank the research affairs section of Razi Vaccine and Serum Research Institute and Gorgan University of Agricultural Sciences and Natural Resources for their support of the research projects. Vikram N. Vakharia and Sunil K. Joshi thank their respective institutes/organizations

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Not AvailableMalignant catarrhal fever (MCF) is a lymphoproliferative multisystemic fatal syndrome of many domestic and wild animals. The present study was conducted on animals of different species (cattle, buffalos and sheep). The clinical examination revealed high persistent fever, corneal opacity, mucoprulent oronasal discharges, mucosal lesions and ulcerative skin lesions. Examined sheep showed no clinical signs. Peripheral blood leukocytic (pb1) samples were collected for laboratory investigations. Semi nested polymerase chain reaction (PCR) assay have been used. Sequencing and phylogenetic analysis were performed for the positive PCR products. Positive staining electron microscopy (EM) and histopathology were also performed. Ovine herpesvirus 2 (OvHV-2) DNA was detected in PBL of 25 animals (12 cattle, 2 buffaloes and 11 sheep). The phylogenetic analysis of the OvHV-2 PCR products revealed 100% identity with the OvHV-2 strains of Brazil, USA and India. The positive staining EM detected herpesviral particles. The histopathological examination showed pansystemic vasculitis with lymphocytic infiltration in lymphoid and non lymphoid organs.Not Availabl