Identifying a few foot-and-mouth disease virus signature nucleotide strings for computational genotyping

<p>Abstract</p> <p>Background</p> <p>Serotypes of the Foot-and-Mouth disease viruses (FMDVs) were generally determined by biological experiments. The computational genotyping is not well studied even with the availability of whole viral genomes, due to uneven evolution among genes as well as frequent genetic recombination. Naively using sequence comparison for genotyping is only able to achieve a limited extent of success.</p> <p>Results</p> <p>We used 129 FMDV strains with known serotype as training strains to select as many as 140 most serotype-specific nucleotide strings. We then constructed a linear-kernel Support Vector Machine classifier using these 140 strings. Under the leave-one-out cross validation scheme, this classifier was able to assign correct serotype to 127 of these 129 strains, achieving 98.45% accuracy. It also assigned serotype correctly to an independent test set of 83 other FMDV strains downloaded separately from NCBI GenBank.</p> <p>Conclusion</p> <p>Computational genotyping is much faster and much cheaper than the wet-lab based biological experiments, upon the availability of the detailed molecular sequences. The high accuracy of our proposed method suggests the potential of utilizing a few signature nucleotide strings instead of whole genomes to determine the serotypes of novel FMDV strains.</p

Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

Currently available vaccines for the pandemic Influenza A (H1N1) 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA) based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI) activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat

Cloning and expression of the cDNA for the major antigen of foot and mouth disease virus type Asia I 63/72

Double-stranded cDNA for the foot and mouth disease virus (FMDV) type Asia I63/72 was prepared and cloned in the expression vector pUR222. The recombinant DNA was transferred into Escherichia coli RRI-DT5 and the transformants were identified as white colonies in the presence of the dye x-gal on agar plates. Many of them gave strong pos. signals on hybridization with 32P-labeled viral RNA. The middle BamHI fragment of the cDNA is known to carry the sequence for a few nonstructural proteins and for the major antigen, VPI. The clones contg. the BamHI fragments of the cDNA and the nearly full length cDNA produced proteins which gave pos. signals in sandwich ELISA, showing that they cross-reacted with the antibodies raised against the virus. This showed that the major antigen is expressed by the cloned cDNA for the FMDV type Asia I

Non-segmented negative sense RNA viruses as vectors for vaccine development

This article intends to cover two aspects of non-segmented negative sense RNA viruses. In the initial section, the strategy employed by these viruses to replicate their genomes is discussed. This would help in understanding the later section in which the use of these viruses as vaccine vectors has been discussed. For the description of the replication strategy which encompasses virus genome transcription and genome replication carried out by the same RNA dependent RNA polymerase complex, a member of the prototype rhabdovirus family - Chandipura virus has been chosen as an example to illustrate the complex nature of the two processes and their regulation. In the discussion on these viruses serving as vectors for carrying vaccine antigen genes, emphasis has been laid on describing the progress made in using the attenuated viruses as vectors and a description of the systems in which the efficiency of immune responses has been tested

Expression in E. coli of the cloned cDNA for the major antigen of foot and mouth disease virus Asia 1 63/72

Double stranded cDNA for the foot and mouth disease virus was prepared, restricted with BamH 1 or ligated to linkers with BamH 1 sticky ends and cloned in BamHI site in the expression vector,pUR222. The cDNA was also cloned at the Pst l site in the same vector by the dC/dG tailing method. They were transferred into E. coli to give colourless colonies in the presence of the dye, X-gal. Many of them showed positive signal on hybridization with $^{32}P$-labelled viral RNA. The middle BomH 1 fragment of the cDNA is known to carry the gene for the major antigen and some non-structural proteins. The clones carrying the recombinant DNA produced proteins which cross-reacted with the antibodies generated against the structural proteins of the virus in an enzyme linked immunosorbent assay, indicating that the cDNA of the major antigen is expressed in the cloned cell

Expression of VP1 protein of serotype A and O of foot-and-mouth disease virus in transgenic sunnhemp plants and its immunogenicity for guinea pigs

Recently, transgenic plants expressing immunogenic proteins of foot-and-mouth disease virus (FMDV) have been used as oral or parenteral vaccines against foot-and-mouth disease (FMD). They exhibit advantages like cost effectiveness, absence of processing, thermostability, and easy oral application. FMDV VP1 protein of single serotype has been mostly used as immunogen. Here we report the development of a bivalent vaccine with tandem-linked VP1 proteins of two serotypes, A and O, present in transgenic forage crop Crotalaria juncea. The expression of the bivalent protein in the transgenic plants was confirmed by Western blot analysis. Guinea pig reacted to orally or parenterally applied vaccine by humoral as well as cell-mediated immune responses including serum antibodies and stimulated lymphocytes, respectively. The vaccine protected the animals against a challenge with the virus of serotype A as well as O. This is the first report on the development of a bivalent FMD vaccine using a forage crop

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Not AvailableIsolation, serological tests and molecular methods were adopted for the diagnosis of brucellosis in sheep flocks with history of reproductive disorders. Three serological tests RBPT, STAT and iELISA were employed to screen 527 sheep from 6 farms. Among serological tests, iELISA detected higher positives (14.80%), than RBPT (5.88%) and STAT (4.17%). Brucella melitensis was isolated from 6 of 33 seropositive sheep and none from seronegative animals. All the isolates were identified as B. melitensis by biochemical tests and confirmed by genus and species specific - AMOS PCR. None of the isolates yielded products of 285bp, 976bp and 498bp specific for B. suis, B. ovis and B. abortus, respectively. Of the 33 seropositive sheep, the bcsp31genus specific amplicon of 223bp was observed in 26 vaginal, 6 serum and 2 in blood samples. Vaginal samples was found suitable clinical sample for the isolation and PCR, followed by serum and blood. Different diagnostic approaches used in the present study are of value as anti Brucella antibodies and antigen were detected confirming the disease in sheep flock. This practically oriented approach is of immense importance, as it enables to institute the control strategies at the earliest.Not Availabl

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Not AvailableThe Anigen rapid immunodiagnostic test was employed to confirm the propagation of Dr. Larghi's strain of rabies virus in BHK 21 cells through it's detection in cell culture harvest. In all, 25 infected (harvests from infected BHK 21 cells) and 15 uninfected controls (spent medium from uninfected BHK21 cells) were tested for the study. The rapid immunodiagnostic test was able to detect rabies virus antigen in the harvests from all the infected BHK 21 cells but not in the culture supernatant (spent medium from uninfected T25 flasks). Further application of reverse transcription-polymerase chain reaction (RTPCR) confirmed the presence of rabies virus in the harvests from infected BHK 21 cells but not in the spent medium from uninfected T25 flasks. The rapid immunodiagnostic test for rabies virus antigen detection is a straightforward test that can be run under laboratory conditions and without a microscope or electricity, and yield results in 5 minutes. This rapid immunodiagnostic test is a quick, inexpensive, and easy to use tool that can identify rabies positive cell culture harvest, enabling the rapid selection of viral source for downstream applications.Not Availabl

Growth kinetics and immune response of chimeric foot-and-mouth disease virus serotype `O' produced through replication competent mini genome of serotype Asia 1, 63/72, in BHK cell lines

Regular vaccinations with potent vaccine, in endemic countries and vaccination to live in non-endemic countries are the methods available to control foot-and-mouth disease. Selection of candidate vaccine strain is not only cumbersome but the candidate should grow well for high potency vaccine preparation. Alternative strategy is to generate an infectious cDNA of a cell culture-adapted virus and use the replicon for development of tailor-made vaccines. We produced a chimeric `O' virus in the backbone of Asia 1 and studied its characteristics. The chimeric virus showed high infectivity titre (>10(10)) in BHK 21 cell lines, revealed small plague morphology and there was no cross reactivity with antiserum against Asia I. The virus multiplies rapidly and reaches peak at 12 h post infection. The vaccine prepared with this virus elicited high antibody titres