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D N Tripathy

Publications and source records attributed to D N Tripathy.

At least 19 recordsLinked to original sources
PubMed2005

Genetic manipulation of two fowlpox virus late transcriptional regulatory elements influences their ability to direct expression of foreign genes.

Fowlpox virus (FWPV) is currently used as a vector to express foreign genes of various poultry and mammalian pathogens. However, due to limited information available about the primary structure of FWPV promoters required for an optimal transcriptional efficiency, the full potential of FWPV as an expression vector has not been completely realized. To dissect such transcriptional regulatory elements at the molecular level, we selected two FWPV promoters dictating contrasting levels of expression of acidic-type inclusion body protein gene (P190) and A15L vaccinia virus homolog of FWPV (P180) for site-directed mutagenesis studies. The transcriptional activity of mutated promoters was analyzed based on their ability to transcribe a reporter gene, lacZ, and translation of the resultant mRNA into functional protein. Replacement of the spacer sequences of P180 with those of P190 resulted in a five-fold increase in mRNA and a 17.6-fold increase in protein over those with its parental promoter, P180. Similarly, replacement of a thymidine after the start codon with guanosine resulted in a 2.3-fold increase in lacZ mRNA and a seven-fold increase in protein. Combining these substitutions in P180SG produced a maximum increase in mRNA and protein of 6.7- and 29.9-fold, respectively, over concentrations with its parental P180 promoter. The promoter activity of P180SG was comparable to that of the strongest natural promoter, P190. The amount of protein per transcript generated by the mutated promoters of P180 increased to at least three times that with the parental P180. In contrast, similar replacements in P190 resulted in a 40-50% reduction in mRNA and protein in all the mutated promoters. We discuss the significance of spacer sequence and the purine after the start codon in the context of a high level of expression.

Animals↗
PubMed2005

The DNA repair enzyme, CPD-photolyase restores the infectivity of UV-damaged fowlpox virus isolated from infected scabs of chickens.

Fowlpox virus (FWPV), an important pathogen of poultry, replicates very efficiently in the featherless areas of skin, and persists in dried and desiccated scabs for prolonged periods. Although the molecular mechanisms underlying the stability of the virus are not completely known, we recently identified the presence of a virus-encoded novel DNA repair enzyme, CPD-photolyase, in FWPV. This enzyme repairs the ultraviolet (UV)-induced pyrimidine dimers, converting them to monomers using photons from white light as a renewable source of energy. In this study, we examined the role of photolyase in the pathogenesis of fowlpox. A comparison of pathogenesis of fowlpox in chickens infected with parental FWPV with that in chickens infected with photolyase-deficient FWPV (Phr(-) FWPV) found no significant differences in terms of replication of virus or formation of secondary lesions. When the virions isolated from infected scabs were exposed to UV light, UV-damaged parental FWPV, unlike Phr(-) FWPV, were rescued through the CPD-photolyase-mediated photoreactivation pathway by at least 48%. However, the mutant virus triggered host's immune response and conferred complete protection against subsequent challenge with virus similar to that conferred by the parental virus. Since the mutant virus is less stable than the parental virus in the infected scabs but is as immunogenic, Phr(-) FWPV might be less persistent in the environment. Furthermore, this particular genetic locus can also be used to insert foreign genes for the development of FWPV recombinant vaccines.

Animals↗
PubMed2001

Fowlpox virus encodes a novel DNA repair enzyme, CPD-photolyase, that restores infectivity of UV light-damaged virus.

Fowlpox virus (FPV), a pathogen of poultry, can persist in desiccated scabs shed from infected hosts. Although the mechanisms which ensure virus survival are unknown, it is likely that some type of remedial action against environmentally induced damage is required. In this regard, we have identified an open reading frame (ORF) coding for a putative class II cyclobutane pyrimidine dimer (CPD)-photolyase in the genome of FPV. This enzyme repairs the UV light-induced formation of CPDs in DNA by using blue light as an energy source and thus could enhance the viability of FPV during its exposure to sunlight. Based on transcriptional analyses, the photolyase gene was found to be expressed late during the FPV replicative cycle. That the resultant protein retained DNA repair activity was demonstrated by the ability of the corresponding FPV ORF to complement functionally a photolyase-deficient Escherichia coli strain. Interestingly, insertional inactivation of the FPV photolyase gene did not impair the replication of such a genetically altered virus in cultured cells. However, greater sensitivity of this mutant than of the parental virus to UV light irradiation was evident when both were subsequently photoreactivated in the absence of host participation. Therefore, FPV appears to incorporate its photolyase into mature virions where the enzyme can promote their survival in the environment. Although expression of a homologous protein has been predicted for some chordopoxviruses, this report is the first to demonstrate that a poxvirus can utilize light to repair damage to its genome.

Amino Acid Sequence↗
PubMed2001

Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk.

Staphylococcus aureus is an economically important and a major mastitis-causing pathogen that also poses food safety and antimicrobial resistance threats. Substances in mastitic milk inhibit the Taq DNA polymerase reaction (Taq PCR) making it of limited use for detecting S. aureus mastitis. In the study reported here, a set of oligonucleotide primers of 21 and 24 bases was used in Taq-PCR to amplify DNA from S. aureus (isolates from bovine mastitis). A specific amplicon of 270 bp was generated as predicted. Replacing Taq DNA polymerase with Thermus thermophilus (Tth) DNA polymerase alone (Tth-PCR) raised the sensitivity of S. aureus detection in milk from experimentally infected cows from 65 to 80%. Combining the use of Tth DNA polymerase and the purification of crude DNA extract using Chelex-100 before PCR raised the sensitivity to 100%. In a random survey involving 100 milk samples from cattle not infected with S. aureus, the test was 100% specific. With milk samples from clinical cases of bovine mastitis, 100% sensitivity and specificity were also observed. It is concluded that Tth-PCR on milk samples with the purification of crude DNA extracts using Chelex-100 is as sensitive as but faster than conventional milk bacteriological culture techniques and is highly specific. The modified PCR correlates with elevated somatic cell counts, detects evidence of chronic and resolving infection based on S. aureus-specific DNA and circumvents the endogenous inhibitory effects of milk.

Animals↗
PubMed2000

Characterization of poxviruses from forest birds in Hawaii.

Two strains of avian pox viruses were isolated from cutaneous lesions in Hawaiian crows (Corvus hawaiiensis) examined in 1994 and a third from a biopsy obtained in 1992 from an infected bird of the Apapane species (Himatione sanguinea) by inoculation of the chorioallantoic membranes (CAM) of developing chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies characteristic of pox virus infection. The pathogenicity of these three viruses in domestic chickens was mild as evidenced by the development of relatively minor lesions of short duration at the sites of inoculation. Their virulence in this host was similar to that of a fowlpox virus (FPV) vaccine strain and contrasted greatly with the ability of two field strains of FPV to produce extensive proliferative lesions. One of the Hawaiian crow pox virus isolates as well as the one originating from the Apapane species could be propagated in two secondary avian cell lines, QT-35 and LMH. A comparison of the restriction fragment length polymorphisms (RFLP) of the genomes of the two cell line-adapted viruses, generated by EcoRI digestion, revealed a limited degree of similarity. Moreover, neither profile was comparable to those of the two field isolates of FPV, which were almost indistinguishable from each other. Thus, based on the genetic distinctness of the two Hawaiian bird viruses, they appear to represent different strains of avipoxvirus.

Animals↗
PubMed1999

Swinepox virus as a vaccine vector for swine pathogens.

Several small and large viruses (e.g., adenovirus, poxvirus, and herpesviruses) have been investigated as vaccine vectors. Each viral system has its advantages and disadvantages. One major advantage for viral vector vaccines is their ability to elicit a protective cell-mediated immunity as well as a humoral response to the antigen delivered by the vector. One major problem to using recombinant viruses as vaccines is the pathogenic potential of the parent virus. Therefore, it is important that along with the optimal expression of the foreign genes and ability to provide protection, the pathogenicity of the vector virus must be reduced during genetic manipulation without affecting its multiplication. The requirements to develop a viral vector, for example, swinepox virus, are a cell culture system that will support the growth of the virus, a suitable nonessential region(s) in the virus genome for insertion of foreign DNA so that virus replication is not affected, a foreign gene(s) that encodes for an immunogenic protein of a swine pathogen, strong transcriptional regulatory elements (promoters) necessary for optimal expression of the foreign genes, a procedure for delivering the foreign gene(s) into the nonessential locus, and a convenient method of distinguishing the recombinant viruses from the parent wild-type virus. Using this methodology, recombinant swinepox virus vaccines expressing pseudorabies virus antigens have been developed and shown to provide protection against challenge. These studies and evidence of local infection of the oral tract by swinepox virus indicate its potential as a recombinant vector for providing immunity against various swine pathogens including those that infect the respiratory and gastrointestinal tracts.

Animals↗
PubMed1998

Detection of Staphylococcus aureus in milk by use of polymerase chain reaction analysis.

OBJECTIVE: To evaluate polymerase chain reaction (PCR) analysis for detection of Staphylococcus aureus (nuc gene) in fresh and formalin-preserved milk. SAMPLE POPULATION: Samples from 80 lactating sheep and 100 lactating dairy cows. PROCEDURE: 4 lactating sheep were inoculated with S aureus by intramammary infusion. A set of primers specific for the nuc gene of S aureus was used to develop a PCR technique, and modification of the rapid boil method was used to isolate bacterial DNA. Milk was obtained from experimentally infected sheep before and after infusion with S aureus, and from the 100 cows and remaining 76 sheep. Samples were screened by bacteriologic culture and PCR. To validate the PCR assay, S aureus or other pathogens were added to distilled water and "normal" sheep milk samples, with and without formalin. RESULTS: The PCR assay was 100% specific for S aureus when known negative and positive samples were tested. Sensitivity was 100% for samples with added S aureus or other pathogens. Sensitivity was lower for samples obtained from experimentally infected sheep, but increased from 53% to 90% with increased washing of target DNA. CONCLUSIONS: The PCR technique based on the nuc gene is able to detect S aureus in sheep milk yields results faster than does traditional culturing, is highly specific, and is able to detect S aureus in formalin-fixed milk samples. CLINICAL RELEVANCE: The assay is particularly suitable for analysis of samples shipped or stored without refrigeration. Although antibiotics in milk may inhibit growth in culture, they should not affect the results of the PCR assay.

Animals↗
PubMed1995

Generation of thymidine kinase-deficient mutants of infectious laryngotracheitis virus.

Current vaccines for the avian respiratory disease infectious laryngotrachetitis consist of naturally attenuated strains of the causative agent--the herpesvirus infectious laryngotracheitis virus (ILTV). Due to the dissemination of these viruses from vaccinated chickens as well as their possible reversion to more pathogenic forms, the use of genetically engineered viral vaccines lacking virulence factors while retaining antigenicity is being considered. Since the thymidine kinase (TK) activity of herpesviruses has been associated with virulence, inactivation of the encoding gene in the ILTV genome should attenuate the virus. Moreover, by analogy to other TK- herpesviruses, the ability of such ILTV mutants to induce a protective response in chickens should not be compromised. Therefore, the deliberate genetic alteration of ILTV was attempted. In order to prevent reversion and also to enable identification of the modified virus, a "marker" transcriptional unit (Escherichia coli lacZ gene fused to a SV-40 3'-polyadenylation signal sequence and regulated by the pseudorabies virus gX gene promoter) was inserted via homologous recombination at one of two loci within the ILTV TK gene. Recombinant viruses were identified and plaque-purified on the basis of their ability to produce beta-galactosidase. Retention of the foreign DNA at the predicted sites in the genomes of the recombinant ILTV was verified by Southern hybridization. Since their replication was unaffected by the thymine analog 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)-5-methyluracil, the recombinants appeared to have a TK- phenotype. Despite this apparent deficiency, prior inoculation of either recombinant virus into chickens afforded the birds protection against a lethal challenge of virulent ILTV. Moreover, the degree of respiratory distress in the chickens vaccinated with the recombinants was relatively mild compared to the severe reaction in birds receiving the parental virus. Thus, ILTV can be genetically attenuated without an accompanying loss of immunogenicity.

Animals↗
PubMed1993

Amplification of rDNA loci to detect and type Neisseria meningitidis and other eubacteria.

In 1991-92, Neisseria meningitidis group C was isolated from the blood of eight students in Urbana, Illinois, USA, and from the cerebrospinal fluid of one student from a nearby community, Decatur, Illinois. These and other bacterial species were analysed by PCR fingerprinting using primers selected from the ribosomal (r)DNA loci. A rDNA primer pair spanning a region within the 16S rDNA amplified a predicted 280 base pair (bp) DNA fragment from Neisseria spp. and fragments of different sizes for other genera. This primer pair specifically detected a carrier of N. meningitidis in a small clinical battery. Identity of the fragment was confirmed by restriction endonuclease analysis. A 600 bp fragment was also amplified from the 16S-23S internal transcribed spacer (ITS) of N. meningitidis; amplification from six other genera yielded different-sized fragments. Digestion of the ITS fragment from N. meningitidis with Alu I revealed three patterns; pattern I was found only for serogroup C isolates, and it was the dominant pattern among recent isolates with the exception of the one from Decatur. The isolate from Decatur yielded pattern III which suggested a non-clonal relationship to the seven isolates from Urbana. Patterns II and III were more prevalent in isolates from the 1960's and 1980's. PCR-based analysis of these loci can complement the techniques which are currently used for the detection and typing of these and other eubacteria.

Adult↗
PubMed1991

Identification and nucleotide sequence of the thymidine kinase gene of swinepox virus.

Using degenerative oligonucleotide probes, representing two different conserved regions of poxvirus and mammalian thymidine kinase (TK) genes, the swinepox virus (SPV) TK gene was mapped to a 1.7-kb BamHI-HindIII fragment of the viral genome. Nucleotide sequencing of this DNA piece revealed that the SPV TK gene was encoded by an open reading frame (ORF) of 177 codons. Immediately downstream of the TK gene was a second ORF with homologues at the same location in both capripoxvirus and leporipoxvirus genomes. A similar gene had translocated to near the left hand terminus of the vaccinia virus (orthopoxvirus) genome. Flanking the two SPV genes were ORFs whose counterparts in other poxvirus genera are located at the same relative positions. SPV appeared to be most closely related to capripoxvirus, based on the organization of the four genes and on the percentage of identical amino acid residues of the respective encoded proteins.

Amino Acid Sequence↗
PubMed1989

Genetic and antigenic differences between fowlpox and quailpox viruses.

The genomes of a fowlpox and quailpox virus isolate were compared by restriction enzyme analysis using BamHI, EcoRI, and HindIII endonucleases. The genetic profiles of the two virus species were very distinct with fragments lacking similar electrophoretic mobilities. In contrast, the patterns of three quailpox virus isolates were very similar with a high proportion of co-migrating fragments. When the immunogenic proteins of two quailpox, three fowlpox, a juncopox, and a pigeonpox virus isolate were examined by immunoblotting, common as well as unique antigens were detected. The greatest disparity was between quailpox virus and the other three species which are genomically conserved. Therefore, on the basis of genetic as well as immunological analysis, quailpox virus is a distinct species of the genus Avipoxvirus.

Animals↗
PubMed1988

A rapid method for identifying the thymidine kinase genes of avipoxviruses.

The thymidine kinase (TK) genes of poxviruses can be rapidly located without using TK- mutants or having to restriction map and clone the viral genomes. Identification of the TK gene is based on in situ gel hybridization with an end-labelled degenerate oligonucleotide probe, representing a consensus sequence near the 3' end of the gene. Restriction fragments of the viral DNAs are electrophoresed in agarose gels and annealed with the probe. Using this method, the TK genes of fowl pox (FPV) and quail pox (QPV) viruses were initially localized to HindIII fragments of approximately 3.8 and 6.7 kb, respectively. After inserting these fragments into pUC 19, recombinant plasmids containing the TK genes were screened by a modified in situ gel annealing procedure. Restriction mapping of the two cloned fragments and subsequent hybridization analysis more precisely placed at least the 3' portion of the FPV and QPV TK genes within a 1.4 kb ClaI-XbaI and 1.7 kb ClaI-PstI fragment, respectively. The site of the FPV TK gene was verified by comparison to the mapped position of the similar gene in an Australian FPV. The location of the QPV TK gene was confirmed by hybridization with the FPV TK gene, despite the apparent divergency of these two genes.

Cloning, Molecular↗
PubMed1988

Genomic and antigenic characterization of avipoxviruses.

A permanent cell line of avian origin, QT-35, was used for the propagation of avipoxvirus isolates, including juncopox, pigeon pox, and field and vaccine strains of fowlpox viruses. The genomes of these avipoxvirus isolates were compared by restriction enzyme analysis using BamHI and HindIII endonuclease digestion and subsequent agarose gel electrophoresis. The genetic profiles of the virus strains were very similar, with a high proportion of comigrating fragments, although most strains could still be distinguished; therefore, these avipoxviruses appear to be closely related. Similar results were obtained when the immunogenic proteins of 6 fowlpox virus strains were examined by immunoblotting. Although the majority of the antigens were common, the strains could be differentiated by unique proteins of differing electrophoretic mobilities.

Animals↗
PubMed1985

Experimental infection of lactating goats with Leptospira interrogans serovars pomona and hardjo.

Pathogenesis of Leptospira interrogans serovars pomona and hardjo was evaluated in 14 lactating goats. Although mild clinical signs of leptospiral infection characterized by pyrexia and reduction in milk yield appeared in some animals, a consistent clinical pattern was not observed in the inoculated animals. The pomona serovar was isolated from the kidney of 1 of the 4 goats inoculated with serovar pomona. The hardjo serovar (strain UI 750) was isolated in the rabbit serum-supplemented bovine albumin polysorbate-80 liquid medium only from the mammary gland of 1 of 4 goats at 13 days after inoculation with serovar hardjo. The positive culture was detected after an 8-month incubation period.

Animals↗
PubMed1985

Experimental infection of pregnant and lactating goats with Leptospira interrogans serovars hardjo and szwajizak.

Pathogenesis of 2 Leptospira serovars, hardjo and szwajizak, was studied in pregnant and lactating goats. Although clinical signs of leptospiral infection were minimal, cultural isolations were made from the mammary gland of 2 goats and the kidney of 1 goat inoculated with serovar hardjo (C846). The isolations were made only on solid bovine albumin polysorbate-80 medium supplemented either with rabbit serum or sodium pyruvate. Cultural isolations of serovar szwajizak were made from kidney, liver, brain, urine, and mammary gland samples of 1 goat and the liver and kidney samples of its kids. These isolations were made in only the solid bovine albumin polysorbate-80 medium which had been supplemented with normal goat serum.

Animals↗
PubMed1980

Growth of hebdomadis group of leptospires in solid medium.

Leptospira hardjo, szwajizak, and sejroe produced colonial growth in bovine albumin polysorbate 80 (BAP80) solid medium which had been supplemented either with sodium pyruvate (100 micrograms/ml) or with 5% rabbit serum. Leptospira hardjo produced two morphologic types of colonies in both supplemented media. Growth did not occur with any of the three serovars in the unsupplemented BAP80 medium. These results indicate that addition of either rabbit serum or sodium pyruvate would aid in promoting growth of some fastidious leptospires.

Culture Media↗