Biomedical subjects

K Venugopal

Publications and source records attributed to K Venugopal.

At least 55 records · Page 3Linked to original sources

PubMed2000

Avian endogenous retrovirus EAV-HP shares regions of identity with avian leukosis virus subgroup J and the avian retrotransposon ART-CH.

The existence of novel endogenous retrovirus elements in the chicken genome, designated EAV-HP, with close sequence identity to the env gene of avian leukosis virus (ALV) subgroup J has been reported (L. M. Smith, A. A. Toye, K. Howes, N. Bumstead, L. N. Payne, and K. Venugopal, J. Gen. Virol. 80:261-268, 1999). To resolve the genome structure of these retroviral elements, we have determined the complete sequence of two proviral clones of EAV-HP from a line N chicken genomic DNA yeast artificial chromosome library and from a meat-type chicken line 21 lambda library. The EAV-HP sequences from the two lines were 98% identical and had a typical provirus structure. The two EAV-HP clones showed identical large deletions spanning part of the gag, the entire pol, and part of the env genes. The env region of the EAV-HP clones was 97% identical to the env sequence of HPRS-103, the prototype subgroup J ALV. The 5' region of EAV-HP comprising the R and U5 regions of the long terminal repeat (LTR), the untranslated leader, and the 5' end of the putative gag region were 97% identical to the avian retrotransposon sequence, ART-CH. The remaining gag sequence shared less than 60% identity with other ALV sequences. The U3 region of the LTR was distinct from those of other retroviruses but contained some of the conserved motifs required for functioning as a promoter. To examine the ability of this endogenous retroviral LTR to function as a transcriptional promoter, the EAV-HP and HPRS-103 LTR U3 regions were compared in a luciferase reporter gene assay. The low luciferase activity detected with the EAV-HP LTR U3 constructs, at levels close to those observed for a control vector lacking the promoter or enhancer elements, suggested that these elements function as a weak promoter, possibly accounting for their low expression levels in chicken embryos.

5' Untranslated Regions↗

PubMed2000

Neoplastic diseases: Marek's disease, avian leukosis and reticuloendotheliosis.

The commercially important neoplastic diseases of poultry are Marek's disease, which is caused by a herpesvirus, and the avian leukoses and reticuloendotheliosis, which are caused by retroviruses. These diseases are responsible for economic loss due to both mortality and depressed performance. Marek's disease virus (MDV) and avian leukosis viruses (ALVs) are prevalent throughout the world, and new strains which arise in particular locations may spread across borders, thereby undermining national disease control measures. Reticuloendotheliosis virus (REV) is also present in many countries. Marek's disease virus is transmitted horizontally only, and international spread in hatching eggs and day-old chicks can be prevented by appropriate hygiene precautions. Transmission of ALV and REV occurs both horizontally and vertically (through the egg), and measures to prevent international spread are more demanding. Marek's disease is controlled by vaccination, whilst avian leukosis is controlled by virus eradication programmes, mainly at the primary breeding level. Similar virus control measures can be applied for reticuloendotheliosis if necessary. No strong evidence exists to suggest that these avian tumour viruses constitute a danger to public health.

Animals↗

PubMed1999

Avian leukosis virus subgroup J: a rapidly evolving group of oncogenic retroviruses.

A strain of avian leukosis virus (ALV) belonging to a new envelope subgroup J was isolated in the UK in 1988 from meat-type chickens. The disease caused by the members of this subgroup has since spread very rapidly worldwide and has become one of the major problems facing the broiler meat industry. Molecular characterisation of HPRS -103, the prototype of subgroup J, has shown that it has a structure of a typical ALV with gag, pol and env genes. However the env gene was distinct from that of other ALV s and was closely related to that of novel endogenous retroviral elements designated EAV - HP. As other regions of the genome were closely related to ALV s, it is believed that ALV-J has evolved by recombination with the env sequences of EAV - HP. ALV-J has a tropism for myeloid cells, a feature that may be associated with its ability to induce myeloid leukosis. Recent data show that ALV -J isolates evolve rapidly resulting in sequence changes within the variable regions of the env gene leading to antigenic variation. Eradication programmes established for other subgroups are proving to be effective in eradicating ALV-J from infected flocks.

Animals↗

PubMed1999

Novel endogenous retroviral sequences in the chicken genome closely related to HPRS-103 (subgroup J) avian leukosis virus.

HPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, is a recently identified retrovirus associated with myeloid leukosis in meat-type chickens. Although this virus shows high sequence identity to other ALV subgroups within the gag and pol genes, its env gene is highly diverged (with only about 40% sequence identity) from other ALV subgroups. On the other hand, the sequence of the env gene of HPRS-103 was 75% identical to that of E51, a member of the EAV family of endogenous avian retroviruses. It is reported here that the chicken genome also contains another EAV-related element, EAV-HP, showing much greater sequence identity (over 97%) to the HPRS-103 env gene. Southern blotting analysis showed that EAV-HP-related sequences were distinct from EAV-O and were present in all lines of chicken examined and in grey jungle fowl, but were absent from several other avian species. The potential role of these endogenous sequences in the evolution of ALV subgroup J viruses is discussed.

Amino Acid Sequence↗

PubMed1998

Development and application of polymerase chain reaction (PCR) tests for the detection of subgroup J avian leukosis virus.

Subgroup J avian leukosis virus (ALV) is a recently identified avian retrovirus associated with myeloid leukosis in meat-type chickens. The env gene of the HPRS-103 strain of ALV, the prototype of this subgroup, differs considerably from that of other subgroups, but shows close homology to the env-like sequences of members of the EAV family of endogenous retroviruses. Polymerase chain reaction (PCR) tests using two sets of primers were developed for the specific detection of the members of this new subgroup along with another pair of primers for detecting other subgroup viruses. The specificity and sensitivity of this detection system was compared with the conventional detection methods in experimentally and naturally infected samples. The use of PCR was found to be rapid, specific and more sensitive than the conventional diagnostic tests for the detection of ALV. Moreover, the two subgroup J ALV-specific PCR tests were found to be capable of differentiating between 'prototype-like' viruses and more recent isolates which show extensive antigenic and sequence variations. The use of this test as a rapid and sensitive method of detection of viruses in epidemiological studies and eradication programs is discussed.

Animals↗

PubMed1998

Antigenic variants of J subgroup avian leukosis virus: sequence analysis reveals multiple changes in the env gene.

HPRS-103, the prototype of avian leukosis virus (ALV) subgroup J, was isolated in 1989 from meat-type chickens from commercial flocks where it induces myelocytic myeloid leukosis (ML). The HPRS-103 env gene differs considerably from other ALV subgroups but shows high identity (75-97%) to env-like sequences of the different members of the EAV family of endogenous avian retroviruses. Recently, we have isolated several viruses related to HPRS-103 from cases of ML. Although these isolates showed properties of ALV subgroup J, the majority of them resisted neutralization by HPRS-103-specific serum, suggesting antigenic variation. The nucleotide sequence of the env gene of the variant viruses showed several substitutions resulting in amino acid changes especially clustered in the variable regions hr1, hr2 and vr3. Analysis of the data suggests that selection pressure, probably from the immune response, is driving the antigenic variation among the isolates. Phylogenetic analysis of the sequences showed the evolutionary relationships of the isolates with HPRS-103 and the EAV family of endogenous avian retroviruses. The epidemiological significance of the antigenic variation and the emergence of variant viruses are discussed.

Amino Acid Sequence↗

PubMed1997

Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs.

The complete nucleotide sequence of two tick-transmitted flaviviruses, Vasilchenko (Vs) from Siberia and louping ill (LI) from the UK, have been determined. The genomes were respectively, 10928 and 10871 nucleotides (nt) in length. The coding strategy and functional protein sequence motifs of tick-borne flaviviruses are presented in both Vs and LI viruses. The phylogenies based on maximum likelihood, maximum parsimony and distance analysis of the polyproteins, identified Vs virus as a member of the tick-borne encephalitis virus subgroup within the tick-borne serocomplex, genus Flavivirus, family Flaviviridae. Comparative alignment of the 3'-untranslated regions revealed deletions of different lengths essentially at the same position downstream of the stop codon for all tick-borne viruses. Two direct 27 nucleotide repeats at the 3'-end were found only for Vs and LI virus. Immediately following the deletions a region of 332-334 nt with relatively conserved primary structure (67-94% identity) was observed at the 3'-non-coding end of the virus genome. Pairwise comparisons of the nucleotide sequence data revealed similar levels of variation between the coding region, and the 5' and 3'-termini of the genome, implying an equivalent strong selective control for translated and untranslated regions. Indeed the predicted folding of the 5' and 3'-untranslated regions revealed patterns of stem and loop structures conserved for all tick-borne flaviviruses suggesting a purifying selection for preservation of essential RNA secondary structures which could be involved in translational control and replication. The possible implications of these findings are discussed.

Animals↗

PubMed1996

Immunisation with DNA polynucleotides protects mice against lethal challenge with St. Louis encephalitis virus.

In vivo transfection by intramuscular injection with plasmids expressing the immunogenic proteins of microbial pathogens has considerable potential as a vaccination strategy against many pathogens of both man and animals. Here we report that weanling mice given a single intramuscular injection of 50 micrograms of a plasmid, pSLE1 expressing the St. Louis encephalitis virus (SLE) prM/E protein under the control of the cytomegalovirus immediate early protein promoter produced SLE-specific antibody and were protected against lethal challenge with the virulent virus. Polynucleotide vaccine technology provides a unique opportunity to produce vaccines against flavivirus diseases of low incidence cheaply and rapidly, and to produce multivalent vaccines such as would be required for immunisation against dengue virus disease.

Animals↗

PubMed1995

An arbovirus cline across the northern hemisphere.

The mode and tempo of arbovirus evolution and dispersal can help to explain the dynamics of pandemics, viral outbreaks, and emerging viruses. By comparing nucleotide and deduced amino acid sequences of their envelope proteins, we describe the continuous distribution of the tick-borne encephalitis (TBE) complex viruses, the most important flaviviruses in Europe, across major geographical areas and the conditions under which mutations occur. The analyses reveal a correlation between the geographical and genetic distances of these viruses. The arthropod host appears to be a key factor for the formation and maintenance of this cline by constraining TBE dispersal and evolution. This is also illustrated by comparisons with mosquito-borne flaviviruses.

Animals↗

PubMed1995

Immunogenic properties of rabbit haemorrhagic disease virus structural protein VP60 expressed by a recombinant baculovirus: an efficient vaccine.

We have constructed a recombinant baculovirus containing the gene encoding the structural protein VP60 from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). Infection of cultured Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of high yields of VP60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. A VP60-dose study showed that the recombinant protein was able to elicit a protective response in rabbits against a nasal challenge with 100 LD50 of RHDV. The effective dose able to protect 50% of the animals in the absence of adjuvant was found to be 10-25 micrograms of recombinant VP60.

Animals↗

PubMed1995

Immunity to St. Louis encephalitis virus by sequential immunization with recombinant vaccinia and baculovirus derived PrM/E proteins.

St. Louis encephalitis (SLE) is an important mosquito-borne disease of great public health concern in parts of the United States. South America and Canada. Protective immunogens of flaviviruses produced in different expression systems have been shown to be effective against virulent virus infection in laboratory animal models. Here we show that the pre-membrane and envelope (PrM/E) of SLE virus expressed in insect and mammalian cell systems using baculovirus and vaccinia virus, respectively, are processed correctly and showed similar antigenic characteristics as the authentic proteins. Immunization with the recombinant proteins individually or in combination resulted in neutralizing and protective immune responses. A schedule consisting of initial immunization with recombinant vaccinia virus followed by a secondary boost with recombinant baculovirus protein resulted in higher levels of neutralizing and protective immune responses. The advantages of the use of such a combined approach as a general immunization strategy are discussed.

Animals↗

PubMed1995

Development of an IgM capture assay for the diagnosis of B19 parvovirus infection using recombinant baculoviruses expressing VP1 or VP2 antigens.

BACKGROUND: The clinical manifestations of human parvovirus B19 infection are often similar to those induced as the result of infection by other infectious agents such as rubella and some bacteria. Although diagnosis of B19 infection is feasible by detection of specific antibodies, the tests require viraemic serum as a source of antigen. This inevitably leads to problems of reproducibility and dependence upon appropriate high quality clinical material. OBJECTIVES: To develop a monoclonal antibody capture ELISA (MACEIA) for detecting anti-B19 IgM antibody in human sera, using recombinant baculoviruses expressing the B19 parvovirus VP1 and VP2 proteins and to compare this with MACEIA using a plasma derived B19 antigen. STUDY DESIGN: Sera from 85 patients with proven B19 infection and the paired convalescent sera from 26 anti-B19 IgM-positive acute samples were examined for B19-specific IgM antibody by a monoclonal antibody capture assay that utilised recombinant baculoviruses expressing B19 proteins in lieu of a plasma-derived B19 antigen. Control samples consisted of 24 anti-rubella IgM, 24 anti-EBV IgM and 102 negative sera from uninfected individuals. RESULTS: Eighty-four of the 85 sera were anti-B19 IgM positive by MACEIA using recombinant baculovirus derived B19 antigen and by indirect immunofluorescence tests, whereas 79 were positive by MACEIA using plasma-derived antigen. Of the 26 convalescent samples which were positive as acute sera, 4 had become negative by 8 weeks post-infection. The expressed recombinant baculovirus antigens had identical molecular weights to the VP1 (84 kDa) and VP2 (58 kDa) proteins of virus purified from human plasma. Recombinant baculovirus-derived VP1 antigen was as effective as VP2 particles at detecting antibodies. CONCLUSIONS: Recombinant proteins VP1 and VP2, obtained from recombinant baculovirus-infected cell lysate, showed equal specificity to and higher sensitivity than, B19 virus purified from human plasma when used in MACEIA to detect B19-IgM antibody.

Journal Article↗

PubMed1995

Intracellular interference of tick-borne flavivirus infection by using a single-chain antibody fragment delivered by recombinant Sindbis virus.

A single-chain antibody fragment that identifies a neutralizing epitope on the envelope protein of louping ill and some other tick-borne flaviviruses was previously expressed in soluble form from bacteria and shown to be functionally active in vitro. To see whether or not the single-chain antibody could bind and inactivate infectious virus in vivo, we have used recombinant Sindbis virus as a delivery vehicle for intracellular expression of the antibody fragment. The variable genes and interchain linker encoding the single-chain antibody were cloned into a double subgenomic Sindbis virus expression vector to generate recombinant Sindbis virus. Infection with this recombinant Sindbis virus provided high-level cytoplasmic expression of the antibody fragment in mammalian cells. We demonstrate (i) that the antibody fragment was antigen binding and (ii) that louping ill virus infectivity was significantly reduced in the presence of intracellular antibody expressed by the superinfecting recombinant Sindbis virus.

Animals↗

PubMed1994

A single chain antibody fragment expressed in bacteria neutralizes tick-borne flaviviruses.

A recombinant single chain antibody fragment (scFv) that identifies a neutralizing epitope on the envelope glycoprotein of louping iII (LI) and tick-borne encephalitis (TBE) virus has been developed using a bacteriophage expression system. The mRNA was extracted from a cloned hybridoma cell culture that produces a mouse monoclonal antibody (MAb 4.2) known to map to amino acids 308-311 of LI and TBE virus, corresponding to domain B on the proposed two-dimensional model of the tick-borne encephalitis virus envelope protein. The V-genes encoding the antigen-binding site of MAb 4.2 were amplified and cloned for expression as a fusion protein to the pIII coat protein of filamentous phage. Solid phase selection of these phage against the LI virus antigen, was necessary to isolate the correct MAb 4.2 scFv fragment which was subsequently produced in soluble form in bacteria and harvested from the culture supernatant medium. The characteristics of this expressed single chain antibody were compared with MAb 4.2. The expressed antibody portrayed the antigenic specificity of MAb 4.2 and also neutralized the infectivity of louping iII and some other tick-borne flaviviruses. The potential of this technique for studying antigen-antibody interactions and for the development of prophylactic reagents are discussed.

Amino Acid Sequence↗

PubMed1994

Recombinant vaccinia virus expressing PrM and E glycoproteins of louping ill virus: induction of partial homologous and heterologous protection in mice.

Recombinant vaccinia viruses expressing either the premembrane/truncated envelope (PrM/TrE) or truncated envelope (TrE) protein of louping ill virus were constructed. Both constructs expressed authentic E proteins as determined by their size and antigenic reactivity with a panel of monoclonal antibodies. The deletion of the C-terminal hydrophobic domain of the envelope glycoprotein resulted in the secretion of E protein into the supernatant culture medium. The immunisation of mice with these recombinant viruses showed that the recombinant expressing PrM/TrE proteins induced neutralising and protective antibodies against challenge with louping ill or tick-borne encephalitis virus, but that the recombinant expressing the E or the TrE protein alone failed to induce any detectable immune responses against homologous or heterologous virus challenge.

Animals↗

PubMed1994

Tick-borne flavivirus NS1 gene: identification of conserved peptides and antigenic analysis of recombinant louping ill virus NS1 protein.

The nucleotide sequence of the NS1 gene of louping ill (LI) virus has been determined. The sequence shows a high degree of homology with other members of the tick-borne serocomplex of flaviviruses and a lower homology with the mosquito-borne flaviviruses. Alignment of the deduced NS1 amino acid sequences with all tick-borne flavivirus NS1 sequences, identified four peptide regions which were conserved for all tick-borne flaviviruses, but were variable amongst mosquito-borne flaviviruses. A dendrogram, derived from the alignment of the NS1 protein sequences, indicated an evolutionary relationship that quite closely reflects the recognised serological classification. The LI virus NS1 protein expressed in Escherichia coli and baculoviruses showed similar antigenic reactivity to the authentic virus-coded protein when tested with NS1-specific monoclonal antibodies, but did not form high molecular weight complexes and was not secreted from cells.

Amino Acid Sequence↗

PubMed1994

Towards a new generation of flavivirus vaccines.

Flavivirus diseases have caused great public health concern for over three centuries, with diseases like yellow fever, dengue, Japanese encephalitis and tick-borne encephalitis causing thousands of deaths. Although yellow fever epidemics can be brought under control by the use of vaccine or mosquito-control measures, there have been many examples of its re-emergence as an epidemic disease. Similarly, the use of vaccines or arthropod-control measures have failed to prevent the spread of other flaviviruses such as Japanese encephalitis virus. There has been rapid growth in the knowledge of molecular information on flaviviruses in the last decade, and on the basis of this information several potential recombinant subunit vaccines are being developed and appear to be effective experimentally. Moreover, the assumption that humoral immunity induced by virus structural envelope glycoproteins is the only effective means of providing protection against flavivirus infection can be questioned. This review attempts to summarize recent thinking in this field and to evaluate the different systems available as potential future flavivirus vaccines in inducing protective immunity.

Flavivirus↗

PubMed1994

Analysis of the structural protein gene sequence shows Kyasanur Forest disease virus as a distinct member in the tick-borne encephalitis virus serocomplex.

Kyasanur Forest disease (KFD) virus is a highly pathogenic member of the family Flaviviridae producing a haemorrhagic disease in infected human beings. Despite this high pathogenicity and potential epidemiological importance, there have been relatively few detailed antigenic or molecular studies on KFD virus. The nucleotide sequences of the genes encoding the structural proteins of the virus have now been determined. From these data we conclude that KFD virus is a distinct member in the tick-borne flavivirus complex with characteristic protease cleavage sites, fusion peptide, signal sequences and hydrophobic transmembrane domains. Comparison of the deduced amino acid sequences of KFD virus showed close relationships with other tick-borne flaviviruses. Among the structural proteins, the E protein showed maximum similarity (77.4% to 81.3%) to tick-borne flaviviruses. Alignment of the amino acid sequence with those of other known tick-borne flaviviruses revealed many conserved regions confirming its identity as a member of the tick-borne encephalitis group, although the genetic marker EHLPTA showed a T-->K substitution in KFD virus. The proposed genetic marker at amino acid positions 232 to 234 (AQE) was unique for KFD virus. A dendrogram derived from the amino acid alignment showed a phylogenetic relationship similar to those obtained on the basis of serological studies. The question of the sudden emergence of KFD virus in India and the possibilities of developing recombinant virus vaccines are discussed.

Amino Acid Sequence↗