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Suresh K. Tikoo - One of the best experts on this subject based on the ideXlab platform.

  • Porcine Adenovirus Type 3 E3 Encodes a Structural Protein Essential for Capsid Stability and Production of Infectious Progeny Virions.
    Journal of virology, 2018
    Co-Authors: Abdelrahman Said, Tekeleselassie A. Woldemariam, Suresh K. Tikoo
    Abstract:

    The Adenovirus E3 region encodes proteins that are not essential for viral replication in vitro The Porcine Adenovirus type 3 (PAdV-3) E3 region encodes three proteins, including 13.7K. Here, we report that 13.7K is expressed as an early protein, which localizes to the nucleus of infected cells. The 13.7K protein is a structural protein, as it is incorporated in CsCl-purified virions. The 13.7K protein appears to be essential for PAdV-3 replication, as mutant PAV13.73A expressing a mutated 13.7K could be isolated only in VIDO AS2 cells expressing the 13.7K protein. Analysis of PAV13.73A suggested that even in the presence of reduced levels of some late viral proteins, there appeared to be no effect on virus assembly and production of mature virions. Further analysis of CsCl-purified PAV13.73A by transmission electron microscopy revealed the presence of disrupted/broken capsids, suggesting that inactivation of 13.7K protein expression may produce fragile capsids. Our results suggest that the PAdV-3 E3 region-encoded 13.7K protein is a capsid protein, which appears to be essential for the formation of stable capsids and production of infectious progeny virions.IMPORTANCE Although E3 region-encoded proteins are involved in the modulation of leukocyte functions (N. Arnberg, Proc Natl Acad Sci U S A 110:19976-19977, 2013) and inducing a lytic infection of lymphocytes (V. K. Murali, D. A. Ornelles, L. R. Gooding, H. T. Wilms, W. Huang, A. E. Tollefson, W. S. Wold, and C. Garnett-Benson, J Virol 88:903-912, 2014), none of the E3 proteins appear to be a component of virion capsid or required for replication of Adenovirus. Here, we demonstrate that the 13.7K protein encoded by the E3 region of Porcine Adenovirus type 3 is a component of progeny virion capsids and appears to be essential for maintaining the integrity of virion capsid and production of infectious progeny virions. To our knowledge, this is the first report to suggest that an Adenovirus E3-encoded protein is an essential structural protein.

  • A novel and simple method for rapid generation of recombinant Porcine adenoviral vectors for transgene expression.
    PloS one, 2015
    Co-Authors: Peng Zhang, Suresh K. Tikoo, Wenbin Wang, Lei Zhang, Zengqi Yang
    Abstract:

    Many human (different serotypes) and nonhuman Adenovirus vectors are being used for gene delivery. However, the current system for isolating recombinant adenoviral vectors is either time-consuming or expensive, especially for the generation of recombinant non-human adenoviral vectors. We herein report a new and simple cloning approach for the rapid generation of a Porcine Adenovirus (PAdV-3) vector which shows promise for gene transfer to human cells and evasion of human Adenovirus type 5 (HAdV-5) immunity. Based on the final cloning plasmid, pFPAV3-CcdB-Cm, and our modified SLiCE strategy (SLiCE cloning and lethal CcdB screening), the process for generating recombinant PAdV-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 clones/ng and zero background (100% accuracy). The recombinant PAdV-3 plasmids could be successfully rescued in Porcine retinal pigment epithelium cells (VR1BL), which constitutively express the HAdV-5 E1 and PAdV-3 E1B 55k genes, and the foreign genes were highly expressed at 24 h after transduction into swine testicle (ST) cells. In conclusion, this strategy for generating recombinant PAdV-3 vectors based on our modified SLiCE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAdV-3 genome as well as other adenoviral genomes.

  • A Porcine Adenovirus with low human seroprevalence is a promising alternative vaccine vector to human Adenovirus 5 in an H5N1 virus disease model.
    PloS one, 2010
    Co-Authors: Ami Patel, Suresh K. Tikoo, Gary P. Kobinger
    Abstract:

    Human Adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent Porcine Adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5.

  • Packaging of viral RNAs in virions of Adenoviruses
    Virology Journal, 2009
    Co-Authors: Li Xing, Suresh K. Tikoo
    Abstract:

    Earlier, we detected viral RNAs packaged in the Porcine Adenovirus (PAdV) -3 virions. Using Southern blot analysis, we further demonstrated that the viral RNAs were predominantly packaged in CsCl purified mature capsids (containing viral genome) than empty/intermediate capsids. Some of the packaged viral RNAs appear to be polyadenylated. Real-time reverse transcription (RT)-PCR analysis indicated that the copy number of the tested viral mRNAs encoding E1B_small and fiber proteins was less than one per full capsid. Moreover, detection of viral RNA packaged in CsCl purified human Adenovirus (HAdV) -5 virions indicates that the viral RNA packaging might be a common phenomenon in members of Adenoviridae family. Further quantitative analysis of viral protein, DNA, and RNA in CsCl purified mature and empty/intermediate capsids of recombinant HAdV-5 expressing enhanced green fluorescent protein indicated that the traceable viral RNA detected in empty/intermediate capsids seems associated with the presence of traceable viral genomic DNA. Taken together, our data suggest that the viral RNAs may be passively packaged in Adenovirus virion during encapsidation of viral genomic DNA in cell nuclei. Thus, viral RNA packaging may be a characteristic feature of adenoviral genomic DNA encapsidation.

  • Porcine Adenovirus type 3 E1B large protein downregulates the induction of IL-8.
    Virology journal, 2007
    Co-Authors: Yan Zhou, Andrew Ficzycz, Suresh K. Tikoo
    Abstract:

    Replication-defective (E1-E3 deleted) Adenovirus vector based gene delivery results in the induction of cytokines including IL-8, which may contribute to the development of inflammatory immune responses. Like other Adenoviruses, E1 + E3 deleted Porcine Adenovirus (PAdV) 3 induces the production of IL-8 in infected cells. In contrast, no IL-8 production could be detected in cells infected with wild-type or mutant PAdV-3s containing deletion in E1A + E3 (PAV211) or E1Bsmall + E3 (PAV212). Expression of PAdV-3 E1Blarge inhibited the NF-κB dependent transcription of luciferase from IL-8 promoter. Imunofluorescence and electrophoretic mobility shift assays suggested that constitutive expression of PAdV-3 E1Blarge inhibited the nuclear translocation of NF-κB and its subsequent binding to DNA. These results suggest that E1Blarge interacts with NF-κB to prevent transcription and down regulate proinflammatory cytokine IL-8 production.

Suresh K. Mittal - One of the best experts on this subject based on the ideXlab platform.

  • Bovine Adenovirus serotype 3 utilizes sialic acid as a cellular receptor for virus entry.
    Virology, 2009
    Co-Authors: Dinesh S Bangari, Anurag Sharma, Suresh K. Mittal
    Abstract:

    Bovine Adenovirus serotype 3 (BAd3) and Porcine Adenovirus serotype 3 (PAd3) entry into the host cells is independent of Coxsackievirus Adenovirus receptor and integrins. The role of sialic acid in BAd3 and PAd3 entry was investigated. Removal of sialic acid by neuraminidase, or blocking sialic acid by wheat germ agglutinin lectin significantly inhibited BAd3, but not PAd3, transduction of Madin-Darby bovine kidney cells. Maackia amurensis agglutinin or Sambucus nigra (elder) agglutinin treatment efficiently blocked BAd3 transduction suggesting that BAd3 utilized alpha(2,3)-linked and alpha(2,6)-linked sialic acid as a cell receptor. BAd3 transduction of MDBK cells was sensitive to sodium periodate, bromelain, or trypsin treatment indicating that the receptor sialoconjugate was a glycoprotein rather than a ganglioside. To determine sialic acid-containing cell membrane proteins that bind to BAd3, virus overlay protein binding assay (VOPBA) was performed and showed that sialylated cell membrane proteins in size of approximately 97 and 34 kDa bind to BAd3. The results suggest that sialic acid serves as a primary receptor for BAd3.

  • Comparative analysis of vector biodistribution, persistence and gene expression following intravenous delivery of bovine, Porcine and human adenoviral vectors in a mouse model.
    Virology, 2009
    Co-Authors: Anurag Sharma, Dinesh S Bangari, Harm Hogenesch, Manish Tandon, Aseem Pandey, Suresh K. Mittal
    Abstract:

    Nonhuman Adenoviruses including bovine Adenovirus serotype 3 (BAd3) and Porcine Adenovirus serotype 3 (PAd3) can circumvent pre-existing immunity against human Adenovirus serotype 5 (HAd5) and are being developed as alternative vectors for gene delivery. To assess the usefulness of these vectors for in vivo gene delivery, we compared biodistribution, persistence, state of vector genome, and transgene and vector gene expression by replication-defective BAd3 and PAd3 vectors with those of HAd5 vector in a FVB/n mouse model following intravenous inoculation. BAd3 vector efficiently transduced the heart, kidney and lung in addition to the liver and spleen and persisted for a longer duration compared to PAd3 or HAd5 vectors. Biodistribution of PAd3 vector was comparable to that of HAd5 vector but showed more rapid vector clearance. Only linear episomal forms of BAd3, PAd3, and HAd5 vector genomes were detected. All three vectors efficiently expressed the green fluorescent protein (GFP) transgene proportionate to the vector genome copy number in various tissues. Furthermore, leaky expression of vector genes, both the early (E4) and the late (hexon) was observed in all three vectors and gradually declined with time. These results suggest that BAd3 and PAd3 vectors could serve as an alternative or supplement to HAd5 for gene delivery applications.

  • Bovine Adenovirus type 3 internalization is independent of primary receptors of human Adenovirus type 5 and Porcine Adenovirus type 3.
    Biochemical and biophysical research communications, 2005
    Co-Authors: Dinesh S Bangari, Anurag Sharma, Suresh K. Mittal
    Abstract:

    Abstract Usefulness of adenoviral vectors derived from human Adenovirus (HAd) type 5 (HAd5) is mainly limited by wide prevalence of preexisting anti-HAd5 immunity as well as non-specific tissue tropism of these vectors. As an alternative, non-human adenoviral vectors including bovine Adenovirus type 3 (BAd3) are currently being investigated. Non-prevalence of BAd3 in humans and its ability to evade preexisting HAd immunity are some of the features that make BAd3 a promising vector for human gene delivery. BAd3 appears to have a tissue tropism distinct from that of HAd5 and also the repertoire of cells efficiently transduced by BAd3 is different. We performed antibody-mediated receptor blocking experiments to show that BAd3 internalization was independent of coxsackievirus–Adenovirus receptor, the primary determinant of HAd5 tropism, or integrin ανβ3, a secondary molecule involved in HAd5 entry. Using homologous and heterologous knob-mediated competition assays with recombinant knobs of HAd5, Porcine Adenovirus type 3 (PAd3), or BAd3, we observed that BAd3 internalization was independent of the primary receptors of HAd5 and PAd3. These results provide support for further exploration of BAd3 vectors for designing targeted vectors for human gene therapy.

  • 152. Coxsackievirus-Adenovirus Receptor (CAR) and |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 Integrin Independent Internalization of Porcine Adenoviral Vectors: Implications in Gene Therapy
    Molecular Therapy, 2005
    Co-Authors: Dinesh S Bangari, Suresh K. Mittal
    Abstract:

    Nonhuman Adenoviruses including Porcine Adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells and circumvents preexisting humoral immunity in humans. Coxsackievirus and Adenovirus receptor (CAR) is the primary receptor and |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 integrin is involved as a secondary receptor for various human Adenovirus (HAd) subtypes including HAd5. In this study we deduced the role of CAR, |[alpha]|v|[beta]|3 or |[alpha]|v|[beta]|5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-|[alpha]|v|[beta]|3 or anti-|[alpha]|v|[beta]|5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant (P

  • Porcine Adenovirus serotype 3 internalization is independent of CAR and αvβ3 or αvβ5 integrin
    Virology, 2005
    Co-Authors: Dinesh S Bangari, Suresh K. Mittal
    Abstract:

    Abstract Nonhuman Adenoviruses including Porcine Adenovirus serotype 3 (PAd3) are emerging vectors for gene delivery. PAd3 efficiently transduces human and murine cells in culture, and circumvents preexisting humoral immunity in humans. The coxsackievirus–Adenovirus receptor (CAR) serves as a primary receptor and α v β 3 or α v β 5 integrin as a secondary receptor for several human Adenovirus (HAd) subtypes including HAd5. In this study, we deduced the role of CAR, α v β 3 or α v β 5 integrin in PAd3 internalization. Transduction experiments were conducted in human mammary epithelial (MCF-10A) cells using replication-defective PAd-GFP (PAd3 vector expressing green fluorescent protein [GFP]) and HAd-GFP (HAd5 vector expressing GFP). MCF-10A cells were treated with or without anti-human CAR, or anti-α v β 3 or anti-α v β 5 integrin antibodies prior to infection with HAd-GFP or PAd-GFP. Significant ( P P v β 3 or α v β 5 integrin-independent and the primary receptor for HAd5 and PAd3 were distinct. CAR- and α v β 3 or α v β 5 integrin-independent entry of PAd3 vectors may have implications in targeting cell types that are not efficiently transduced by other adenoviral vectors.

Eva Nagy - One of the best experts on this subject based on the ideXlab platform.

  • Sequence Analysis of Porcine Adenovirus Serotype 5 Fibre Gene: Evidence for Recombination
    Virus Genes, 2002
    Co-Authors: Miklós Nagy, Eva Nagy, Tamas Tuboly
    Abstract:

    The nucleic acid and deduced amino acid sequence of the fibre gene of the HNF-61 strain of Porcine Adenovirus serotype 5 (PAdV-5) was determined and compared to that of the HNF-70 strain of the same serotype (Nagy et al., J Gen Virol 82 , 525–529, 2001) and also to Adenovirus fibre genes from the genera MastAdenovirus and AtAdenovirus . The putative HNF-61 and HNF-70 proteins were similar to each other, with 90% amino acid identity. Conserved amino acid sequences described for mastAdenovirus fibre shafts were identified in the shaft regions of both PAdV-5 fibres, except for the so-called TLWT motif. The head regions of the PAdV-5 fibre did not resemble any of the known mastAdenovirus fibre heads, but they showed characteristics of the fibre head protein sequences of viruses grouped in the proposed genus AtAdenovirus (Benkö et al., Virus Taxonomy , Seventh Report of the International Committee on the Taxonomy of Viruses, Academic Press, New York, San Diego, 2000, pp. 227–238). The findings suggested recombination between viruses of different Adenovirus genera.

  • The complete nucleotide sequence of Porcine Adenovirus serotype 5.
    Journal of General Virology, 2001
    Co-Authors: Miklós Nagy, Eva Nagy, Tamas Tuboly
    Abstract:

    The complete nucleotide sequence of Porcine Adenovirus serotype 5 (PAdV-5) has been determined and the putative genomic map was constructed. The size of the genome was found to be 32621 nucleotides. Twenty-eight putative ORFs were identified by their homology to other Adenovirus or other virus and eukaryotic genes. Several special protein sequence motifs were identified by their homology to similar protein motifs. The putative promoter regions, polyadenylation and splice sites were predicted and the early and late transcription units were determined. Based on sequence analysis and RNA secondary structure prediction, sequences for virus-associated RNA could not be recognized. Phylogenetic analysis showed that PAdV-5 was more closely related to certain bovine Adenoviruses than to other Porcine Adenoviruses.

  • construction and characterization of recombinant Porcine Adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene
    Journal of General Virology, 2001
    Co-Authors: Tamas Tuboly, Eva Nagy
    Abstract:

    Five recombinant Porcine Adenoviruses of serotype 5 (PAdV-5) carrying the full-length or the 5' 2.2 kb half of the transmissible gastroenteritis virus (TGEV) spike (S) gene were generated by homologous recombination in E. coli strain BJ5183 cells and subsequent transfection of swine testicle cells. The foreign genes were inserted into the E3 region of PAdV-5. One recombinant virus had no deletion in the E3 region, whereas a 1.2 kb fragment was removed from the E3 region in the remainder of the recombinant viruses. One stable construct with a 4.4 kb insertion had a genome size of 109.6% of the wild-type genome, the largest reported for any recombinant Adenovirus. Only those viruses that carried the S gene in the left to right orientation expressed the S gene. Three recombinant viruses were tested by oral immunization of pigs and both antibody response and virus shedding were monitored. None of the pigs showed clinical signs and the virus was recovered from rectal swabs until 6-7 days post-infection. Viruses expressing the S gene induced TGEV- and PAdV-5-specific virus-neutralizing antibodies. Moreover, TGEV-specific secretory IgA was detected in the small intestine and in the lungs of the immunized animals.

  • Sequence analysis and deletion of Porcine Adenovirus serotype 5 E3 region.
    Virus research, 2000
    Co-Authors: Tama´s Tuboly, Eva Nagy
    Abstract:

    A 3000 basepair (bp) region corresponding to the E3 region, the flanking pVIII and part of the fiber protein genes, of the two prototype strains (HNF-61 and HNF-70) of Porcine Adenovirus serotype five (PAdV-5) was sequenced. A potential E3 promoter and poly-A signals were identified. The size of the E3 region was 2039 (strain HNF-61) and 2020 bp (strain HNF-70) the largest E3 so far reported among PAdVs. Three open reading frames (ORF2-4) were identified within the E3 region. Based on the predicted amino acid (aa) sequences ORF2 was similar to other Adenovirus E3 ORFs, ORF3 showed some similarity to a bovine Adenovirus (BAdV-1) ORF. ORF4 was unique to PAdV-5. E3 mRNA transcripts were detected early in infection by Northern blot analysis. Genomic clones of HNF-70 with a 1505 or 1237 bp deletions in the E3 region were constructed to map non-essential regions. After transfection of the DNA into swine testicle cells, virions were recovered for only the shorter 1237 bp deletion. At least 60% of the E3 region was not essential for virus replication, bringing the theoretical vector capacity of a helper independent PAdV-5 to 2.9 kb.

  • Characterization of the Early Region 4 of Porcine Adenovirus Type 3
    Virus Genes, 1997
    Co-Authors: P. Seshidhar Reddy, J. Brian Derbyshire, Neeraja Idamakanti, Eva Nagy
    Abstract:

    The nucleotide sequence of a 3028 bp DNA segment, located between map co-ordinates 100 and 92 in the genome of Porcine Adenovirus type 3 (PAV-3), was determined. The segment includes the entire early region 4 (E-4) and the right inverted terminal repeat sequences. There were two TATA boxes and one canonical polyadenylation signal on the l strand. Homology searches of the GenBank data base for the predicted amino acid sequences revealed that, of the eight open reading frames (ORFs) on the l strand, and four ORFs on the r strand, only ORF 8 on the l strand showed homology with the 34 kDa E-4 protein of human Adenovirus types 2, 12 and 34. Northern blot analysis showed that transcription from the E-4 region of PAV-3 began 4 h after infection, peaked at 8 h and declined after 10 h, before DNA replication began 16 h after infection. The E-4 region of PAV-3 was further characterized by 5′ and 3′ end mapping of the transcription unit.

M. A. Johnson - One of the best experts on this subject based on the ideXlab platform.

  • Porcine Adenovirus as a delivery system for swine vaccines and immunotherapeutics
    Veterinary journal (London England : 1997), 2005
    Co-Authors: Jef M. Hammond, M. A. Johnson
    Abstract:

    Abstract Porcine Adenovirus (PAdV) has many qualities which make it an ideal choice for use as a delivery vector in swine. It is a low grade pathogen, present almost world-wide in a number of serotypes varying in their virulence and tissue tropism, which may allow for serotype specific vaccine targeting. PAdV is species specific having only been isolated from swine, reducing the possibility of its spread to other animals or man following administration. When engineered to contain a foreign gene, recombinant PAdV (rPAdV) can be grown to high titres in tissue culture cells making it cheap to produce. Knowledge of the complete nucleotide sequence of the PAdV genome has enabled rationally directed insertions of foreign genes which remain stably inserted in the genome and can be expressed at high levels following delivery to the target host. Importantly, recombinant PAdV can be administered by injection or by the oral route in feed or drinking water. We have delivered a range of antigens and immunomodulatory molecules to commercially available pigs using rPAdV and found it to be a very effective delivery system. Significantly, recombinant PAdV serotype 3 is highly effective as a delivery vehicle even when administered in the face of high levels of artificially induced serotype specific neutralising antibody to the vector.

  • Protection of pigs against 'in contact' challenge with classical swine fever following oral or subcutaneous vaccination with a recombinant Porcine Adenovirus.
    Virus research, 2003
    Co-Authors: Jef M. Hammond, E. S. Jansen, C. J. Morrissy, A. L. M. Hodgson, M. A. Johnson
    Abstract:

    Abstract A recombinant Porcine Adenovirus expressing the classical swine fever virus (CSFV) gp55 gene (rPAdV-gp55) was administered to commercially available outbred pigs via the subcutaneous or oral route and their susceptibility to ‘in contact’ challenge with classical swine fever determined. Animals vaccinated subcutaneously with a single dose of recombinant vaccine and challenged by ‘in contact’ exposure were protected from disease, whereas pigs given an equivalent single oral dose did not survive challenge. However, pigs given two oral doses of rPAdV-gp55, 22 days apart, were completely protected from disease. In addition, two doses of rPAdV-gp55 given subcutaneously was shown to boost CSFV neutralising antibody compared with a single dose, but neither a single dose nor two doses given orally induced detectable neutralising antibody responses.

  • Vaccination of pigs with a recombinant Porcine Adenovirus expressing the gD gene from pseudorabies virus.
    Vaccine, 2001
    Co-Authors: Jef M. Hammond, Suresh K. Tikoo, Lorne A. Babiuk, E. S. Jansen, C. J. Morrissy, M. M. Williamson, Winsome V Goff, Brenda Van Der Heide, M. A. Johnson
    Abstract:

    Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant Porcine Adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2-3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge.

  • Vaccination with a single dose of a recombinant Porcine Adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever.
    Vaccine, 1999
    Co-Authors: Jef M. Hammond, R. J. Mccoy, E. S. Jansen, C. J. Morrissy, A. L. M. Hodgson, M. A. Johnson
    Abstract:

    Abstract A recombinant Porcine Adenovirus (rPAV) with the gp55 (E2) gene from the classical swine fever virus (CSFV) ‘Weybridge’ strain inserted into the right hand end of the PAV serotype 3 (PAV3) genome was constructed. Expression of gp55 was directed by the major late promoter and tri-partite leader sequences located and cloned from PAV3. No compensatory deletions of PAV DNA sequences were made. Vaccination of outbred pigs with a single dose of the recombinant virus (rPAV-gp55) resulted in complete protection from lethal challenge with CSFV. No adverse clinical signs were observed in vaccinated animals following administration of rPAV-gp55 and following challenge, no clinical signs of CSF were observed prior to, or at, post mortem. The insert made into the rPAV increased the genome length to 106.8% of wild type and therefore exceeded the expected maximum insert size for a stable recombinant by almost 2%. Thus rPAV-gp55 contains the largest stable insertion made into a non-deleted Mastadeno virus recombinant so far reported.

  • Nucleotide and Amino Acid Sequence Analysis of the 100K Protein of a Serotype 3 Porcine Adenovirus
    DNA sequence : the journal of DNA sequencing and mapping, 1997
    Co-Authors: R. J. Mccoy, M. Sheppard, M. A. Johnson
    Abstract:

    The genomic region between map units 69 and 78 of a type 3 Porcine Adenovirus (PAV3) was sequenced and analysed. An open reading frame (ORF) of 2514 nucleotides encoding a polypeptide of 838 amino acids and approximately 94.1 kDa was found. The size and location of the ORF suggested it was the PAV3 homo-logue of the 100K gene and this was confirmed by nucleotide sequence comparison with the 100K of human Adenovirus type 2. Amino acid sequence alignment of the predicted polypeptide with the sequences of the 100K proteins of four human Adenoviruses and type 10 fowl Adenovirus revealed sequence identities of between 31% and 52%. Although amino acid conservation was present throughout the entire sequences compared, lower identity was noted in both the amino-and carboxy-termini.

P. Seshidhar Reddy - One of the best experts on this subject based on the ideXlab platform.

  • Porcine Adenovirus-3 as a helper-dependent expression vector.
    Journal of General Virology, 1999
    Co-Authors: P. Seshidhar Reddy, Neeraja Idamakanti, Lorne A. Babiuk, Majid Mehtali, Suresh K. Tikoo
    Abstract:

    Porcine Adenovirus has been proposed as a potential vector for generating novel and effective vaccines for pigs. As a prerequisite for the generation of helper-dependent Porcine Adenovirus-3 (PAV-3) vectors, two E1-complementing Porcine cell lines expressing E1 proteins of human Adenovirus-5 (HAV-5) were made. These cell lines could be efficiently transfected with DNA and allowed the rescue and propagation of a PAV-3 recombinant, PAV201, containing a 0.597 kb E3 deletion and a 0.803 kb E1A deletion. Our data demonstrate that E1A proteins of HAV-5 have the capacity to transform foetal Porcine retina cells and complement for the E1A proteins of PAV-3. The green fluorescent protein (GFP) gene placed under the control of a cytomegalovirus immediate early promoter was inserted into the E1A region of the PAV201 genome. Using these cell lines, a helper-dependent PAV-3 recombinant expressing GFP, PAV202, was constructed and characterized. The wild-type PAV-3 and the recombinant PAV202 expressing GFP were used to determine the ability of the virus to enter and replicate in cells of human and animal origin under cell culture conditions. Our results suggest that PAV-3 enters but does not replicate in dog, sheep, bovine and human cells.

  • Nucleotide sequence and transcription map of Porcine Adenovirus type 3.
    Virology, 1998
    Co-Authors: P. Seshidhar Reddy, Suresh K. Tikoo, Neeraja Idamakanti, Jae-young Song, Joongbok Lee, Bang-hun Hyun, Jong-hyeon Park, Sang-ho Cha, Yong-tae Bae, Lorne A. Babiuk
    Abstract:

    Abstract The complete nucleotide sequence of Porcine Adenovirus type 3 was determined and a transcriptional map for the genome was constructed. The size of the genome is 34094 bp in length with an unusually high G + C content (63.7%), the highest thus far reported for any Adenovirus. Overall organization of the genome is similar to that for previously sequenced adenoviral DNAs, but there also were distinct differences. The late regions genes are organized into six families, instead of five as they are in human Adenovirus type 2. In contrast to bovine Adenovirus type 3 and ovine Adenovirus, which lack virion-associated RNA genes, the nucleotide sequence analysis of the viral genome indicates that it encodes one short VA RNA species. With the exception of the fiber and a 33-kDa nonstructural protein, the predicted amino acid sequences of the open reading frames in the late regions and the E2 region and IVa2 exhibited a high level of homology, whereas the deduced amino acid sequences of ORFs in E1, E3, and E4 regions, and the pIX showed a lesser homology with the corresponding proteins of other Adenoviruses. The proteins V, VII, and IX are unusually long, and the protein VII lacks the consensus protease cleavage site. Genomic and cDNA sequence analysis has identified promoters, cap sites, intron–exon boundaries, polyadenylation signals, and polyadenylation sites in the viral genome.

  • Characterization of the Early Region 4 of Porcine Adenovirus Type 3
    Virus Genes, 1997
    Co-Authors: P. Seshidhar Reddy, J. Brian Derbyshire, Neeraja Idamakanti, Eva Nagy
    Abstract:

    The nucleotide sequence of a 3028 bp DNA segment, located between map co-ordinates 100 and 92 in the genome of Porcine Adenovirus type 3 (PAV-3), was determined. The segment includes the entire early region 4 (E-4) and the right inverted terminal repeat sequences. There were two TATA boxes and one canonical polyadenylation signal on the l strand. Homology searches of the GenBank data base for the predicted amino acid sequences revealed that, of the eight open reading frames (ORFs) on the l strand, and four ORFs on the r strand, only ORF 8 on the l strand showed homology with the 34 kDa E-4 protein of human Adenovirus types 2, 12 and 34. Northern blot analysis showed that transcription from the E-4 region of PAV-3 began 4 h after infection, peaked at 8 h and declined after 10 h, before DNA replication began 16 h after infection. The E-4 region of PAV-3 was further characterized by 5′ and 3′ end mapping of the transcription unit.

  • Porcine Adenoviruses types 1, 2 and 3 have short and simple early E-3 regions.
    Virus research, 1996
    Co-Authors: P. Seshidhar Reddy, J. Brian Derbyshire, Neeraja Idamakanti, Eva Nagy
    Abstract:

    Abstract The nucleotide sequence of the E-3 region genes, the hexon associated protein pVIII genes, and part of the fiber genes coding for the N-terminal tail regions, of Porcine Adenovirus (PAV) types 1 and 2 were determined. The sizes of the E-3 regions were found to be 1162 and 1222 bp, respectively. The five open reading frames (ORF) encoded within the sequenced regions of PAV types 1 and 2 shared a high degree of homology with the published sequences of the corresponding ORFs of PAV-3. The E-3 regions of PAV types 1, 2 and 3 were further characterized by Northern blot analysis and 5′ and 3′ end mapping of the transcripts by S1 nuclease analysis. The results of these experiments indicated that the E-3 regions in these three viruses are shorter and simpler in organization than the E-3 regions of human Adenoviruses. A potential promoter for the E-3 regions of these PAVs was identified.

  • Comparison of the Inverted Terminal Repetition Sequences from Five Porcine Adenovirus Serotypes
    Virology, 1995
    Co-Authors: P. Seshidhar Reddy, Tamas Tuboly, J. Brian Derbyshire, John R. Dennis, Eva Nagy
    Abstract:

    The nucleotide sequences of the region of inverted terminal repetition from representative strains of all five Porcine Adenovirus (PAV) serotypes were determined and analyzed. The first 17 nucleotides of this region were identical in PAV-1 to 3 and PAV-5, and 10 bp of identical sequence was found in all the PAVs. The closest relationships were among PAV-1 to 3, which shared more common sequences than the other serotypes. PAV-4 had the longest inverted terminal repeat reported for any Adenovirus. The proximal 54-bp AT-rich region was partially conserved and the distal GC-rich region was less well conserved among all five serotypes.