Characterization of the early region 4 of porcine adenovirus type 3.

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 1 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 1 strand, and four ORFs on the r strand, only ORF 8 on the 1 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.

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.

Partial transcriptional mapping of the fowlpox virus genome and analysis of the EcoRI L fragment.

Several fowlpox virus (FPV) DNA fragments were selected by differential hybridization using cDNA of transcripts that were strongly transcribed early and/or later after infection of QT-35 cells. The EcoRI L fragment contained three strongly transcribed FPV genes: L1L, a late 1452 bp partial (amino end) ORF; L2R, an early/late 522 bp ORF; and L3R, a late 948 bp ORF. The protein products of L1L, L2R and L3R shared homology with the products of vaccinia virus (VV) genes H4L (RAP94), H5R (Ag35) and H6R (topoisomerase), respectively, suggesting a conservation of gene structure and order between VV and FPV. The 5' upstream non-coding sequences of L1L and L3R were A + T rich and the sequence 5' TAAATG 3' overlapped the predicted translation start codon. Primer extension analysis of the L2R transcript mapped the transcriptional start sites of early and late mRNAs 14 nt downstream of a VV early promoter-like critical region sequence, AAAATTGAA-AAAAAAA. A VV-like TAAAT late transcriptional element was present 20 nt upstream of the L2R ATG translational start codon. A plasmid with the putative early L2R promoter cloned upstream of the Newcastle disease virus haemagglutinin-neuraminidase (HN) cDNA as a reporter gene was at least 6-fold more effective in generating HN MRNa than plasmids containing the P7.5 or P11 VV promoters in transient expression assays in FPV-infected CEF cells treated with cytosine arabinoside. The L2R promoter was also able to express an amount of HN mRNA equal to that expressed by the VV promoters late in infection.

Comparison of the inverted terminal repetition sequences from five porcine adenovirus serotypes.

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.

Restriction endonuclease analysis and physical mapping of the genome of porcine adenovirus type 5.

The HNF61 and HNF70 isolates of porcine adenovirus type 5 (PAV-5) were cultivated in PK-15 cells, and viral DNA was extracted from the infected cells by a modified Hirt procedure. The DNAs were digested by each of 9 restriction endonucleases, and fragments representing the entire genomes were cloned. Based on the sizes of the restriction enzyme fragments, the genome of each isolate was estimated to be 33.2 kb. Physical maps for the 9 restriction endonucleases were constructed. The physical maps of the two isolates were identical for 5 of the restriction endonucleases, but 4 enzymes revealed differences in restriction sites occurring mainly between map units 78 and 83, which may include the E3 region of the genome. There were no similarities between the physical maps of PAV-5 and those described for the other 4 serotypes of PAV.

Antiviral action of interferon-alpha against porcine transmissible gastroenteritis virus.

Swine testis (ST) cell cultures were treated with various doses of recombinant human interferon-alpha 2a (IFN), and assayed for 2',5' oligoadenylate synthetase (2-5 A synthetase) activity. Treatment with 100 or 1000 units/ml of IFN resulted in increased 2-5 A synthetase activity, but there was no significant response to 1 unit/ml of IFN. Titres of porcine transmissible gastroenteritis virus (TGEV) were reduced between 6 and 15 hours post-infection in ST cells treated with 1000 or 2500 units/ml of IFN. Polyacrylamide gel electrophoresis of lysates of TGEV-infected ST cells, and of lysates immunoprecipitated with anti-TGEV antibodies, revealed that the synthesis of the N and S proteins of TGEV was reduced in cells treated with 100 or 1000 units/ml of IFN. Viral RNA production, as determined with a probe which hybridized to the S gene of TGEV, was found to be reduced in ST cells treated with 1000 units/ml of IFN, but not in cells treated with 100 units/ml. It was concluded that, in IFN-treated ST cells, TGEV protein production may be decreased in the absence of reduced viral RNA production, and that 2-5 A synthetase may not be a significant factor in the antiviral activity of IFN against TGEV.

Interferon induction in swine lymphocyte antigen-defined miniature pigs.

Interferon was induced in two groups of swine lymphocyte antigen (SLA)-defined miniature pigs with polyinosinic: polycytidylic acid complexed with poly-L-lysine and carboxymethylcellulose. The group 1 pigs were low antibody-response phenotypes (SLAa/a, SLAa/c, SLAc/c), and the group 2 pigs were high antibody-response phenotypes (SLAd/d, SLAd/g, SLAg/g). Six hours after induction the antiviral tires were not influenced by the SLA group, but higher titres were observed in females. Higher antiviral titres were found in group 2 pigs before treatment and 24 hours after treatment, and higher titres were found in female pigs. The antiviral titres before and after treatment were also influenced by the sire. Group 2 pigs had a lower total leucocyte counts before treatment, and there was a significant reduction in leucocyte numbers in both groups six hours after induction, due mainly to a large reduction in lymphocyte counts.

Sequence analysis of putative pVIII, E3 and fibre regions of porcine adenovirus type 3.

Sequence analysis of a region of the genome of porcine adenovirus type 3 from map unit 79.5 to map unit 92 was performed. Homology studies revealed genes coding for the hexon-associated protein pVIII on the left and for the fibre protein on the right of the sequenced region. By analogy with the genomic organization of other adenoviruses, the 1179 bp sequence between the pVIII and fibre open reading frames, extending from map unit 81.3 to map unit 84.7, was identified as the equivalent of the E3 region of human adenoviruses. The deduced amino acid sequence of one of the three open reading frames of the putative E3 region showed homology with the 13.3K E3 protein of canine adenovirus type 2. The primary structure of the putative fibre protein was similar to that described for human adenovirus types 2 and 5, with a 14 pseudorepeat motif in the shaft region of the fibre. A 742 bp tandem repeat starting in the middle of the fibre gene and extending beyond the termination codon of this gene was observed.

Cellular receptors for transmissible gastroenteritis virus on porcine enterocytes.

The activity of aminopeptidase-N (APN), reported to be a major receptor for porcine transmissible gastroenteritis virus (TGEV), in enterocyte fractions harvested from the jejunal villi and crypts of newborn and weaned piglets, did not correspond with the levels of saturable virus binding previously demonstrated for the same fractions. Plasma membranes prepared from enterocytes harvested from the jejunal villi of a newborn piglet were used in the preparation of a monoclonal antibody (MAb) which blocked the binding of TGEV, but not that of the porcine respiratory coronavirus (PRCV), to ST cells. This MAb immunoprecipitated a 200 kDa non-glycosylated protein from lysates of ST cells, which was not precipitated by an anti-APN MAb. The 200 kDa protein was shown by immunostaining and fluorescence activated cell scanning to be present on ST cells and on villous enterocytes from newborn piglets, but not on MDBK cells or enterocytes from weaned piglets. APN was demonstrated by the same techniques to be present on villous enterocytes from both newborn and weaned piglets, as well as on ST cells. It was concluded that the 200 kDa protein may be a second receptor for TGEV, contributing to the high susceptibility of newborn piglets to the virus.

Passive protection of piglets by recombinant baculovirus induced transmissible gastroenteritis virus specific antibodies.

Sera of pigs immunized with parts of the transmissible gastroenteritis virus (TGEV) spike (S) protein expressed by recombinant baculoviruses were tested, together with a TGEV hyperimmune antiserum, for their abilities to protect three-day-old piglets against TGEV infection. The piglets were infected with virulent TGEV and the sera were given orally 3 h before infection, together with the virus, and every 6 h postinfection during the 30 h of the experiment. Virus shedding was monitored by TGEV isolation from rectal swab samples. The sera containing antibodies induced by the complete S protein or the amino terminal half of the S protein showed protective properties, indicated by delayed onset of clinical signs and virus shedding, similar to the TGEV hyperimmune serum. Those immune sera containing antibodies induced by shorter recombinant proteins were not protective.

Molecular cloning and physical mapping of porcine adenovirus types 1 and 2.

The 25R strain of porcine adenovirus type 1 (PAV-1) and the A47 strain of PAV-2 were propagated in ST cells, and DNA was extracted from the infected cells by a modified Hirt method. The DNA of each virus was digested by each of nine restriction endonucleases, and restriction enzyme fragments representing the entire genome were cloned. The genomic size of each virus was approximately 33 kb. Physical maps for the nine restriction endonucleases were constructed from the results of double digestion and Southern blot hybridization experiments, and oriented with respect to the PAV-3 genome. PAV-1 and PAV-2 were found to be related genetically to PAV-3, and there was a closer relationship between PAV-1 and PAV-3 than between PAV-1 and PAV-2 or between PAV-2 and PAV-3.

Evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs.

Aminopeptidase-N (APN) has been identified [B. Delmas, J. Gelfi, R. L'Haridon, L. K. Vogel, H. Sjostrom, O. Noren, and H. Laude, Nature (London) 357:417-420, 1992] as a major receptor for porcine transmissible gastroenteritis virus (TGEV). Binding of TGEV to villous enterocytes from the jejuna of newborn pigs is saturable and at a higher level than that of binding of virus to newborn cryptal enterocytes or to enterocytes from older piglets (H. M. Weingartl and J. B. Derbyshire, Vet. Microbiol. 35:23-32, 1993). The distribution of APN in enterocytes in the jejuna of neonatal and 3 week-old-piglets, as determined by the measurement of enzymatic activity and by labeling of the cells with an anti-APN monoclonal antibody, did not correspond with the reported distribution of saturable binding sites on enterocytes. Monoclonal antibodies, which were prepared against plasma membranes derived from enterocytes harvested from the upper villi of newborn pigs, blocked the replication of TGEV, but not the porcine respiratory coronavirus, in ST cells and immunoprecipitated a 200-kDa protein in ST cell lysates. This protein was demonstrated by immunohistochemistry and by fluorescence-activated cell scanning to be present on the villous enterocytes of newborn pigs but to be lacking on the cryptal enterocytes of newborn pigs and on the villous and cryptal enterocytes of 3-week-old piglets. Since this distribution of the protein corresponds to the previously demonstrated distribution of saturable binding sites, we conclude that the 200-kDa protein may be an additional receptor for TGEV which is restricted to the villous enterocytes of newborn pigs and which contributes to the age sensitivity of these animals to the virus.

Vaccination of chickens with a recombinant fowlpox virus containing the hemagglutinin-neuraminidase gene of Newcastle disease virus under the control of the fowlpox virus thymidine kinase promoter.

When chickens were vaccinated with a recombinant fowlpox virus (FPV) containing the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) cDNA under the control of the thymidine kinase (TK) promoter and inserted into the FPV TK gene, the FPV antibody response to the recombinant virus was similar to the response to vaccination with standard FPV, and the recombinant virus protected chickens against challenge with virulent FPV. While the presence of the NDV HN cDNA was demonstrated in the recombinant virus, which was stable on serial passage, expression of HN was not detected by hemagglutination, Western blot analysis or immunoprecipitation of infected cell lysate. Chickens vaccinated with the recombinant virus failed to mount an NDV hemagglutination-inhibition antibody response, and they did not resist challenge with velogenic NDV. It was concluded that the TK promoter was too weak to drive the HN gene, but that the insertion into the FPV TK gene did not reduce the immunogenicity of the virus.

Development and evaluation of a non-isotopically labeled DNA probe for the diagnosis of infectious laryngotracheitis.

A digoxigenin-labeled cloned infectious laryngotracheitis virus (ILTV) DNA fragment was evaluated as a nonradioactive alternative probe in the diagnosis of infectious laryngotracheitis. The dot-blot hybridization protocol was optimized and was capable of detecting 40 pg of purified ILTV DNA and as few as 50 ILTV-infected chicken embryo liver cells. The utility of this approach for diagnostic use was evaluated through four ILTV inoculation trials using a mild field isolate, a virulent challenge strain, a tissue-culture-origin vaccine, and an egg-origin vaccine. Birds were examined for clinical signs of ILT, and conjunctival and pharyngeal swabs from inoculated and sentinel birds were tested for ILTV by the digoxigenin-labeled probe and by virus isolation. In general, higher numbers of ILTV-positive samples were detected by both assays from conjunctival swabs. For the non-vaccine strains, detection by dot-blot hybridization was equivalent to that for virus isolation. However, for the two vaccine strains, there was some lack of correlation between the dot-blot results and the virus-isolation results. The kappa values between virus-isolation results and dot-blot results for the tissue-culture-origin vaccine, egg-origin vaccine, Ont 1598 field isolate, and virulent strain were 0.00, 0.16, 0.39, and 0.24, respectively, for pharyngeal samples and 0.19, 0.29, 0.58, and 0.48, respectively, for conjunctival samples.

Studies of fowlpox virus recombination in the generation of recombinant vaccines.

A p7.5/beta-galactosidase (7.5 lacZ) gene construct, cloned adjacent to the fowlpox virus (FPV) thymidine kinase (tk) gene was used as a marker to identify the products of recombination as 'blue' FPV plaques. The rFPVs were detected as early as 4 h after the introduction of plasmid DNAs and by 72 h post-infection (p.i.) for one transfer vector comprised 0.48% of the viral population. The proportion of rFPV increased linearly from 0.073% to 0.62% as the cumulative length of homologous sequences in the transfer vector increased from 0.73 to 4.5 kb. Two approaches using a second reporter gene, the Newcastle disease virus haemagglutinin-neuraminidase (NDV HN) gene were tested to differentiate between single and double cross-over events. In one, the HN gene was cloned into the FPV tk gene and the 7.5 lacZ cloned outside of the homologous region. Progeny of a single cross-over with FPV DNA generated an unstable plaque containing the HN gene and subsequent intramolecular recombination resulted in excision of the 7.5 lacZ and the generation of a stable 'white' plaque. For virus grown in CEF cells (tk+) in the presence of 5-bromo-deoxyuridine, only those viruses which contained a tk gene disrupted by the HN gene formed plaques. This approach allowed us to easily identify rFPV containing the HN gene but lacking 7.5 lacZ or other bacterial sequences. In a second approach, a double cross-over between rFPV DNA containing a stably expressed beta-galactosidase gene cloned into the tk gene (blue plaque) and plasmid DNA containing the HN gene flanked by tk sequences would allow transplacement of the 7.5 lacZ gene with the HN gene, and generating a white plaque. We were unable to generate recombinant viruses with the HN gene and which generated a white plaque, indicating that double cross-over events do not occur at a sufficiently high frequency in FPV.

Immunogenicity of the S protein of transmissible gastroenteritis virus expressed in baculovirus.

Seven fragments of the spike (S) gene cDNA of transmissible gastroenteritis virus (TGEV), as well as the full length cDNA, were cloned and expressed in baculovirus vectors. Piglets were immunized with cells infected with the recombinant viruses. Each of the recombinants induced TGEV-specific antibodies detected in a fixed cell enzyme immunoassay. The amino terminal half of the S protein, containing all four major antigenic sites (A, B, C and D), and encoded by a 2.2 kb fragment of the S gene, induced virus neutralizing (VN) antibody titers comparable with those induced by the complete S protein. Recombinant proteins lacking the A antigenic site, or with a deletion including the putative receptor binding sites and the D antigenic site, were not capable of inducing levels of VN antibodies similar to those induced by the whole S protein.

Antiviral activity of interferon against transmissible gastroenteritis virus in cell culture and ligated intestinal segments in neonatal pigs.

Segments of jejunum in 5 to 6 days old piglets were surgically ligated, inoculated with transmissible gastroenteritis virus (TGEV) and 18 hours later the segments were fixed for histology or suspensions were prepared for plaque assay in swine testis (ST) cell cultures to determine the yield of virus. When the virulent Purdue strain of TGEV was used, villous atrophy was seen and TGEV antigen was demonstrated immunohistochemically in the villous enterocytes. The Miller M6 strain of virus produced less extensive lesions in the segments, but since it was titratable by plaque assay it was used in the subsequent yield reduction assays to determine the antiviral activity of interferon. When intestinal segments were inoculated simultaneously with either 3200 units of natural porcine interferon-alpha or up to 1000,000 units of recombinant human interferon-alpha 2 a, and TGEV, there no reductions in virus yield, although the same cytokines exerted an antiviral effect in ST cells treated in a similar way. However, virus yields were significantly reduced in intestinal segments in piglets treated parenterally with the synthetic interferon inducer polyinosinic: polycytidylic acid 6 hours before challenge of the segments with TGEV. There was also a trend for the antiviral effects of interferon induction before challenge to be augmented by the inclusion of interferon with the virus inoculum. It was concluded that interferon would be ineffective as a therapeutic for TGEV, although it might be useful prophylactically.

Restriction endonuclease analysis of a porcine isolate of bovine herpesvirus type 1.

Deoxyribonucleic acid (DNA) was extracted from bovine herpesvirus type 1 (BHV-1) isolated from a stillborn porcine fetus, from the Cooper reference strain of BHV-1, and from an Ontario bovine respiratory isolate. Each DNA was digested with the restriction endonucleases HindIII, EcoRI, HpaI and BamHI. Except for very minor differences in the patterns produced after digestion with EcoRI and HindIII, the DNA of the porcine isolate reacted in a similar manner to the bovine viruses, and it was concluded that the porcine virus is genetically similar to bovine isolates of BHV-1.