- Volume 67, Issue 7, 1986

The morphological aspects of Breda virus serotype 2 replication in intestinal cells of gnotobiotic calves were investigated by electron microscopy. Ultrastructural findings suggest a morphogenetic pathway involving cytoplasmic vesicles, the Golgi apparatus and the cell nucleus. Virus uptake probably occurs via a receptor-mediated endocytosis-like mechanism. Endocytotic vesicles then carry virus to an as yet undetermined site of uncoating. Masses of tubules having the same diameters as Breda virion cores are found in nuclei, suggesting a role for the cell nucleus in replication of nucleocapsids. Similar tubules, as well as complete virions, were found in the Golgi region, the apparent site of virus assembly. Virus-containing Golgi vesicles then presumably move to cell surfaces where they fuse with apical and baso-lateral cell membranes to release virions in a way that permits more than one viral replicative cycle to occur without damage to host cell integrity. Virions are elongated with rounded ends and measure 42 × 100.5 nm. The morphogenesis and replication of Breda virus most closely resembles that of Berne virus of the proposed family Toroviridae.

In equine dermis cells infected with Berne virus particles were first detected 10 h after infection. Virions were encountered in all parts of the Golgi system and, infrequently, in the rough endoplasmic reticulum. A unique form of budding of preassembled rigid tubular nucleocapsids was demonstrated. Masses of tubular nucleocapsids of a lesser diameter and electrondensity were prominent in the cytoplasm and the nucleus of infected cells. Within the Golgi system and cytoplasmic cisternae virions appeared as straight or slightly curved rods. Extremely long, aberrant virions (250 nm) were occasionally seen. The proper torovirion morphology was observed in extracellular particles and in vacuoles near the cell surface.

Monoclonal antibodies reacting with the herpes simplex virus (HSV)-encoded major DNA-binding protein defined an intracellular filamentous network. This network was associated predominantly with the infected cell nucleus and occurred in cells infected with HSV type 2. It did not co-distribute with microfilaments, microtubules or intermediate filaments, and DNA synthesis was required for its formation. We suggest explanations for the occurrence and function of this novel filamentous network structure.

Rat cytomegalovirus (RCMV) DNA was cleaved by restriction endonuclease EcoRI into 24 fragments ranging in mol. wt. from 34 × 106 to 0.20 × 106, of which 18 fragments could be cloned in plasmid pACYC 184. Restriction endonuclease XbaI cleaved the RCMV genome into 28 fragments, ranging in size from 44 × 106 to 0.81 × 106, of which 24 fragments were cloned in plasmid pSP62-PL. Among the restriction fragments that could not be cloned were two major terminal colinear fragments, EcoRI-A (34 × 106) and XbaI-A (44 × 106). Thus, the complete sets of recombinant plasmids spanned about 70% of the RCMV genome. Our mapping results including determination of the termini of the genome, characterization of double digestion products of restriction fragments and cross-hybridization of 35S-labelled (cloned) EcoRI and XbaI fragments to Southern blots of EcoRI-, XbaI- or BglII-cleaved RCMV DNA, allowed us to construct the EcoRI and XbaI restriction maps of RCMV DNA. Since no cross-hybridization between internal fragments was seen, it is concluded that the RCMV genome consists of a long unique sequence of 224 kilobases without internal inverted repeat sequences, which is similar to the structures of murine and guinea-pig CMV DNA but unlike that of human CMV DNA. In a minor population (approx. 20%) of the RCMV DNA, one terminus was found to be larger by 0.35 × 106 mol. wt. The nature of this fragment is unclear at the moment.

Polypeptides synthesized in Vero cells infected with African swine fever virus (ASFV) can be divided into early and late classes on the basis of their sensitivity to cytosine arabinoside. As ASFV does not inhibit cell protein synthesis until late in infection, immunoprecipitation was used to identify virus-specific polypeptides. Eighteen early and 15 late polypeptides were detected by polyacrylamide gel electrophoresis. Early polypeptides can be further divided into those which are transiently expressed at early times and the majority which are synthesized throughout infection. In vitro translation products of RNAs from infected cells at different stages of infection were compared with in vivo products after immunoprecipitation. A good correspondence was observed between the invivo and in vitro patterns. Alle arly RNAs translated in vitro were synthesized in the presence of cytosine arabinoside and cycloheximide and can therefore be classified as immediate early RNAs. Only two polypeptides transcribed from early RNA were not present among late RNA products. No evidence was obtained for a discrete class of delayed early RNAs.

A full length cDNA copy of the NS mRNA of the Missouri strain (Hazelhurst subtype, New Jersey serotype) of vesicular stomatitis virus (VSV) has been cloned and sequenced. The mRNA is 856 nucleotides long (excluding polyadenylic acid) and encodes a protein of 274 amino acids (mol. wt. 31000). Comparison with the NS gene of the Ogden strain (Concan subtype, New Jersey serotype) showed 15% difference at the nucleotide level and 10% difference at the amino acid level; the majority of the changes were located in the 3′ half of the mRNA. Comparison with the NS genes of two strains representing the Indiana serotype showed about 50% nucleotide and 33% amino acid sequence homology between the serotypes. In a four-way comparison of the proteins, two regions of higher homology were noted which may be of functional importance. Eighteen potential phosphorylation sites (Ser or Thr) were conserved between the four proteins; five of these sites correspond to the residues which have been suggested to be constitutively phosphorylated and may be essential for NS activity.

The dominant neomycin resistance gene (neoR) was introduced into the genome of the myeloproliferative sarcoma virus (MPSV), a replication-defective retrovirus carrying the mos oncogene. The resulting selectable neoR-MPSV virus did not lose its acute transforming property, unlike the results of attempts by other groups to insert marker genes into oncogenic viruses. NeoR-MPSV DNA was used to generate infectious virus by transfection followed by rescue with Friend or Moloney murine leukaemia virus. Infection of fibroblasts with this virus resulted in morphologically transformed cells which were resistant to the neomycin analogue G418. Segregation of the two functions (transformation and G418 resistance) was not observed in more than 500 independent viral transfers to fibroblasts. Furthermore, neoR-MPSV retained the leukaemogenesis-inducing properties of the wild-type virus. Myeloproliferation and G418-resistance transfer did not segregate after passage in mice.

The 28000 mol. wt. polypeptide (p28) of adult T-cell leukaemia-associated antigen encoded by the 24S defective human T-cell leukaemia virus (HTLV-I) is associated with protein kinase activity. We have determined the nucleotide sequence of this defective HTLV-I provirus and found that it contains a portion of the gag gene (p19 and part of p24), the pX region, and two long terminal repeats, one at each end. The predicted p28 gag-pX fused protein consists of 190 amino acids and its mol. wt. was calculated as 21055. The results of peptide mapping analysis showing that p28 contains p19 supported the nucleotide sequence data. That p28 was encoded by this defective provirus was also demonstrated by transient expression of p28 polypeptide in COS 7 cells transfected with a recombinant plasmid containing a simian virus 40 early promoter and the p28-coding region of the 24S HTLV-I.

Monoclonal antibodies (MAbs) were used to delineate the antigenic relationship between the three morbillivirus types: measles virus (MV), canine distemper virus (CDV) and rinderpest virus (RPV). Panels of six to 31 MAbs against the haemagglutinin (H), fusion (F), nucleocapsid protein (NP), phosphoprotein (P) and matrix (M) proteins of MV and the H, F, NP and P proteins of CDV were employed. Nine strains of MV, three strains of CDV and four strains of RPV were examined by radioimmunoprecipitation assay and immune fluorescence for reactivity with the heterologous MAbs. Overall, the NP and in particular the F proteins of the morbilliviruses showed a high degree of epitopic homology; the P and M proteins showed a partial epitopic homology, with the greatest variation between the M proteins of CDV and MV; the H proteins showed a low degree of epitopic homology and then only between MV and RPV. These data indicate that the major cross-protecting antigen in heterotypic vaccination amongst morbilliviruses is the F antigen. The epitopic relationships found between morbilliviruses as identified by the MAbs were classified as follows. (i) Group-specific epitopes were present on all strains of the three morbillivirus types. (ii) Group-cross-reactive epitopes were present on only some of the strains from each morbillivirus type (these epitopes identified the presence of intratypic strain variation in all proteins of all three virus types). (iii) Type-specific epitopes, i.e. MV unique or CDV unique, were found only on the homologous morbillivirus type. (iv) CDV-RPV intertypic and MV-RPV intertypic epitopes were, respectively, epitopes shared by CDV and RPV but not with any MV strain, and epitopes shared by MV and RPV but not with any CDV strain. These cross-reactivities and type-specific reactions were obtained with the internal viral proteins (M, P and NP). The epitopes of the F proteins were mainly group-specific and no CDV-RPV or MV-RPV intertypic epitopes were found. The epitopes of the H protein were either type-specific or MV-RPV intertypic. These data support the proposed evolutionary relationship between the morbilliviruses.

Previously, a panel of monoclonal antibodies recognizing epitopes in four antigenic sites on the haemagglutinin—neuraminidase (HN) glycoprotein of the Australia-Victoria strain of Newcastle disease virus were used in strain comparisons. Epitopes in three sites were found to be conserved while the epitope recognized by the single antibody to site 3 was not. A new panel of antibodies is described, two of which bind to epitopes in site 3 and six of which bind to a site (site 1,4) that overlaps with sites 1 and 4 as determined by analyses of variants, temperature-sensitive mutants, and strains by assays of neutralization of infectivity and binding in a radioimmunoassay. Neutralization of heterologous strains with the panel of antibodies revealed that both new site 3 epitopes are also highly divergent, while three additional epitopes outside site 3 (those in site 1,4) are highly conserved. The new site 3 antibodies can bind to virions of several heterologous strains without neutralizing infectivity. Thus, of the 10 epitopes we have now examined, all of three in site 3 are specific with respect to neutralization of infectivity for th ehomologous strain, while all of seven in other sites are conserved in heterologous strains. This suggests that the strain specificity originally described for a single site 3 epitope is, instead, a property of a much more extensive, poorly conserved domain on the HN molecule.

The antigenic structure of the peplomer glycoprotein E2 of the porcine transmissible gastroenteritis coronavirus (TGEV) was explored using a panel of 23 hybridoma antibodies (MAbs). The topography of the epitopes was established by means of a competition radioimmunoassay. Four main antigenic sites, termed A, B, C and D, were thus clearly delineated. Most of the neutralization-mediating determinants were found to cluster in the A-B area, which has been shown to be highly conserved among TGEV strains. Cooperative enhancement of binding to sites B and D was observed following attachment of MAbs relevant to site A. Additional epitopes were identified on E2 by MAbs that selectively recognized its intracellular precursor. Functional mapping was also performed using neutralization-resistant variants. Analysis of their reactivity confirmed part of the epitope linkages defined by the first approach. The overall lower frequency of such variants altered at site A suggested that some of the epitopes may play an essential function.

Pulse labelling of cells with [35S]methionine at different times after infection followed by SDS-PAGE was used to resolve and to identify polypeptides designated as specific to transmissible gastroenteritis virus (TGEV)-infected swine testicular (ST) cell cultures. The major TGEV structural proteins, with apparent molecular weights of 200000 (200K), 47K and 30K were detected in radiolabelled cell extracts by 6 h post-infection. Additionally, a 17K major polypeptide was present in infected cells but not in mock-infected control cultures. Labelling with [3H]glucosamine revealed only the 200K and 30K proteins to be glycosylated. TGEV-primed porcine lymphocytes, secondarily stimulated in vitro with sucrose gradient-purified virus, produced antibody only to the two glycoproteins (gp) indicating that the 17K polypeptide is not a surface feature of the virion. Two pigs were infected oronasally with the virulent Miller strain of TGEV and their sera were analysed by immunoprecipitation. At 25 days post-infection convalescent sera responded strongly to gp30 and gp200 and there was a weak initial response to the 17K polypeptide. Serum immunoglobulins at 60 days post-infection reacted strongly to the 17K protein while the antibody response to gp30 was significantly reduced and that to gp200 was slightly reduced.

The live infectious bronchitis (IB) vaccine, H120, protected chickens against intranasal challenge with a mixture of Escherichia coli strains (E. coli Pool) and IB virus (IBV) strains of the same (Massachusetts) serotype as H120; it usually also protected against challenge with the E. coli Pool and IBV strains of other serological types. When these challenge strains were themselves used as vaccines they usually protected against challenge with a mixture of the E. coli Pool and an IBV strainof the Massachusetts serotype (VF69-149) or an IBV strain not of the Massachusetts serotype (HVI-116). Poor protection, when observed, was most common inthose experiments involving a minority of the IBV strains that had been incriminated in recent outbreaks of disease in vaccinated flocks of chickens. Much lower concentrations of IBV strain VF69-149 and E. coli O18 were found in the nose, trachea and spleen of H120-vaccinated chickens killed at different times after they were given a mixture of these organisms than were found in these sites in similarly challenged unvaccinated chickens. Some protection against challenge with IBV and the E. coli Pool was also observedin chickens vaccinated with an inactivated IBV strain; it was much less effective than that obtained following vaccination with the corresponding live IBV strain.

Avian infectious bronchitis coronavirus (IBV) inactivated by β-propiolactone induce partial protection of the trachea in up to 40% of chickens following one intramuscular inoculation 4 to 6 weeks prior to challenge. Retention of an intact tracheal ciliated epithelium 4 days after challenge was the criterion of protection. There was no correlation between protection and serum titres of virus-neutralizing (VN) and haemagglutination-inhibiting (HI) antibody, which were maximal at about 4 weeks after inoculation. Virus from which the S1 but not the S2 (spike-anchoring) spike glycopolypeptide had been removed by urea did not induce protection or VN or HI antibody. Four intramuscular inoculations of monomeric S1 induced VN and HI antibody in two and four chickens respectively. These results indicate that VN and HI antibodies are induced primarily by S1, that intact spikes are a major requirement for the induction of protective immunity and that this propertyis probably associated with S1.

Urea has been used to remove the S1 spike glycopolypeptide from avian infectious bronchitis virus (IBV) strains M41 and Beaudette, without removing the S2 spike-anchoring glycopolypeptide. Reduction of the pH to 2.9 did not cause release of S1 although some S1 was released spontaneously from IBV Beaudette at pH 7.4. Virus that lacked S1 was no longer infectious or able to cause haemagglutination (HA). However, radiolabelled IBV that lacked S1 attached to erythrocytes and chick kidney cells to the same or similar extent as did intact virus. Treatment of IBV with a phospholipase C preparation, required to make IBV cause HA, did not increase binding of IBV to erythrocytes. The results indicate that while the attachment to cells of virus that lacks S1 is qualitatively different from that of intact virus, the decline in infectivity is the consequence of the loss of some other spike function.

Tissues of human primary hepatocellular carcinoma (PHC) from six patients infected with hepatitis B virus (HBV) were propagated in nude mice, as well as a strain of hepatitis B surface antigen-positive PHC (PLC/PRF/5). Integration of viral DNA into chromosomal DNA of tumour cells was evaluated by the capacity to hybridize with radiolabelled DNA probes, each representing fundamental partsof the HBV genome, that is S and C genes and regions pre-S and X. All PHC cells possessed region X integrated in their chromosomes. However, integration of the S gene, C gene and region pre-S was found in only six of the seven PHCs. Based on these findings, the integration of region X seems to be most closely associated with carcinogenesis in HBV infection.

Fibroblast cell lines infected in vitro with different strains of gibbon ape leukaemia virus or the related woolly monkey virus (SSAV) synthesized two RNA species of approximately 8=.4 kb and 2.9 kb. The former, a complete RNA, represents the gap-pol mRNA, while the latter is a spliced transcript lacking gag and pol, and represents the env mRNA. In contrast, RNA from one T-lymphoid cell line derived from a gibbon ape T-lymphocytic leukaemia (UCD-144) expressed a viral mRNA in addition to gag-pol and env mRNA. This RNA is 6.4 kb and lacks at least 3=.0 kb of sequences derived from the internal region of the viral genome, including most or all of the pol gene. These data, as well as data from Southern blots of UCD-144 DNA, suggest that the 6.4 kb mRNA could represent a transcript from a defective recombinant provirus and may contain cell-derived sequences.

The gene coding for the most abundant glycoprotein (gp58) of human cytomegalovirus (HCMV), strain AD169, was physically mapped on the viral genome. A monospecific rabbit antiserum against gp58 was used to screen a cDNA library that was constructed from poly(A)+ RNA of HCMV-infected cells in the prokaryotic expression vector λ gt11. A cDNA clone was identified which synthesized part of the glycoprotein. It allowed localization of the coding region within the right terminal sequence of the HindIII-F fragment between map coordinates 0.344 and 0.380 of HCMV virion DNA.