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Replicating viruses generally induce type 1 immune responses, with high interferon (IFN)-gamma levels and antibodies of the IgG2a isotype. In the present study we demonstrate the intrinsic ability of non-replicating virions to induce comparable immune responses in the notable absence of any adjuvant. Injection of inactivated pseudorabies virus, an alphaherpesvirus, by various routes into mice resulted in the generation of T helper (Th) 1 type immune response. Co-delivery of inactivated pseudorabies herpesvirus (iPRV) with protein redirected IgG1-dominated tetanus toxoid-specific responses towards an IgG1/IgG2a balanced response. Also inactivated preparations of viruses from the paramyxo- (Newcastle disease virus), rhabdo- (rabies virus), corona- (infectious bronchitis virus) and reovirus (avian reovirus) families led to IgG2a antibody responses; however, the genetic background of the host did result in considerable variation. Because disrupted virions also induced type 1 immune responses, we conclude that structural elements of virions inherently contribute to IFN-gamma-dependent isotype switching by inactivated viruses. Strikingly, immunizations in gene-disrupted mice showed that a functional IFN-alpha/beta, IFN-gamma or interleukin (IL)-12 pathway was not required for the generation of a polarized Th1 type immune response initiated by inactivated virus particles. These findings have a bearing on the understanding of immune responsiveness to virus structures and the design of vaccines containing virus components.

Cell-mediated immunity is thought to play a decisive role in protecting cats against feline infectious peritonitis (FIP), a progressive and lethal coronavirus disease. In view of the potential of DNA vaccines to induce cell-mediated responses, their efficacy to induce protective immunity in cats was evaluated. The membrane (M) and nucleocapsid (N) proteins were chosen as antigens, because antibodies to the spike (S) protein of FIP virus (FIPV) are known to precipitate pathogenesis. However, vaccination by repeated injections of plasmids encoding these proteins did not protect kittens against challenge infection with FIPV. Also, a prime-boost protocol failed to afford protection, with priming using plasmid DNA and boosting using recombinant vaccinia viruses expressing the same coronavirus proteins. Because of the role of IL-12 in initiating cell-mediated immunity, the effects of co-delivery of plasmids encoding the feline cytokine were studied. Again, IL-12 did not meet expectations - on the contrary, it enhanced susceptibility to FIPV challenge. This study shows that DNA vaccination failed to protect cats against FIP and that IL-12 may yield adverse effects when used as a cytokine adjuvant.

The envelope is of cardinal importance for the entry of feline immunodeficiency virus (FIV) into its host cells, which consist of cells of the immune system including macrophages. To characterize the envelope glycoprotein determinants involved in macrophage tropism, chimeric infectious molecular clones were constructed containing envelope gene sequences from isolates that had been propagated in peripheral blood mononuclear cells (PBMC). The progeny virus was examined for growth in PBMC and bone marrow-derived macrophages and viruses with different replication kinetics in macrophages were selected. Envelope-chimeric viruses revealed that nucleotide sequences encoding variable regions 3 and 4 of the surface glycoprotein, SU, are involved in macrophage tropism of FIV. To assess the biological importance of this finding, the phenotypes of envelope proteins of viruses derived from bone marrow, brain, lymph node and PBMC of an experimentally FIV-infected, healthy cat were examined. Since selection during propagation had to be avoided, provirus envelope gene sequences were amplified directly and cloned into an infectious molecular clone of FIV strain Petaluma. The viruses obtained were examined for their replication properties. Of 15 clones tested, 13 clones replicated both in PBMC and macrophages, two (brain-derived clones) replicated in PBMC only and none replicated in Crandell feline kidney cells or astrocytes. These results indicate that dual tropism for PBMC and macrophages is a common feature of FIV variants present in vivo.

The alphaherpesvirus glycoproteins gE and gI form a hetero-oligomeric complex involved in cell-to-cell transmission. The gI-deficient recombinant feline herpesvirus (FHV), FHVdeltagI-LZ, produces plaques that are only 15% the size of those of wild-type FHV. Here, we have complemented FHV(delta)gI-LZ allotopically by expressing intact gI and C-terminally truncated gI derivatives from the thymidine kinase locus. The effect on gE-gI-mediated cell-to-cell spread was assessed by plaque assay employing computer-assisted image analysis (software available at http://www.androclus.vet.uu.nl/spotter/spotter.htm+ ++). Allotopic complementation with intact gI fully restored plaque size. Deletion of the C-terminal 11 residues of gI did not affect cell-to-cell spread, whereas deletion of the complete cytoplasmic tail reduced plaque size by only 35%. Mutants expressing gI166, roughly corresponding to the N-terminal half of the ectodomain, displayed a small-plaque phenotype. Nevertheless, their plaques were reproducibly larger than those of matched gI-deficient controls, indicating that the gE-gI166 hetero-oligomer, though crippled, is still able to mediate cell-to-cell spread. Our data demonstrate that plaque analysis provides a reliable and convenient tool to measure and quantitate gE-gI function in vitro.

Coronaviruses infect humans and animals through epithelial cells of the gastrointestinal and respiratory tracts that serve as their primary target. When studying infections in cultured polarized epithelial cells, we found previously that coronaviruses are released from specific plasma-membrane domains; thus, mouse hepatitis virus (strain A59; MHV-A59) leaves murine epithelial kidney cells from the basolateral surface, whereas release of transmissible gastroenteritis virus from porcine epithelial kidney cells is confined to the apical membrane. This observation begged the question whether a particular coronavirus is consistently shed through the same membrane, irrespective of the nature of the epithelial cell. We therefore extended our studies with MHV-A59 to Madin-Darby canine kidney (MDCK) strain I and human colon carcinoma (Caco-2) cells, both of which are naturally refractory to MHV-A59 but were made susceptible to infection by transfection with recombinant MHV receptor cDNA. The release of MHV-A59 from Caco(MHVR) cells occurred preferentially from the basolateral side, consistent with our previous observations. In contrast, release from MDCK(MHVR) cells occurred almost exclusively from the apical surface. Because of this difference, we studied MHV-A59 infection of MDCK(MHVR) cells in more detail. The virus entered the cells preferentially from the apical side, a situation similar to that in murine epithelial cells, where the highest density of MHV receptor glycoprotein was found. The results from this and previous studies show that targeting of vesicles containing MHV-A59 to a specific side of epithelial cells may vary in different epithelial cell types.

We analysed the production of transforming growth factor beta (TGF-beta) during a cytomegalovirus (CMV) infection in a rat model system. Splenocytes from immunocompetent rats infected with rat CMV (RCMV) released increased amounts of TGF-beta1. TGF-beta production was also evident in RCMV-infected radiation-immunosuppressed rats; their sera inhibited the interleukin 2-induced proliferation of T cells, which could be restored by anti-TGF-beta antibodies. In addition, TGF-beta production could be visualized immunohistologically in the lungs, spleen, liver and bone marrow of radiation-immunosuppressed infected rats. The virus directly induced this cytokine since TGF-beta was produced upon RCMV infection in vitro. The induction of TGF-beta production may contribute to immunosuppression during CMV infection.

We have cloned, sequenced and expressed the spike (S) gene of canine coronavirus (CCV; strain K378). Its deduced amino acid sequence has revealed features in common with other coronavirus S proteins: a stretch of hydrophobic amino acids at the amino terminus (the putative signal sequence), another hydrophobic region at the carboxy terminus (the membrane anchor), heptad repeats preceding the anchor, and a cysteine-rich region located just downstream from it. Like other representatives of the same antigenic cluster (CCV-Insavc-1 strain, feline infectious peritonitis and enteric coronaviruses, porcine transmissible gastroenteritis and respiratory coronaviruses, and the human coronavirus HCV 229E), the CCV S polypeptide lacks a proteolytic cleavage site present in many other coronavirus S proteins. Pairwise comparisons of the S amino acid sequences within the antigenic cluster demonstrated that the two CCV strains (K378 and Insavc-1) are 93.3% identical, about as similar to each other as they are to the two feline coronaviruses. The porcine sequences are clearly more divergent mainly due to the large differences in the amino-terminal (residues 1 to 300) domains of the proteins; when only the carboxy-terminal parts (residues 301 and on) are considered the homologies between the canine, feline and porcine S polypeptides are generally quite high, with identities ranging from 90.8% to 96.8% . The human coronavirus is less related to the other members of the antigenic group. A phylogenetic tree constructed on the basis of the S sequences showed that the two CCVs are evolutionarily more related to the feline than to the porcine viruses. Expression of the CCV S gene using the vaccinia virus T7 RNA polymerase system yielded a protein of the expected M(r) (approximately 200K) which could be immunoprecipitated with an anti-feline infectious peritonitis virus polyclonal serum and which was indistinguishable from the S protein synthesized in CCV-infected cells.

The production of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6, all proinflammatory cytokines, was investigated in radiation-immunosuppressed rats infected with rat cytomegalovirus (RCMV). At day 7 post-infection, when the animals showed disease signs, high TNF-alpha levels were detected in the serum and in homogenates of various organ tissues. In contrast, IL-1 and IL-6 levels were not significantly elevated. Moreover, replication of RCMV induced TNF-alpha expression in different types of cells grown in vitro. When frozen tissue sections were examined by immunohistology, TNF-alpha-producing cells were found in areas with extensive pathology in the lungs, spleen and liver. Both lymphocytes and RCMV-infected cells were identified as the sources of TNF-alpha. Its abundance in RCMV-infected rats suggests an important role for TNF-alpha in CMV pathogenesis.

In vivo administration of exogenous cytokines may influence elicited immune responses, and hence may change the efficacy of a vaccine. We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) on the immune response elicited by inactivated rabies virus vaccine in a mouse model. Each of the cytokines increased virus-specific IgG responses after primary and after secondary immunization. A single dose of 1.3 ng TNF-alpha or IL-1 alpha, when injected shortly before vaccination, only marginally stimulated resistance to challenge infection (four- and seven-fold, respectively) without enhancing virus neutralizing antibody (VNAb) responses. In contrast, a single injection of 10(3) units of IFN-gamma or five daily injections of 1.6 micrograms IL-2 increased vaccine dilutions protecting 50% of mice (PD50 values) 77- to 50-fold, respectively, with a concomitant enhancement of VNAb. At a 1:10,000 dilution of a standard inactivated rabies vaccine preparation both IFN-gamma and IL-2 increased protective immunity without enhancing VNAb responses; in non-vaccinated animals this treatment had no effect on resistance to challenge. Combined administration of IFN-gamma and IL-2 synergistically enhanced VNAb responses. In contrast to the other cytokines tested, IFN-gamma preferentially stimulated virus-specific IgG2a production. It also augmented the vaccine-induced priming of rabies virus-specific splenocyte proliferation. These results document that certain cytokines alone or in combination are potent immunological adjuvants which may direct and modulate immunization-induced antiviral immune responses.

Infection with the mouse hepatitis coronavirus (MHV) provides an excellent model for the study of viral diseases of the central nervous system and the gastrointestinal tract. With the ultimate aim of studying mucosal immunity to MHV we have cloned the genes encoding the structural proteins of MHV strain A59 (MHV-A59) into the E3 region of a human adenovirus type 5 vector. Infection of HeLa cells with the resulting recombinant adenoviruses AdMHVS, AdMHVN and AdMHVM revealed the correct expression of the spike (S), nucleocapsid (N) and membrane (M) proteins, respectively. Intraperitoneal inoculation of BALB/c mice with the recombinant viruses elicited serum antibodies which specifically recognized the respective MHV proteins in an immunoprecipitation assay. Only antibodies to the S protein neutralized MHV-A59 in vitro but titres were low. When analysed by ELISA or by immunofluorescence only the antibody response to the N protein was significant; weak responses or no detectable response at all were found for S and M, respectively. Upon intracerebral challenge with a lethal dose of MHV-A59 we found that a significant fraction of animals vaccinated with adenovirus vectors expressing either the S protein or N protein were protected. This protective effect was significantly stronger when the animals were given a booster immunization with the same vector prior to challenge. No protection was induced by AdMHVM. Interestingly, enhanced protection resulted when AdMHVS and AdMHVN were applied in combination as compared to survival after single immunizations. The results indicate that both the N and S proteins generate a protective immune response and suggest that this response is enhanced by combined expression of the two proteins.

Defective interfering (DI) RNAs of Berne virus (BEV) were generated by serial undiluted passaging of the virus in embryonic mule skin cells. Two DI RNAs of 1.0 and 1.4 kb (designated DI1000 and DI1400) were characterized in more detail. Isokinetic sucrose gradient analysis showed that these DI RNAs are specifically packaged into particles with smaller S values than standard virions. Both DI RNAs were cloned and sequenced. Three genomic cDNA clones were identified using probes complementary to the 5' end of a DI RNA, which are thought to be derived from the 5'-terminal region of the BEV genome. A non-translated region of about 700 nt and the 5' end of the putative BEV replicase gene were identified in the consensus nucleotide sequence. Both DI RNAs were shown to contain sequences from the 5' and 3' ends of the BEV genome. A conserved sequence motif, which has been postulated to be involved in sub-genomic RNA transcription, was also identified just downstream of the extreme 5' ends of DI1000 and DI1400.

The individual and synergistic antiviral effects of cytokines released by infiltrating immune cells or by cells of the nervous system may play an important role in inhibiting virus spread during infections of the central nervous system (CNS). We examined the antiviral activity against the neurotropic pseudorabies virus (PRV) of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), and combinations of these cytokines, as compared to that of IFN-beta, in rat nervous tissue cells. PRV replicated efficiently in all neural cell types tested, including neurons, astrocytes and oligodendrocytes. The inhibitory effects were determined by quantifying the inhibition of virus plaque formation, yields of infectious virus at various times after infection and synthesis of viral proteins. At a low m.o.i., IFN-gamma and IFN-beta inhibited viral plaque formation in all cell types; TNF-alpha was effective only in astrocytes but showed synergy with IFN-gamma. At a higher m.o.i., IFN-beta inhibited yields of infectious virus more effectively than IFN-gamma, whereas TNF-alpha had no effect on virus yields and was only marginally synergistic with the antiviral activity of IFN-gamma. The yield-reduction assays correlated well with cytokine-induced inhibition of viral protein synthesis. Our results show that both IFN-gamma and IFN-beta can induce a state of antiviral resistance in neural cells whereas TNF-alpha is effective only in astrocytes at low m.o.i.; they suggest an antiviral role of cytokines in the immune response to virus infections of the CNS.

Feline immunodeficiency virus (FIV) grown in cat lymphocyte and thymocyte cultures was labelled with L-[35S]methionine or [3H]glucosamine and virus-coded proteins were identified using immunoprecipitation. Polypeptides with apparent Mr values of 15K, 24K, 43K, 50K, 120K and 160K were detected. An additional polypeptide of 10K was detected by Western blot analysis. The two highest Mr species sometimes appeared as one band, of which only the 120K polypeptide was glycosylated. In the presence of tunicamycin gp120 was no longer detectable and a non-glycosylated precursor of 75K was found instead. Pulse-chase experiments suggested that the smaller polypeptides p24 and p15 are cleavage products of both p160 and p50. Western blot analysis using a rabbit serum directed against p26 of equine infectious anaemia virus (EIAV) and an anti-EIAV horse serum from a field case of infection revealed a cross-reactivity with p24 of FIV. Cat sera collected late after experimental FIV infection recognized p26 of EIAV, indicating a reciprocal cross-reactivity.

In infected embryonic mule skin cells, Berne virus directs the synthesis of two main polypeptides (22K, 20K); in addition, virus-specific proteins with apparent molecular weights of greater than 200K, 80K to 120K, 32K and 17K were detected after radioimmune precipitation. The replication of Berne virus was reduced more than 1000-fold by actinomycin D, when the drug (0.1 to 1.0 micrograms/ml) was added during the first 8 h after infection; alpha-amanitin (25 micrograms/ml) produced a similar though less pronounced effect. U.v. preirradiation of the cells for greater than or equal to 5 s led to a dramatic decrease in the production of extracellular virus. The results presented support our suggestion that Berne virus is a representative of a new family of animal viruses.

Growth of feline infectious peritonitis virus in a continuous feline cell line is described and evidence for the macrophage-like character of these cells is presented. Under one-step growth conditions, cytopathic changes and giant cell formation were observed 12 h after infection; more than 99% of the virus remained cell-associated 15 h after infection. Viral proteins at the surface of infected cells were detected by immunofluorescence. The exposed antigens were localized on four proteins with molecular weights of 225.5K, 175K, 138K and 25K using radioiodination followed by immunoprecipitation. Another viral polypeptide of 44K (the nucleocapsid protein) was only labelled when the cell membranes had been disrupted. Expression of viral antigens on the cell surface may be a significant factor in the immune pathogenesis of feline infectious peritonitis.

Pike fry rhabdovirus (pfr) rna has been characterized as a single-stranded non-segmented molecule of about 4 X 10(6) daltons. In sucrose gradients in 0-1 M-NaC1 it had a sedimentation coefficient of 40 to 45S, somewhat lower than that Of Semliki Forest virus RNA. The sedimentation velocity of the RNA is strongly influenced by the salt concentration and divalent cations. It shows a buoyant density of 1-65 g/ml in caesium sulphate. The base composition of the RNA is 21-6% G, 25-I% A, 22-4% C and 30-9% U. It is thus comparable to the RNAs of other rhabdoviruses.

The neurovirulence of eight temperature-sensitive (ts) mutants of mouse hepatitis virus strain A59 in 4-week-old BALB/c mice was investigated. Whereas a dose of 100 p.f.u. of wild-type virus killed mice within a week, a 1000-fold higher dose of ts mutants did not. Three ts mutants induced demyelinating disease in the central nervous system (CNS). The pathology of the demyelinating disease caused by one mutant, designated ts-342, was studied in detail. Pathological changes, starting 3 days post-inoculation (p.i.), were characterized by inflammation and demyelination in the CNS. Antibody responses directed against all virus-specific structural proteins were present at 7 days p.i. No virus particles were observed by electron microscopy at 14 days p.i. However, macrophages and lymphocytes were abundant in the areas of demyelination. The growth kinetics in vivo of wild-type virus, ts-342 and a revertant of ts-342 were compared. Wild-type virus and the revertant replicated rapidly in the brain and spread to the liver causing a lethal hepatitis. Ts-342, however, replicated to a much lesser extent within the brain and could not be detected in the blood or liver. The ts lesion in the genome of ts-342 seems, therefore, to determine the outcome of the infection.

In Felis catus whole foetus D cells infected with feline infectious peritonitis virus (FIPV), strain 79-1146, six virus-specific, poly(A)-containing RNA species of about 20, 9.6, 5.2, 3.8, 2.8 and 1.6 kb were found. By translation in vitro the 3.8 and 2.8 kb RNAs were shown to encode the 25K envelope protein and the 45K nucleocapsid protein, respectively. The partial map of the FIPV genome was compared with genomic maps of porcine, murine and avian coronaviruses. Differences in these maps suggest that transcription units have been lost or gained during coronavirus divergence.

From 380S particles of Berne virus (proposed family Toroviridae) one species of polyadenylated RNA was isolated. Using agarose gel electrophoresis its length was estimated as 20 kb or greater. When assayed under hypertonic transfection conditions genomic RNA was found to be infectious; RNase treatment destroyed the infectivity. The positive polarity of the molecule was confirmed by filter spot hybridization using cDNA prepared against poly(A)-selected RNA from infected cells. In embryonic mule skin cells infected with Berne virus the presence of five virus-specific, polyadenylated RNA species of 7.5, 2.1, 1.4, 0.8 and at least 20 kb was demonstrated. In vitro translation of the 7.5, 2.1 and 0.8 kb RNAs followed by immunoprecipitation showed that they encode a 151K product (possibly the precursor to the peplomer proteins), the envelope protein and the nucleocapsid protein, respectively.

Twelve Dutch isolates and the M41 strain of infectious bronchitis virus (IBV), a coronavirus of chickens, were characterized by cross-neutralization and T1 finger-printing to elucidate their evolutionary relationship. The T1 fingerprinting showed that the Dutch isolates formed two clusters. The first cluster contained strains H52, H120, D387, V1259, V1385 and V1397; the estimated sequence homology is 99%. Cluster two comprised strains D207, D274, D212, D1466, D3128 and D3896, which have about 95% sequence homology. The M41 virus did not belong to either cluster. The four different serotypes which arose in the late 1970s belonged to cluster two and appeared to be different from the vaccine strains (H52 and H120) used at that time. This indicates that the strains were newly introduced and could have arisen from a common virus. On the other hand, three recently isolated field strains were genetically closely related to the vaccine strains H120 and H52 (cluster one), suggesting that these live vaccine strains themselves could have given rise to these serologically altered field isolates. The data are relevant to the development of new vaccine strategies.

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 electron density 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.

Treatment of Wag/Rij rats with recombinant rat interferon gamma (rRIF-gamma) resulted in complete protection against a lethal pseudorabies virus (PRV) infection. To investigate whether the protection resulted from direct inhibition of virus replication or from a stimulation of immune mechanisms, we tested rRIF-gamma activity in naturally immunocompromised and artificially immunosuppressed rats. The antiviral effect of rRIF-gamma was not abolished in silica- and carrageenan-treated, phagocyte-depleted rats. Immunologically immature newborn and T cell-deficient nude rats were also protected under a regime of rRIF-gamma treatment as well as whole body gamma-irradiated rats. Sera of the protected rats were devoid of PRV-neutralizing antibodies. Our results indicate that the protective activity of rRIF-gamma is based on direct inhibition of virus replication; stimulation of the immune system is not required but may be responsible for protection upon challenge several weeks after infection.

Seventeen temperature-sensitive mutants of equine arteritis virus, a nonarthropod-borne togavirus, have been isolated. 5-Fluorouracil, o-methylhydroxylamine and ethyl methanesulphonate were used as mutagens. The mutants were characterized by their ability to synthesize virus RNA and virus proteins at the permissive (35 degrees C) and restrictive temperature (40 degrees C) using autoradiography of cells labelled with 3H-uridine in the presence of actinomycin D and immunofluorescence respectively. Among the mutants, four were unable to synthesize virus RNA and virus proteins at 40 degrees C (RNA-/protein-). The other mutants were RNA-/protein+ (3); RNA +/-/protein- (2); RNA+/protein+ (6) and RNA+/protein- (1).

In Berne virus-infected cells and in gradient-purified virions two major proteins with mol. wt. of 20K and 22K were detected. The 22K species is thought to represent the main envelope polypeptide; in infectious culture media it was present in a low envelope polypeptide; in infectious culture media it was present in a low density substructure which could be quantitatively converted into slowly sedimenting material by detergent treatment. The 20K polypeptide (accounting for about 80% of the 14C-amino acid label in the virion) was phosphorylated, occurred in an intracellular substructure of higher density than the virion (rho = 1.36 g/ml in CsCl) and was the only viral protein possessing RNA-binding properties; it was recognized preferentially by heterologous animal sera in immune precipitation. The 20K species is therefore identified as the main capsid protein. Two additional polypeptides (19K and 17K) were regularly detected in extracts of infected cells; they appeared to share oligopeptides with the 20K protein and are interpreted as being proteolytic cleavage products. The nucleocapsid of Berne virus was visualized after ether treatment as a flexible bacilliform structure with conspicuous transverse striation. Demonstration of a 20K nucleocapsid protein further supports the authors' proposal that Berne virus is a representative of a new family of enveloped RNA viruses (Toroviridae).

In Berne (Switzerland), a virus was isolated from a horse which was found to be serologically unrelated to known equine viruses. Its growth was unaffected by iododeoxyuridine and it was inactivated by organic solvents. A purification procedure involving ammonium sulphate precipitation and sucrose gradient equilibrium centrifugation was developed and viral activities were monitored using infectivity and enzyme-linked immunosorbent assays. Purified virions of density 1.16 g/ml were shown by negative staining electron microscopy to be roughly spherical and to measure 120 to 140 nm in diameter; projections (peplomers, about 20 nm long) were identified on the virion surface. In thin sections, an envelope and an elongated core structure could be distinguished. The core, measuring about 23 nm across and 104 nm in length, appears to assume a rod-, crescent- or open ring-shape within the envelope. It has a tubular structure and shows a transverse striation (periodicity 4.5 nm). Binding at the plasma membrane was observed. Berne virus is considered as a representative of a hitherto undefined family of widespread animal viruses serologically related to recent bovine isolates in Ames, Iowa (U.S.A.) and Lyon (France).

The nucleotide sequence of the nucleocapsid (N) protein gene of Berne virus (BEV; proposed family Toroviridae) was determined from two independent clones of a cDNA library. From the deduced amino acid sequence a basic protein of 18.3K was predicted. In vitro transcription and translation, followed by immunoprecipitation, were used to identify the gene. The identification was confirmed by metabolic labelling, using the knowledge that cysteine residues are absent from the amino acid sequence of the N protein. Smaller N-related polypeptides encountered in BEV-infected cell lysates were shown to be probable products of aberrant translation, due to initiation on AUG codons further downstream in the N protein gene.

Using [3H]glucosamine and [3H]mannose labels, two virus-specific glycosylated polypeptide species with Mr values of about 200,000 (200K) and in the 75K to 100K range, respectively, were recognized in Berne virus-infected embryonic mule skin cells. In purified virions only the latter glycoprotein occurred. Concanavalin A was bound to the virion as evidenced by reduction in infectivity. Analyses using SDS-PAGE, blotting and glycoprotein identification with concanavalin A and horseradish peroxidase showed coincidence of the virion glycoprotein signals with the maximum infectivity and haemagglutinating activity in an isokinetic sucrose gradient. Polyclonal rabbit immune serum and a neutralizing and haemagglutination-inhibiting monoclonal antibody raised against Berne virus recognized both the 75K to 100K and the '200K' glycoproteins. Using tunicamycin, a concentration-dependent inhibition of infectivity was noted; however, non-infectious particles containing the two major polypeptides (20K and 22K) were released from the cells in small quantities. The glycoproteins were absent from cytoplasmic extracts and a novel polypeptide of about 150K was identified instead. Translation of poly(A)-selected intracellular RNA from infected cells in a rabbit reticulocyte cell-free system also resulted in the appearance of a new high Mr polypeptide (about 170K). Using pulse-chase labelling and radioimmunoprecipitation, suggestive evidence for a precursor-product relationship between the intracellular '200K' and the virion glycoproteins has been obtained. These experiments identify the N-glycosylated proteins in the 75K to 100K range as constituents of the peplomeric envelope projection of Berne virus; they probably arise by post-translational processing of a 150K to 170K precursor molecule involving glycosylation and subsequent cleavage.

Intracellular virus-specific proteins induced by equine arteritis virus (EAV) have been compared with in vitro translation products of virion and intracellular EAV RNAs. In infected BHK-21 cells, the two major virion proteins (C and E1) and polypeptides with mol. wt. of 60,000 (p60), 42,000 (p42) and 30,000 (p30) were found. There were no indications that the viral proteins were processed from a larger precursor as shown by pulse-chase, amino acid analogue and protease inhibitor experiments. The six polyadenylated RNAs that occur in EAV-infected cells were isolated and translated in an mRNA-dependent reticulocyte cell-free system. Translation of RNA6 resulted in the appearance of a product having the mol. wt. (14,000) of the nucleocapsid protein (C). EAV genomic RNA was translated into proteins of mol. wt. 30,000 and 200,000, while RNA1, the intracellular homologue of genomic RNA only encoded p30. The absence of large precursor molecules in infected cells and the results from the in vitro translation experiments both suggest that at least some of the proteins are primary translation products.

Lactic dehydrogenase virus (LDV) was purified from infectious ascites fluid of mice bearing Ehrlich tumours using Sepharose gel filtration and rate zonal and isopycnic sedimentation. In glycerol gradients, a sedimentation coefficient of about 200S and a buoyant density of 1-14 g/ml was determined for the virus particle. Spherical particles with diam. between 62 and 80 nm, depending on the method of fixation and staining, have been identified electron microscopically. The virus particle consists of a spherical nucleocapsid wrapped into a double-layered envelope. The nucleocapsids, isolated by treatment with NP40 and purified by centrifuging on sucrose gradients had a sedimentation coefficient of 176S. Electron micrographs show spherical particles with a diam of 35 plus or minus nm. Classification of LDV as a member of the togaviridae family is discussed.

The peplomer gene of feline infectious peritonitis virus (FIPV) strain 79-1146 was isolated from a genomic cDNA library by differential hybridization with RNA 2 and 3 as probes. From the nucleotide sequence a primary translation product of 1452 residues (Mr 160,472) was predicted, containing an N-terminal signal sequence, a C-terminal transmembrane segment and 35 potential N-linked glycosylation sites. By S1 nuclease analysis the 5' end of the presumptive RNA 2 body was located at about 30 nucleotides upstream from the initiating AUG codon. At approximately the same position a nine nucleotide sequence ACUAAACUU was found, which was also present 37 nucleotides downstream from the open reading frame. Comparison of the sequences of the FIPV, murine hepatitis virus and infectious bronchitis virus peplomer proteins showed about 27% overall homology, with most conservation in the C-terminal half.