- Volume 69, Issue 2, 1988

Foot-and-mouth disease virus (FMDV) RNA, isolated as virion RNA from purified virus particles or as total RNA from infected cells, has been microinjected into nuclei and cytoplasms of BHK cells. When injected directly into the nucleus FMDV RNA was not infectious, whereas cytoplasmic injection resulted in a high proportion of productive infections. Infectivity microinjection assays on dilution series of various FMDV RNAs showed that both single-stranded positive sense 35S RNA and double-stranded replicative form (Rf) RNA have an infectivity close to 1 p.f.u. per molecule, although only a minor fraction of BHK cells appeared able to support plaque formation following microinjection of Rf FMDV RNA. The infectivity of Rf FMDV RNA was not sensitive to inhibition by actinomycin D. The results are discussed in relation to the high virus particle to p.f.u. ratios observed for FMDV.

Interferon (IFN) response mutants were selected from mouse L929 fibroblast cells and their specific resistance to is-1, an IFN-sensitive mutant of mengovirus, was studied. The standard L cell subline used in our laboratory (G3), is resistant to is-1 infection after pretreatment with low levels of IFN. Two clonal sublines that support the growth of is-1 in the presence of IFN (AS-4 and TA-6) were isolated from it, and two revertant lines (AS-4R1 and TA-6R1) were subsequently selected from AS-4 and TA-6. The kinetics of is-1 growth in the presence of IFN were found to vary in each of these sublines. Specific resistance to is-1 cannot be accounted for by enhanced induction of IFN, ability to bind IFN, or increased 2′-5′-oligo(A)-dependent endonuclease activity. AS-4 and TA-6 appear to have arisen through loss of one or more whole chromosomes. The origin of TA-6R1 is unclear.

A cloned cDNA probe derived from coxsackie B4 virus-infected cell RNA was shown to hybridize to the RNA of a number of different enteroviruses including coxsackie A and B viruses, echoviruses and poliovirus. The probe was used to detect virus-specific RNA sequences in cardiac tissue obtained from patients diagnosed as having a coxsackievirus infection. Virus RNA was detected using the technique of in situ hybridization in 46% (6/13) cases, but none was found in normal, control, cardiac samples. Two distinct patterns of infection were observed. The significance of these differences and the possible uses of the technique are discussed.

The fusion (F) glycoprotein, large glyco- (G) protein, phospho- (P) protein and 22K protein of respiratory syncytial (RS) virus A2 strain were purified by a combination of immunoaffinity adsorption and preparative SDS-PAGE. All four proteins elicited serum antibody in mice after repeated inoculation in adjuvant, although the magnitude of the response as measured by ELISA varied from mouse to mouse. The F protein generated neutralizing antibodies in only 50% of the mice determined to be seropositive by ELISA. The G protein also induced neutralizing antibodies but in this instance neutralization tests and ELISA titres were more closely correlated. No neutralizing activity was detected in mice immunized with the P or 22K proteins although all produced antibody detectable by ELISA. Mice immunized with either the F or the G protein were found to be protected against subsequent RS virus challenge, whether they had developed neutralizing antibody or not. Mice inoculated with the P or 22K proteins were not protected.

In this study we aimed to define the protein and viral subtype specificities of helper Th cells to respiratory syncytial virus (RSV). BALB/c mice were primed by infection with RSV, or with vaccinia viruses (VV) containing genes encoding several individual RSV proteins. Priming for Th cell memory was assayed by stimulating spleen cells in vitro with different RSV isolates and measuring RSV-specific interleukin 2 (IL-2) release by T cells into supernatants using an IL-2-dependent CTLL cell line. Splenocytes from mice primed intranasally with RSV exhibited RSV-specific Th cell memory, whereas those from unprimed mice did not. Th cell recognition was in part specific to the strain of RSV used in priming and in part cross-reactive between RSV strains. Intraperitoneal priming with RSV fusion protein-expressing VV or nucleoprotein-expressing VV induced a stronger RSV-specific Th cell response than the attachment glycoprotein-expressing VV which produced only slight Th recognition. No Th cell recognition of two non-structural proteins (1A and 1B) could be demonstrated.

The kinetics of synthesis of [35S]methionine-labelled respiratory syncytial virus-specific proteins were studied in CV-1 cells infected at high multiplicity. Immunoprecipitated viral proteins resolved by SDS-PAGE were quantified by scanning fluorographs of protein bands. The nucleocapsid (N) protein was detectable by 2 h post-infection (p.i.), whereas the phospho- (P), matrix (M) and fusion (F0) proteins and Vp24 (a matrix-like protein) were first detected between 4 and 6 h p.i. Synthesis of the glyco- (G) protein was first detected at 6 h p.i. and reached its peak synthesis rate at 10 h p.i. Virus-specific P, M and Vp24 proteins were phosphorylated in infected cells. The P protein was highly phosphorylated in purified virions whereas phosphorylated species of the M and Vp24 proteins were minor components. The phosphorylated form of the P protein was detected by monoclonal antibody precipitation, confirming the identity of this protein. The N protein was not phosphorylated in infected cells or in virions. Synthesis of [35S]methionine-labelled proteins preceded detectable 32Pi labelling by several hours. The putative phosphorylated M protein was detected at 6 h p.i. before phosphorylated forms of P and Vp24 were seen. The timing of appearance of the phosphorylated species of P and Vp24 proteins in infected cells corresponded to the release of infectious virions from infected cell monolayers at 10 to 12 h p.i.

The morphogenesis of yellow fever virus replication was examined in infected Vero cell cultures. Penetration and uncoating occurred by endocytosis with the formation of coated vesicles, similar to that demonstrated for other enveloped and unenveloped viruses. Inclusion bodies associated with newly formed nucleocapsids were evident in the perinuclear region during the growth cycle. No evidence of RNA synthesis in the vicinity of the inclusion bodies was obtained by autoradiography, suggesting that genome replication and assembly of viral nucleocapsids occur at separate cytoplasmic sites. An excessive proliferation of membrane-bound organelles involving both vacuoles and endoplasmic reticula was the most striking feature of virus-infected cells late in infection. No morphological changes in the appearance of nuclei or mitochondria were detected. Virus release appeared to occur by movement of nascent virions through the proliferated endoplasmic reticula followed by exocytic fusion of virus-containing vesicles with the plasmalemma. A possible mechanism whereby the internal nucleocapsid acquires an outer envelope is discussed.

Rio Bravo (RB) virus has been assigned to the family Flaviviridae on the basis of its antigenic relatedness to other members of this family. RB virus, unlike most members of the Flaviviridae, is believed not to have an arthropod vector. We examined biochemical and biophysical characteristics of RB virus to determine whether it should be assigned to the Flaviviridae and to compare it with arthropod-borne flaviviruses. Purified RB virus banded at a density of 1.18 g/ml in sucrose and had a sedimentation coefficient of about 200 S. Virions, negatively stained with ammonium molybdate, were spherical, had diameters of 42 nm, and appeared to be surrounded by envelopes bearing surface projections. The loss of infectivity after infectious virus was incubated with diethyl ether or sodium deoxycholate confirmed the presence of envelopes. Partially purified RB virions contained single-stranded RNA, lacking 3′ poly(A) tracts, that sedimented in a 15% to 30% sucrose gradient as one discrete band with a sedimentation coefficient of about 40 S. Most of the viral proteins in preparations of purified virus and in immunoprecipitates had similar electrophoretic mobilities and glycosylation patterns to known flavivirus proteins. Therefore, they were assigned the following tentative designations using the nomenclature for flavivirus proteins: gp52 and gp47, envelope proteins; gp46, non-structural protein 1;p25, gp20(prM), precursor to membrane protein; gp < 18K. Putative core and membrane proteins were not identified. These physical and biochemical characteristics of RB virus are remarkably similar to those of the arthropod-borne members of the Flaviviridae and they confirm the classification of RB virus in this family. This is the first report of biochemical and physical properties of a non-arthropod-borne member of the Flaviviridae.

Canine parvovirus (CPV) strains were compared for haemagglutination (HA) activity and antigenicity and were divided into two types by HA activity. Strains Cp49 and 29-F showed temperature-dependent HA, like feline panleukopenia virus (FPLV) and mink enteritis virus (MEV), whereas strains Sp-80 and Y-1 showed temperature-independent HA with erythrocytes from eight species of animals. The results of a cross HA inhibition test using immunized rat sera suggested that of the two types of CPV those showing temperature-dependent HA were antigenically like FPLV and MEV whereas those showing temperature-independent HA were not. This antigenic difference between the two types was confirmed by a HA inhibition test with monoclonal antibodies. A chronological survey revealed that CPV isolates from earlier years have HA activity and antigenicity similar to those of FPLV and MEV, whereas current CPV isolates do not. There are some exceptional isolates from the transitional period which have similar antigenicity to FPLV and MEV but different HA activity. These results suggest that the haemagglutinin of CPV altered from one form resembling that of FPLV to a somewhat different structure during passage in dogs in nature.

We have previously described blocks to the viral lytic cycle at two different levels in cytomegalovirus (CMV)-infected non-permissive cells. BALB/c-3T3 cells express only the predominant immediate early (IE) nuclear phosphoproteins (IE68 or IE94) of human CMV (HCMV) or simian CMV (SCMV) and do not replicate the input viral genomes. However, in human teratocarcinoma stem cells and 293 cells, expression of the HCMV IE68 gene (but not the SCMV IE94 gene) is blocked at the transcriptional level. Here we report the results of an extensive comparison of the level of permissiveness for HCMV, SCMV and murine CMV (MCMV) in a variety of additional cell types of human, monkey and mouse origin. We also describe a subtle change in the tryptic peptide pattern of the IE68 polypeptide produced in BALB/c-3T3 cells compared to permissive human foreskin fibroblasts. Neither the IE68 nor IE94 proteins could be detected by biochemical labelling procedures in infected mouse Ltk- or F9 teratocarcinoma stem cells, although IE94 was synthesized after retinoic acid-induced differentiation of the F9 cells. Synthesis of [35S]methionine-labelled IE94 protein, but not that of HCMV IE68, was detected in infected Vero cells and in human peripheral blood leukocyte cultures. The failure to synthesize detectable IE68 protein in infected Vero cells appeared to be unrelated to a lack of entry of viral DNA and to a lack of appropriate transcription factors. Indeed, immunofluorescence assays showed that the IE68 antigen was expressed efficiently in DNA-transfected Vero cells and in a small fraction of infected Vero cells. Overall, two clear host range trends emerged. First, whilst all three viruses showed a tendency for repression of IE expression in transformed cell lines, the effect was severe for HCMV and only minimal for SCMV. Secondly, progression of infection to the viral DNA synthesis level in non-transformed fibroblast cell types occurred in a much wider range of host species cell types for SCMV and MCMV than for HCMV.

BALB/c mice were infected with herpes simplex virus type 2 by inoculation into the footpad. Three months or more after infection, and in the total absence of any evidence of illness, latent virus could be reactivated from both ganglia and footpad tissue but only after explantation and culturing for a week or more. Virus recovery from the footpad could not be prevented by treatment of the infected mice with acycloguanosine for up to 6 months in their drinking water at 40 mg/kg, nor by femoral and sciatic nerve section, nor by combining both treatments.

Herpes simplex virus type 2 has been reactivated from the latent state in the footpad and dorsal root ganglia of acycloguanosine-treated BALB/c mice. Virus was also recovered from the footpad tissue but not from the ganglia of denervated, latently infected mice. Treatment in vitro of explanted footpad cultures with acycloguanosine or phosphonoacetic acid did not affect the rate of virus reactivation. In all the isolates examined the virus was found to be acycloguanosine-sensitive. Recovery of virus from footpad tissue of mice after a long period of acycloguanosine treatment supports the theory that virus had been truly latent in the footpad and not in a state of persistent infection.

Electron microscope studies been made of mock-infected or herpes simplex virus (HSV) type 1 (strain 17)- or HSV-2 (strain HG52)-infected Flow 2002 cells grown in the absence or presence of various concentrations of cicloxolone sodium (CCX). Fifty nonserial, thin sections of mock-infected or HSV-infected cells, which contained a portion of the nucleus, were examined by transmission electron microscopy. With increasing drug concentration, counts of capsid structures both in the nucleus and cytoplasm showed a decrease in the number of virions per cell section, an increase in the ratio of nuclear to cytoplasmic virus, a relative reduction in the percentage of cytoplasmic enveloped virus particles, but no effect on the ratio of empty to core-containing capsid structures. The high particle to p.f.u. ratio induced by CCX treatment is thus not explained through failure to assemble morphologically mature core-containing capsids. In part it can be explained by non-envelopment, but in addition, specific effects on other virion proteins (tegument and envelope) must be involved. The extracellular virus particle yield was unaffected, indicating that CCX treatment enhances the egress of HSV. In the presence of CCX the encapsidation of HSV DNA into DNase-resistant structures was unaffected.

Human monocytes appear to be very important in the pathogenesis of dengue infection. They are thought to be the most active sites of virus replication during dengue infection. We have analysed interferon (IFN) production by dengue virus from peripheral blood mononuclear cells (PBMC). IFN activity was first detected at 12 h after infection of monocytes and reached a maximum level by 48 h. Non-adherent PBMC depleted of monocytes did not produce detectable levels of IFN, and did not contain dengue antigen-positive cells after exposure to dengue virus. The IFN produced was characterized as IFN-α by neutralization tests using specific antisera to HuIFN-α, HuIFN-β and HuIFN-γ, and by radioimmunoassay. The culture fluids of dengue virus-infected monocytes, which contained IFN-α, were able to inhibit infection of human monocytes by dengue virus. These results suggest that IFN-α produced by dengue virus-infected monocytes may play an important role in controlling primary dengue virus infection.

We analysed serotype-specific antigens of human rotavirus (HRV) by the use of neutralizing monoclonal antibodies (N-MAbs). The reactivity patterns of 12 serotype-specific N-MAbs against 15 HRV strains in a neutralization test revealed great intraserotypic antigenic variation especially those belonging to serotypes 2, 3 and 4. On the basis of the protein specificity of the antibodies examined, it was suggested that whereas serotype 2-specific neutralization epitopes were present on both VP3 and VP7 outer capsid proteins, serotype 1-, 3- and 4-specific neutralization epitopes were located on VP7. Serotype-specific neutralization epitopes on VP7 of the serotype 1 HRV KU strain were analysed further using mutants of the KU strain resistant to different serotype 1-specific N-MAbs. The result suggested the presence of at least five operationally overlapping neutralization epitopes on VP7 of the KU strain, which collectively constituted a single large neutralization domain.

The construction of the first stable human cell lines that express and secrete authentic hepatitis B virus surface antigen (HBsAg), using a BK virus (BKV) episomal plasmid vector, is described. The amount of HBsAg produced by BKV vectors (up to 600 ng/107 cells) was comparable to other eukaryotic vector systems. The level of HBsAg expression remained the same regardless of the orientation of the HBsAg gene, substitution of the HBsAg gene promoter with the mouse metallothionein I gene promoter or the tissue origin of the human cell lines used to establish stable cellular transformants. Northern blot analysis also indicated synthesis of normal HBsAg transcripts. Surprisingly, however, the vectors were maintained at far lower than expected copy number (one to five copies/cell). Reasons for this are discussed.

Human fibroblasts derived from four individuals with various deletions in the short arm of one chromosome 11 were susceptible to morphological transformation by early region BK virus DNA, whereas diploid human fibroblasts were not. This difference in susceptibility to transformation by early region BK virus DNA might be explained by a putative ‘transformation suppressor’ locus situated within the deleted region on the short arm of chromosome 11.