- Volume 67, Issue 3, 1986

Intracardial inoculation of large quantities (200 µg viral protein/kg body weight) of infectious or u.v.-inactivated purified influenza viruses into ferrets resulted in a rapid febrile response which was significantly lower for two recently isolated H1N1 viruses, A/USSR/90/77 and A/Fiji/15899/83, than for two virulent clones, 7a and 64c, of the A/Puerto Rico/8/34-A/England/939/69 (H3N2) reassortant virus system. These results, which are in accord with the severity of fever produced by these strains in intranasally infected ferrets, show that influenza virus strains can differ in their capacity to induce fever (probably reflecting a differential capacity to induce endogenous pyrogen from phagocytes) and indicate, since u.v.-inactivated strains are pyrogenic, that this may be due to differences between strains in the nature or amount of certain virion components.

During surveillance of the swine population in Israel a haemagglutinating agent was isolated and identified as a paramyxovirus (PMV). Serological studies based on haemagglutination and neuraminidase inhibition (HI and NI) tests displayed a close antigenic relationship between the isolate and the prototype strain of a variety of the avian PMV serotype 3 (PMV-3). However, comprehensive HI and NI cross-reaction tests between the isolate and PMV-3 reference strains, on the one hand, and the other eight avian PMV serotypes, on the other hand, showed that the viruses compared differed in terms of their antigenic interrelationships with the other avian PMVs and also in the quantitative values of the cross-reactivity. This is interpreted to mean that the swine isolate and the avian PMV-3 prototype strain are different viruses, although very closely related, but are not different isolations of the same virus from avian and mammalian hosts.

BHK-21 cells readily produce tumours in athymic nude mice, but BHK-21 cells persistently infected with wild-type vesicular stomatitis virus (VSV) do not. However, rare persistently infected virus-shedding tumours (VSV-P tumour cells) were independently derived by in vivo selection on three different occasions. Cloned viruses isolated from each of these (VSV-P virus mutants) carried mutations determining the VSV-P phenotype because they all allowed growth of virus-shedding tumours in nude mice when they were used to persistently infect normal (unselected) BHK-21 cells. Treatment of nude mice with anti-asialo-GM1 allowed BHK cells persistently infected with wild-type VSV to form tumours, and BHK cells persistently infected with VSV-P were resistant to natural killer (NK) cells in vitro; this implicates NK cells in the in vivo rejection of persistently infected tumours and in the selection of the VSV-P variant. In this paper, we have sequenced the glycoprotein (G protein), matrix (M) and non-structural (NS) proteins of three independently derived VSV-P type mutants to find mutations associated with in vivo passage of persistently infected nude mouse tumours and with resistance to NK cells. We found extensive mutation in the G protein of VSV-P but relatively few mutations in the M and NS proteins. This suggests but does not prove a role for the G protein in NK cell killing of infected cells.

Amino acid sequences coded for by the preS region of the hepatitis B virus (HBV) envelope gene are present both in HBV and in subviral hepatitis B surface antigen (HBsAg) particles. Consequently, anti-preS-specific antibodies are elicited during the course of HBV infection. Such antibodies are virus-neutralizing. Therefore, it is important to determine whether or not vaccination with HBsAg also induces an anti-preS-specific immune response. We describe here an enzyme-linked immunosorbent assay applicable for the screening of sera from vaccinated individuals for anti-preS antibodies. IgG from serum specimens was adsorbed to staphylococcal Protein A on a superparamagnetic support and subsequently mixed with a synthetic peptide analogue [preS(120–145)] covalently linked to β-lactamase. The presence of anti-preS in serum specimens resulted in binding of the conjugated β-lactamase to the magnetic support. The adsorbed enzyme was quantified colorimetrically.

A single intraperitoneal injection of 250 µg dextran sulphate 500 (DS500) reduced the susceptibility of mice to scrapie given by the same route. A lower dose (25 µg) was less effective but still produced significant incubation period lengthening, while a high dose (2.5 mg) further increased the degree of prolongation. This reduced susceptibility occurred with DS500 administered up to at least 4 weeks prior to intraperitoneal scrapie inoculation and up to at least 2 weeks after scrapie inoculation. A reduced average effect, but more variable between mice, was obtained with DS500 given 1 month or 2 months after scrapie. The effective scrapie titre was reduced by 90% when DS500 was injected either 72 h before or 7 h after ME7 scrapie. Using a relatively lower but normally still fatal dose of the 22A strain of scrapie approximately 50% of the treated mice survived. The effective 90% loss of titre was consistent with either of these strains of scrapie in 11 different inbred strains of mice (BALB/c, BSC, BRVR, C3H, C57BL, IM, LM, MM, RIII, VL and VM). No significant increase in the prolongation effect was obtained using multiple DS500 doses in two different time combinations. DS500 causes long-term interference in both the early processing and the replication of scrapie agent, unlike those immunomodulators which increase susceptibility.

Complementary DNA clones to 90% of the Newcastle disease virus (NDV) genome have been produced and mapped. These clones cover the entire HN, F and M genes, most if not all of the L gene and parts of the NP and P genes. The map of overlapping clones gives the gene order 3′-NP-P-M-F-HN-L-5′ for NDV, identical to the gene order of Sendai virus, on the assumption that the NP gene of NDV is at the 3′ end of the genome as previously suggested by inactivation of NDV transcription by u.v. light. The nucleotide sequence of 453 bases covering the junction between the HN and L genes has been determined. There is nucleotide sequence homology to the consensus polyadenylation and mRNA start sites of Sendai virus and vesicular stomatitis virus. The deduced amino acid sequence of the C terminus of the HN protein of NDV shows homology to the C-terminal amino acid sequences of the HN proteins of simian virus 5 and Sendai virus. An explanation for the presence of HN0, the precursor to HN in some strains of NDV, is suggested by the presence of a long non-coding region at the 3′ terminus of the mRNA encoding the HN protein of NDV that could, by mutation, allow synthesis of a larger polypeptide.

A group of dogs with naturally occurring canine distemper developed prodromal systemic symptoms followed by neurological disorders. The post-infection courses of these diseases lasted approximately 2 months. A varying degree of demyelination and inclusion body formation was found mostly in the cerebella of virologically confirmed cases with little or no inflammatory response. The distribution of canine distemper virus antigen coincided with the histopathological lesions. The animals had moderate to high neutralizing titres to the virus in their sera and a low level of interferon-like activity in their cerebrospinal fluids. Isolation of viruses was most successful by the cocultivation method for brain specimens, but was possible by the direct method using lung homogenates. In infected Vero cells, the isolates derived from brain caused the formation of distinct plaques consisting of multinucleate giant cells, but the isolates from lung included a cytopathic effect mainly consisting of cell rounding which eventually spread throughout the culture. The former infection produced less extracellular virus than the latter. The synthesis of the viral surface proteins H and F, and of M, was markedly reduced compared with that of the internal viral proteins such as NP, P and L. The SDS-PAGE migration pattern of the P protein varied from case to case, but was similar when isolates from different tissues of the same case were compared. In the affected tissues, the amount of viral polypeptides decreased markedly relative to that of the NP and there was also an absolute decrease compared to their abundance in Vero cells. This decrease was more obvious in the brain than in the lung. The relevance of these results is discussed.

At least four distinct epitopes are described on the respiratory syncytial virus fusion protein (VP70) using 13 monoclonal antibodies in solid-phase competitive binding studies. Two, and possibly three, fusion-inhibiting epitopes, one non-fusion-inhibiting neutralizing epitope, and one non-neutralizing epitope are described. All but the latter site demonstrated partial overlap, suggesting possible topographical proximity of these epitopes. Polyclonal rabbit sera to VP70, which neutralized virus but did not inhibit fusion of infected cells, blocked the binding of all fusion-inhibiting monoclonal antibodies to VP70 in the solid-phase assay but did not inhibit their effect in vitro. Western blot analysis of these monoclonal antibodies demonstrates that one fusion-inhibiting epitope is localized on the 48K fragment of VP70 and is resistant to denaturation by heat, 2-mercaptoethanol and SDS.

An analysis of the plasma membrane proteins of human embryonic fibroblasts (HEF) infected with human cytomegalovirus strain AD169 (HCMV) was performed using in vitro radioactive labelling techniques followed by PAGE. Of the 12 virus-induced proteins detected in infected cells, glycoproteins of mol. wt. 34000 (34K), 53K to 55K, 60K to 63K, 70K to 72K, 98K to 103K and 145K to 150K and proteins of 130K to 133K and 260K to 270K were considered significant. The 60K to 63K, 70K to 72K and 130K to 133K components were detectable at early stages of infection (8 h), although only the latter two were labelled by surface iodination. The others only appeared in the membrane from 48 h to 80 h after infection. Serological studies indicated that the 34K, 70K to 72K, 98K to 103K and 145K to 150K components may be HCMV-specified virion constituents, as these glycoproteins reacted with antibodies raised against virions and extracted envelope glycoproteins. Of immunological importance was the exposure on the cell surface of the protein moieties of 70K to 72K and 130K to 133K proteins at 8 h and 53K to 55K, 60K to 63K, 70K to 72K and 145K to 150K components at 80 to 90 h after infection. Pooled human immune sera contained antibodies which reacted with these exposed proteins, as well as with three other virus-induced membrane components of 230K to 240K, 98K to 103K and 78K to 80K.

Virus particles were isolated from hypertrophied salivary glands of the tsetse fly, Glossina pallidipes collected near Mombasa, Kenya. Purified virus particles were rodshaped, 57 nm wide by 700 to 1300 nm long. Particle lengths fell into two size classes, with ‘short’ particles averaging 869 nm and ‘long’ particles 1175 nm. The virus particles morphologically resembled elongated baculovirus nucleocapsids although, unlike baculoviruses, no fully enveloped virions were found in purified preparations. The particles contained double-stranded DNA which appeared to be linear when analysed by electrophoresis in agarose gels, ethidium bromide-caesium chloride gradient centrifugation or electron microscopy (EM). There was some evidence for the DNA being heterogeneous in size from EM studies and from the observation that restriction enzyme analysis failed to provide a clear profile of DNA fragments. Protein from purified virions contained at least 12 polypeptides with a major component of 39000 mol. wt. These results suggest that the virus cannot be placed in any of the existing taxonomic groupings of DNA viruses.

A virus closely related to duck hepatitis B virus (DHBV) was isolated from serum and liver samples of wild migratory ducks (mallards) caught in two separate wildlife reserve parks in France. In the first one (Dombes region) 12% of wild mallards were positive for DHBV, and in the second (River Somme) 3% of mallards were found positive. The DHBV isolated from the serum of wild mallards was also associated with an endogenous DNA polymerase activity capable in vitro of completing a partially double-stranded viral DNA into a fully double-stranded DNA of 3 kb. The various replicative DNA forms reported for DHBV were also detected in the liver of wild viraemic mallards. The DNA restriction enzyme pattern of the wild mallard strain differed from that of American and French strains of DHBV. The wild mallard strain DHBV was experimentally transmitted to mallard and Pekin ducklings and induced a chronic viraemia in both varieties of infected birds. This strain might be the common ancestor of all DHBV strains isolated from domestic ducks world-wide. The discovery of a DHBV-related virus in the natural wild population might be an important clue in the study of the different roles of environmental, host and viral factors in the pathogenesis of DHBV infection, and their possible oncogenic action in ducks.

Colostrum-deprived lambs experimentally infected with an atypical ovine rotavirus isolated from naturally scouring animals, and a naturally infected colostrum-deprived lamb, were examined by immunofluorescence and immunoperoxidase labelling, and electron microscopy. A hyperimmune serum to the virus was produced in a gnotobiotic lamb and used to demonstrate antigen in the villous epithelial cells of the small intestine from infected animals. Scanning and transmission electron microscopy of tissues from infected animals revealed giant multinucleate syncytia composed of fused epithelial cells. Infected cells contained intracytoplasmic virus particles which resembled group A rotaviruses in morphology and morphogenesis. Small numbers of infected cells in MA104 cultures inoculated with ovine atypical rotavirus could be detected by immunofluorescence but virus growth could not be maintained by passage. Virus particles were seen by thin-section electron microscopy but their morphology and morphogenesis were abnormal; they were each composed of a single electron-dense shell, with no core, and were associated with envelopes of smooth membrane rather than rough endoplasmic reticulum.

Oral inoculation of newborn mice with the MET strain of human rotavirus produced transient diarrhoeal disease. Light and scanning electron microscopy showed typical rotavirus-induced morphological lesions in the villous epithelium of the small intestine consisting of extensive cytoplasmic vacuolation, villous necrosis and atrophy. Virus recovered from intestinal suspensions of infected mice showed the typical electrophoretic profile of the genome of the inoculated strain. Rotavirus antibody appeared in infected mice 10 to 20 days after inoculation but not in controls or nursing dams. The availability of a small animal model for experimental infection with human rotaviruses should prove useful for virulence and protection studies.

The presence of human papillomavirus type 16 (HPV16) DNA in 34 genital tract tumours of Italian female patients was investigated by Southern blot hybridization in high stringency conditions. HPV16 DNA was detected in 16 neoplasias, including cervical invasive and intraepithelial lesions as well as vulvar intraepithelial neoplasias and, to a lesser extent, vulvar invasive carcinomas. Appropriate control tissues included in the study were negative. The data suggest that integration of viral DNA had occurred in most tumours, both in invasive and in intraepithelial lesions. HPV16 variants or defective genomes, lacking the BamHI restriction site, were detected in three tumours.

The capacity of monoclonal antibodies to protect mice passively against yellow fever (YF) virus infection was investigated. Both neutralizing (54K-specific) and non-neutralizing (54K- and 48K-specific) antibodies protected mice against challenge with the RMP substrain of YF virus. Average survival times of mice inoculated intracerebrally with a standard lethal dose of YF virus differed according to the strain used: thus mice inoculated with the most neurovirulent viruses, FNV and Asibi, survived for 6.50 and 7.65 days respectively, and those with RMP virus survived for 15.75 days. The capacity of antibodies to protect mice passively against virus challenge was directly related to virus neurovirulence. Possible mechanisms and the significance of protection by antibodies against non-structural proteins that do not mediate neutralization, are discussed.

In particles of turnip yellow mosaic virus (TYMV), the interactions between proteins are particularly strong when compared to those between proteins in some other icosahedral viruses. Intact RNA is released from TYMV particles on freezing and this process has been studied by examining several parameters that influence cryodenaturation such as dehydration, pressure, aggregation and the presence of protective agents (glycerol and ammonium sulphate). Pressure of 1.5 × 108 Pa had no effect on virus particles whereas dehydration of a virus suspension had a drastic effect. Aggregate formation resulting from freezing of solutions containing high virus concentrations seems to be a prerequisite for RNA release; cryoprotective agents hindered RNA release.