- Volume 71, Issue 5, 1990

Tick-borne virus transmission was examined using guinea-pigs and hamsters previously infested with ticks. Guinea-pigs developed immunity to Rhipicephalus appendiculatus after a single exposure to the ticks. Nymphal and adult stages that fed on resistant guinea-pigs had increased mortality during feeding, and reduced engorged weights. Egg production from female ticks fed on resistant hosts fell by at least 50%. Guinea-pigs maintained high levels of immunity to tick infestation for at least 210 days after the initial exposure. In contrast, hamsters did not develop resistance to ticks even after three or four infestations. R. appendiculatus adults infected with Thogoto (THO) virus (donors) were allowed to co-feed with uninfected nymphs (recipients) on either resistant or naive guinea-pigs. The number of recipient ticks that acquired virus was significantly reduced on resistant guinea-pigs. In contrast, feeding on pre-infested hamsters did not affect tick-borne transmission of THO virus. Host resistance to tick infestation, if prevalent in nature, may severely limit the spread of tick-borne viruses. Such an effect could result directly from a reduction in the number of ticks that acquire virus, or indirectly from poor egg production (in the case of viruses maintained in ticks by vertical transmission) and reduced survival of ticks fed on resistant hosts.

Aedes triseriatus mosquitoes were orally infected with two different California serogroup bunyaviruses (La Crosse and snowshoe hare viruses) and high frequency reassortment occurred in these mosquitoes. Increased viral replication and subsequent gene segment reassortment was noted in the ovaries of mosquitoes that had ingested multiple blood-meals. To determine whether newly generated reassortant viruses could be transmitted transovarially to progeny mosquitoes, adult female mosquitoes were inoculated with the two temperature-sensitive (ts) parental viruses, and allowed to blood-feed and oviposit. Of 58 infected progeny mosquitoes assayed, six (10 %) contained non-ts viruses, and three of these transmitted non-ts viruses to a susceptible host. Selected viruses of the non-ts phenotype, which were isolated from mosquitoes and from mice fed upon by the mosquitoes, were demonstrated to be reassortant viruses by oligonucleotide fingerprinting.

Heterogeneity in polydnavirus DNA was exploited as a means of following the transmission of viral genomes in parasitoid populations. Parasitoid lines isogenic for viral DNA markers were established from both a braconid (Cotesia melanoscela) and an ichneumonid (Hyposoter fugitivus) species. In crossing experiments these markers routinely segregated in Mendelian (chromosomal) fashion, suggesting that the structure of polydnavirus genomes is probably determined by the integrated form of viral DNA, rather than by extrachromosomal molecules.

Recombinant plasmids for intracellular synthesis of mature porcine interferon alpha 1 (IFN-α1) in Escherichia coli were constructed. High amounts of antiviral activity were obtained [up to 4 × 105 international units (IU) per ml of bacterial culture]. Recombinant porcine IFN-αl (rIFN-αl) was purified to homogeneity by monoclonal antibody immunoaffinity and was found to have the expected M r (17·5K) and N-terminal sequence (except for the apparent lack of an N-terminal methionine). Its specific antiviral activity was 5 × 107 to 10×l07 lU/mg MDBK cells. In vitro biological properties of this purified rIFN-α1 were compared to those of virus-induced porcine leukocyte interferon: the two interferons shared similar antigenic determinants and had the same ability to induce a cytocidal effect on primary cultures of pig kidney epithelial cells. However, rIFN-α1 was at least six times more active in inducing an antiviral state on homologous porcine cells. These properties are discussed in the light of a possible in vivo use of the purified recombinant molecule.

The gene encoding the spike glycoprotein of the human coronavirus HCV 229E has been cloned and sequenced. This analysis predicts an S polypeptide of 1173 amino acids with an Mr of 128600. The polypeptide has 30 potential N -glycosylation sites. A number of structural features typical of coronavirus S proteins can be recognized, including a signal sequence, a membrane anchor, heptad repeat structures and a carboxy-terminal cysteine cluster. A detailed, computer-aided comparison with the S proteins of infectious bronchitis virus, feline infectious peritonitis virus, transmissible gastroenteritis virus and murine hepatitis virus, strain JHM is presented. We have also done a Northern blot analysis of viral RNAs in HCV 229E- infected cells using synthetic oligonucleotides. On the basis of this analysis, and by analogy to the replication strategy of other coronaviruses, we are able to propose a model for the organization and expression of the HCV 229E genome.

An ELISA was developed for the determination of antibodies to rabbit haemorrhagic disease virus (RHDV) in whole blood and blood serum of rabbits. Naturally acquired antibodies were detected in 19·4% of blood samples collected from 1461 rabbits in 43 farms apparently free of the disease, 19·7% samples were doubtful and 60·9% of the rabbits were free of antibodies to RHDV. Their presence has a considerable effect on the resistance of rabbits to infection with RHDV. Antibodies were also found in rabbit blood serum samples collected up to 12 years before the first outbreaks of RHD were reported. Up to 14 viral protein antigens were determined by PAGE and Western blot analysis, of which three with M r values of 6IK, 38K and 52K were major proteins, the 61K being dominant. Our hyperimmune sera, a Chinese reference serum and sera with positive antibody titres, including those collected several years before the first outbreaks of RHD, reacted identically with these antigens in the Western blot analysis. The data obtained suggest that naturally acquired antibodies are a product of a specific response to prior infection with an avirulent strain of the virus.

Disoxaril (5-[7-[4(4,5-dihydro-2-oxazolyl)phenoxy]- heptyl]-3-methylisoxazole) protects poliovirus procapsids against alkaline dissociation and thermal denatur- ation up to 42 °C. When added during the purification of procapsids, it enhances their yield and antigenic quality. Disoxaril increases the immunogenicity of both purified virions and procapsids in mice. Whereas untreated procapsids mainly elicit H-specific antibodies, disoxaril-treated procapsids yield high titres of neutralizing antibodies. The prospect of using disoxaril-treated procapsids in vaccines is discussed.

The rotavirus genome consists of 11 segments of dsRNA that are replicated asymmetrically with plus strand RNA serving as the template for minus strand RNA synthesis. In this study, we have used nondenaturing gel electrophoresis to examine subviral particles that synthesize dsRNA (replicase particles), for possible changes in structure during RNA replication. Analysis of SVPs purified from simian rotavirus SA11-infected MA104 cells and resolved on 0·6% agarose gels containing 50 mm-Tris-glycine pH 8·8 showed that the overall size of particles able to synthesize dsRNA in a cell-free system was 100 nm or more. Electrophoretic analysis of the size of replicase particles as a function of length of incubation in the cell-free system demonstrated that replicase particles decreased in size with increasing length of incubation. However, after 60 to 90 min of incubation, replicase particles no longer changed in size but were similar in size to the rotavirus single-shelled (75 nm), core (60 nm) and precore (45 nm) replicative intermediates which have been described previously. As the size of replicase particles decreased with increasing length of incubation, the number of newly made genome-length dsRNAs in the particles increased. Analysis of the RNA products detected in replicase particles showed that RNA replication is regulated such that the synthesis of full-length dsRNAs in the replicase particle proceeds from the smallest to the largest genome segments. Treatment of replicase particles with single-strand-specific RNase reduced their size to that of replicative intermediates and interfered with their ability to synthesize dsRNA, thus indicating that the plus strand RNA template for replication extends from the replicase particle. This study showed that replicase particles undergo a continuous change in size during RNA replication due apparently to plus strand RNA templates moving into the replicase particle during the synthesis of dsRNA.

Canine parvovirus (CPV)-specific T cell clones were generated by culturing lymph node cells from CPV- immunized BALB/c mice at limiting dilutions in the presence of CPV antigen and interleukin-2 (IL-2). All isolated T cell clones exhibited the cell surface phenotype Thyl+, CD4+, CD8− and proliferated specifically in response to CPV antigen. After stimulation with CPV antigen in culture the T cell clones produced IL-2 and proliferated in the absence of exogenous IL-2. Naive mice to which CPV-specific T cell clones had been adoptively transferred developed a CPV-specific delayed type hypersensitivity reaction upon simultaneous intracutaneous injection of CPV in their ears. The ability of recombinant viral fusion proteins, representing the VP2 capsid protein of the antigenically closely related feline panleukopenia virus and of synthetic peptides derived from the amino acid sequence of the VP2 of CPV, to stimulate these T cell clones enabled the identification of T cell epitopes.

The polymerase chain reaction (PCR) technique was employed in searching for human T cell lymphotropic virus type I (HTLV-I) gag, env and pol sequences in samples of DNA prepared from two HTLV-I seropositive patients with tropical spastic paraparesis (TSP), the Swedish multiple sclerosis (MS) patients who recently have been reported to be PCR-positive for HTLV-I gag and env sequences, and eight healthy individuals. Precautions were taken in order to reduce the risk of cross-contamination in the PCR. In the two TSP patients strong signals were obtained with gag, env and pol amplification primers and detection probes. In MS patients and healthy individuals, no signals were obtained with gag and env. In occasional experiments, weak signals were seen for the pol segment for a single MS patient and/or healthy individuals, but these signals were not reproducible in subsequent experiments. Thus, the present data do not confirm the presence of HTLV-I sequences in MS patients.

We recently reported the enhanced immunogenicity of a peptide epitope when it was presented as a fusion protein with hepatitis B core antigen. In those experiments the fusion protein was expressed in vaccinia virus. We have now refined the system so that large amounts of highly immunogenic particles can be produced using a simple bacterial expression system. We describe the expression of three different viral epitopes as chimeric particles that induce good antibody responses to each epitope after one dose of low amounts of antigen. Finally we demonstrate that the immunogenicity is a reflection of both T helper cell sites within the core protein and also the particulate nature of the immunogens.

Transcript mapping of the equine herpesvirus 1 (EHV- 1) glycoprotein B (gB) gene homologue by Northern blot, SI nuclease and primer extension analyses indicated that two overlapping transcripts of 3·4 and 4·6 kb originated from the same strand and were transcribed from left to right between coordinates 0·40 and 0·43 of the EHV-1 genome. The 3·4 kb transcript encoded EHV-1 gB and the 5′ RNA terminus was located approximately 30 bases downstream from a probable TATA element. The coding region of the gB gene homologue was reconstructed from two subclones using oligonucleotide mutagenesis and inserted into vaccinia virus by homologous recombination. Cells infectedwiththerecombinantvirussynthesizedEH V -1 gB antigen, which was detectable in the cytoplasm and on the cell surface by immunofluorescence using an EHV-1 neutralizing horse serum and EHV-1 monoclonal antibodies. On Western blots, bands of 138K to 143K, 80K to 90K and 55K to 57K were identified in recombinant virus-infected cells, by both EHV-1 monoclonal antibodies and the polyclonal horse serum. These were similar in M r to bands identified by these sera in EHV-1-infected cells. Mice vaccinated with the recombinant virus produced antibodies which recognized proteins of the same M r as EHV-1 gB, on Western blots, but did not have in vitro neutralizing activity.

An infection was established in adult BALB/c mice by means of intranasal inoculation of the AB4 strain of equine herpesvirus type 1 (EHV-1). The acute infection was confined to the respiratory tract and blood. Virus was shown to replicate in the nasal mucosa, trachea and lung for several days producing clinical signs of disease. Viraemia was also detected and a small proportion of peripheral blood cells contained virus at the peak of the infection. Histological and electron microscopic evidence were obtained which proved that productive virus replication occurred in the ciliated epithelial cells lining the bronchi and in pneumocytes in the lung, resulting in the destruction of these cells. Both humoral and cell-mediated responses to the infection were detected and monitored. By means of immunoprophylaxis or chemotherapy it was possible to modify the course of the infection. This infection model has many striking features in common with that observed in the natural host and the observations suggest that the mouse is a convenient and relevant model in which to study both host responses to EHV-1 infection and modification of the pathogenesis by means of immunoprophylaxis or therapy.

Overlapping fragments of the gene encoding glycoprotein gl of pseudorabies virus (PRV; herpesvirus suis 1) were expressed in bacteria. Using the fusion proteins and a panel of monoclonal antibodies (MAbs) against gI as well as swine sera we found that the N-terminal part of gI (residues 33 to approximately 100) contains a highly antigenic and immunogenic domain. Transfer of antibodies binding to this region as well as vaccination with fusion proteins containing the N terminus of gI are able to confer protection to mice against a lethal challenge of virus. The results show that gI, which is non-essential for virus replication in tissue culture, can induce neutralizing and protective antibodies. The potential suitability of fusion proteins encompassing N-terminal parts of gI as diagnostic tools is demonstrated.

The large (L) protein subunit of unsegmented negative-strand RNA virus polymerases is thought to be responsible for the majority of enzymic activities involved in viral transcription and replication. In order to gain insight into this multifunctional role we compared the deduced amino acid sequences of five L proteins of rhabdoviruses (vesicular stomatitis virus and rabies virus) or paramyxoviruses (Sendai virus, Newcastle disease virus and measles virus). Statistical analysis showed that they share an atypical amino acid usage, outlining the uniqueness of the negative-strand virus life style. Similarity studies between L proteins traced evolutionary relationships in partial disagreement with the present taxonomic arrangement of this group of viruses. The five L proteins exhibit a high degree of homology along most of their length, with strongly invariant amino acids embedded in conserved blocks separated by variable regions, suggesting a structure of concatenated functional domains. The most highly conserved central block contains the probable active site for RNA synthesis. We tentatively identified some other functional sites, distributed around this central core, that would naturally work together to assure the polymerase activity. This provides detailed guidelines for the future study of L proteins by site-directed mutagenesis.

A cDNA library was constructed in λgt10 using mRNA purified from cells infected with parainfluenza virus type 2 (PIV2). Virus-specific clones were identified by screening the library with 32P-labelled cDNA probes made from randomly primed vRNA. Clones containing the haemagglutinin-neuraminidase (HN) gene were identified by sequence comparisons with known parainfluenza virus HN gene sequences. The largest HN clone isolated had a nucleic acid sequence of 2065 bp with a single long open reading frame encoding a protein of 571 amino acids. The HN protein has nine predicted glycosylation sites and an amino-terminal membrane-spanning region. The PIV2 HN protein shares 43 % amino acid identity with the HN protein of simian virus 5 and 40% with mumps virus, 30% of the amino acids being common to all three viruses.

A nucleoprotein (NP) preparation purified from the chorioallantoic membrane of chicken eggs infected with fowl plague virus (A/FPV/Rostock/34, H7N1) yielded, in addition to the commonly known 56K protein, a 42K component that could not be detected in virus particles. After testing with a series of NP-specific monoclonal antibodies it was found that some reacted with both proteins and others were bound only by the 56K protein. Among both types of NP-specific monoclonal antibodies only a limited number were bound to infected murine cells. Some antibodies bound to cells infected with a given subtype failed to react with the surface of cells infected with a different subtype. Binding was demonstrated by cellular ELISA, radioimmunoassay and immunofluorescence. The results indicate that only restricted antigenic domains of the native NP and perhaps NP fragments are exposed at the surface of infected murine cells. Additionally, the purified NP preparation was used to immunize mice in order to determine the protective capacity of cell- associated NP. In parallel, and as a relevant control, mice were immunized with a vaccinia virus recombinant containing the gene for NP prior to challenge with infectious virus. High levels of monospecific antibodies and a cytotoxic T cell activity was found in mice immunized with purified NP or infected with the vaccinia recombinant after secondary restimulation in vitro. After treatment with specific antibodies the cytotoxic cells were shown to be classical CD8+ cytotoxic T lymphocytes. Despite the elicitation of a humoral and a cellular immune response by the forms of NP employed mice were not protected from influenza virus infection.

A number of different influenza C virus strains were tested for their fusion properties using a resonance energy assay which allows direct monitoring of fusion between virus membranes and artificial lipid vesicles. The fusion pH of various strains was found to range between 5.6 and 6.1. Haemolytic activity of the different strains with chicken erythrocytes was observed at slightly lower pH values and varied between 5.1 and 5.7. Studies of the kinetics of influenza C virus fusion showed distinct characteristics in fusion activity. A lag before onset of fusion was found with influenza C virus which was not observed for influenza A or B viruses. In addition, studies on the rate of conformational change of the influenza C virus glycoprotein, as determined by morphological changes and endogenous tryptophan fluorescence, suggest that the conformational change is rate-limiting in the fusion process, whereas for influenza A viruses the glycoprotein conformational change is fast and a later step in the fusion process is rate-limiting. Monitoring the conformational change of influenza C virus glycoprotein by the onset of trypsin susceptibility showed, however, that membrane fusion occurred in some cases without onset of trypsin susceptibility, indicating that the trypsin-susceptible conformation is a post-fusogenic conformation.

Monoclonal antibodies (MAbs) to the haemagglutinin—neuraminidase (HN), fusion (F) and matrix (M) proteins of Newcastle disease virus were prepared and characterized. At least three non-overlapping or partially overlapping antigenic sites were delineated on the HN, three on the F and three on the M proteins by competitive binding assays. Antigenic sites on the HN and F proteins roughly represented functional domains defined by serological tests. Two antigenic sites on the F protein were involved in virus neutralizing and haemolysis-inhibiting activity. These antigenic determinants were readily affected by treatment with certain surfactants and acetone. Proteolytic cleavage of the HN and F proteins was associated with conformational change, revealed by altered reactivity with MAbs and by altered topological arrangements of some epitopes. None of the anti-M MAbs inhibited any biological activities of the virus.