- Volume 69, Issue 12, 1988

Macrophages (Mφ) harvested from the peritoneal cavities of mice after thioglycollate stimulation could be infected with murine cytomegalovirus (MCMV), although the efficiency of infection was low. Sequential measurements of interferon (IFN) production by virus-infected Mφ were performed in an attempt to explain the characteristics of MCMV infection in the cell cultures. Infected Mφ produced moderate amounts of IFN, which was completely neutralized by anti-IFN-α/β serum. The IFN was detectable in cultures as early as 8 h after infection and was produced only by exposing Mφ to infectious virus. Production increased until 48 to 72 h and preceded virus production, which was initially detected 72 h after infection. Treatment of the Mφ cultures with anti-IFN-α/β resulted not only in a marked increase in virus production, as well as a shortening of the long eclipse period of MCMV infection, but also induced increases in the number of Mφ releasing MCMV (VR-Mφ). Thus, the IFN produced in MCMV-infected Mφ (MCMV-Mφ IFN) appeared to suppress the production and spread of MCMV. The increase in the number of VR-Mφ observed was more resistant to anti-IFN-α/β treatment than the production of infectious virus. The antiviral effect of MCMV-Mφ IFN on MCMV infection in mouse embryo fibroblasts was similar to that induced by IFN-α/β. Therefore, MCMV-Mφ IFN appeared to be more active in protecting against the spread of cell-free MCMV than of cell-associated virus. These differences in sensitivity to IFN action suggest that Mφ may have a role in the latency of MCMV and that their production of IFN may facilitate the generation of latent infection.

Bovine interferon-α11 (IFN-α11) and porcine interferon-γ (IFN-γ) inhibited African swine fever virus replication in both porcine monocytes and alveolar macrophages. The most potent antiviral activity was observed with IFN-γ-treated alveolar macrophages. The production of both a virulent (CC83) and a non-virulent (BA71) isolate of the virus was inhibited. Bovine tumour necrosis factor α did not show antiviral activity in either monocytes or alveolar macrophages. Rather, an increase of African swine fever virus production in tumour necrosis factor α-treated monocytes was found. An analysis of viral protein synthesis in IFN-α1l- and IFN-γ-treated alveolar macrophages showed an inhibition of synthesis of some viral proteins. The inhibition of late proteins was very pronounced in IFN-γ-treated cells, and it was probably a consequence of the inhibition of African swine fever virus DNA polymerase activity.

The genomes of African swine fever virus isolates collected from soft ticks (Ornithodoros moubata) inhabiting warthog burrows in four areas of Zambia were compared by restriction enzyme site mapping. Isolates from different areas showed considerable diversity. The regions of genomes that differed between isolates were distributed throughout the virus genome, although some more conserved regions were identified, such as the right-hand third of the genome. The genomes of seven isolates from neighbouring warthog burrows within Livingstone Game Park in southern Zambia were more similar to each other than those from different areas. However, a number of differences were observed even between the genomes of isolates from the same warthog burrow. The variation between these latter isolates probably resulted from point mutations located at various positions along the genome, in addition to small additions or deletions at both terminal regions. Restriction enzyme site mapping indicated that one isolate may have originated by earlier recombination between two distinguishable viruses.

The complete nucleotide sequence of a cloned copy of the HindIII K fragment of the WR strain of vaccinia virus has been determined. Eight open reading frames (ORFs) have been identified, on the basis of size and codon usage. The predicted amino acid sequences of the putative genes have been compared to the Protein Identification Resource and to published vaccinia virus sequences. One gene, predicted to encode a 42.2K protein, is highly related to the family of serine protease inhibitors. It shows approximately 25% identity to human antithrombin III and 19% identity to the cowpox virus 38K protein gene which is also related to serine protease inhibitors. The product of another gene shows a similar high level of identity to the 37K vaccinia virus major envelope antigen. The existence of viable deletion mutants and recombinants containing foreign DNA inserted into both these genes indicates that they are non-essential.

cDNA molecules encoding the structural proteins of the virulent Trinidad donkey and the TC-83 vaccine strains of Venezuelan equine encephalitis (VEE) virus were inserted under control of the vaccinia virus 7.5K promoter into the thymidine kinase gene of vaccinia virus. Synthesis of the capsid protein and glycoproteins E2 and E1 of VEE virus was demonstrated by immunoblotting of lysates of CV-1 cells infected with recombinant vaccinia/VEE viruses. VEE glycoproteins were detected in recombinant virus-infected cells by fluorescent antibody (FA) analysis performed with a panel of VEE-specific monoclonal antibodies. Seven E2-specific epitopes and two of four E1-specific epitopes were demonstrated by FA.

Protection of mice from fatal neuroadapted Sindbis virus encephalitis can be accomplished by passive transfer of monoclonal antibodies (MAbs) to either the E1 or E2 glycoprotein of Sindbis virus. Both neutralizing and non-neutralizing MAbs can be protective. To define further the characteristics of MAbs that provide protection from fatal disease, antigenic epitopes on the E1 and E2 glycoproteins were identified using a competitive binding enzyme immunoassay. Four distinct epitopes on E1 and three on E2 were defined. MAbs to all E1 epitopes, both neutralizing (three) and non-neutralizing (one) protected mice from fatal encephalitis. MAbs to the E2 neutralizing epitopes (two) protected mice from fatal encephalitis while those to the non-neutralizing epitope did not. The efficiency of protection from fatal Sindbis virus encephalitis of four neutralizing and non-neutralizing protective anti-E1 and anti-E2 MAbs representing different epitopes was compared. The neutralizing MAbs (against epitopes E2-ab, E2-c and E1-c) gave 50% protection at lower doses (2 to 20 µg) than the non-neutralizing MAb representing epitope E1-e (150 µg) when given before virus challenge. When given after virus challenge, MAbs to E2-ab and E2-c protected at lower doses (0.03 to 0.3 µg) than did either MAbs to E1-c (> 100 µg) or E1-e (10 µg). The MAbs to E1-e, E2-ab and E2-c were required in larger amounts to afford protection before than after challenge, while the opposite was true for MAb to E1-c.

Six interspecific hybridomas (heterohybridomas) secreting bovine monoclonal antibodies (MAbs) against respiratory syncytial (RS) virus were produced. Four of the heterohybridomas were formed using the mouse myeloma cell line NS1 as the fusion partner, one using NS0, and the remaining heterohybridoma was formed using a bovine × murine hybridoma as the fusion partner. Five heterohybridomas secreted bovine IgG1 and one secreted IgG2. All six MAbs recognized human subtype A and B viruses as well as bovine RS virus. They were specific for the fusion glycoprotein and reacted with a 140K dimer and a 70K monomer in a Western blot of native antigen; three also bound to the 46K F1 component and its 22K cleavage product in a blot of reduced antigen. Two of these MAbs neutralized RS virus infectivity, inhibited virus-induced fusion, lysed RS virus-infected cells in the presence of complement and protected mice against RS virus challenge.

A cDNA library was synthesized using RNA from bovine papillomavirus type 4 (BPV-4)-induced papillomas. The major viral transcript was characterized by sequencing of its cDNA, primer extension mapping and S1 nuclease protection studies. The transcript initiates at multiple sites between nucleotide (nt) 777 and nt 902, contains a splice junction between nt 1016 and nt 3376 and terminates at nt 4034, using the early polyadenylation signal at nt 4009. Sequencing of viral cDNAs and mRNA revealed deviations from the reported genomic sequence between nt 3412 and nt 3460. These resulted in a temporary frameshift, abolished the translation termination codon of the E5a open reading frame (ORF), and introduced a new termination codon in the E4 ORF. The new uninterrupted E5 ORF was found to have greater homology to the E4 ORFs of other papillomaviruses than to their E5 ORFs. The E5 ORF of BPV-4 is joined to a five codon ORF in the leader exon of the major transcript, in the same way as the E4 ORF in the major transcripts of BPV-1 and human papillomavirus type 11. On this basis, it has been redesignated E4, and the viral genomic sequence revised. Two novel transcriptional promoters of BPV-4 were defined: a putative controller of the multiple major RNA start sites around nt 870 and a minor TATA box at nt 691. In addition, minor early region transcripts were mapped which initiate between nt 3071 and nt 3152. None of these transcripts utilizes the splice site at nt 3376. These messages may express E2-encoded functions.

Cottontail rabbit papillomavirus (CRPV) genomic sequences coding for virus early functions were introduced into a retroviral vector in order to produce cDNAs of the viral early region. Two constructs differing in the length of control sequences preceding the E6 open reading frame were transfected into Psi-2 cells and the released retroviral stock was used to infect NIH3T3 cells. The proviral sequences were rescued from antibiotic G418-resistant virus-infected cells after fusion with Cos cells, amplified as plasmids in Escherichia coli and analysed. Nucleotide sequencing showed that the splicing signals used in the construct containing only the early coding region are the same as in CRPV-expressing tumours, linking the beginning of E1 to the middle of E2. On the other hand, in a construct including most of the long control region a splice donor site located in the 5′ end of this region, at position 7810, was very efficiently used, totally excluding the use of the donor site at position 1371. None of the constructs containing CRPV sequences transcribed from Moloney murine leukaemia virus promoter was able to transform mouse fibroblasts after DNA transfection.

In situ hybridization with synthetic oligonucleotides was used to fine map the extent and continuity of herpes simplex virus type 1 (HSV-1) latency-related RNA (LR RNA) in trigeminal ganglia of latently infected humans. A series of 34 20 residue oligonucleotides were employed to cover a 3 kb region of the HSV-1 genome to which the latency-related gene had previously been mapped. The 5′ end of the main LR RNA transcript began approximately 1210 nucleotides downstream from the 3′ end of the mRNA from the immediate early gene ICP0. The 3′ end of the LR RNA overlapped the 3′ end of the ICP0 mRNA by approximately 1000 nucleotides. A potential small intron was detected near the 3′ end of the LR RNA.

Reports on the arrest of herpes simplex virus type 1 (HSV-1) replication by interferon (IFN) are inconsistent. By the use of immunofluorescence and immunoblot assays with monoclonal and polyclonal antibodies, effective arrest of viral translation by human IFN-α in human fibroblasts was detected for the HSV-1 strains KOS and McIntyre. In HeLa cells which are less sensitive to IFN inhibition and in 444 cells, a HeLa-fibroblast hybrid cell line, the inhibition was less pronounced. These results confirm earlier observations that IFN or polyinosinic-polycytidylic acid block the replication of HSV-1 in human, monkey and mouse cells no later than the immediate early phase of infection.

Tumour necrosis factor (TNF) induces antiviral activity in HEp-2 cells. Virus yield reduction assays with vesicular stomatitis virus as challenging virus demonstrated that the antiviral state was more pronounced in confluent cultures under low serum conditions. A significant enhancement of the antiviral state was obtained by combining TNF with low concentrations of either interferon (IFN)-β1 or IFN-γ. The reduction in virus yield was significantly higher than that expected from summation of the independent antiviral activities of either substance alone, i.e. TNF and IFN acted synergistically as antiviral agents. Synergism of TNF with IFN-β or IFN-γ appeared to be mediated by different pathways, since different requirements for pretreatment and different effects on oligo-2′,5′-adenylate synthetase (2-5AS) induction were observed. Induction of 2–5AS by TNF could be shown to be an indirect event that was sensitive to an antiserum against natural IFN-β1.

Flaviviruses encode seven non-structural proteins for which functions have not yet been described. The identification of the viral and possible host proteins which may be involved in flavivirus replication has been impeded by the fact that the viral replication complexes are tightly associated with endoplasmic reticular membranes within infected cells and that in vitro polymerase activity is associated with large membrane fragments. To facilitate further study of flavivirus replication complexes, selected ultrapure detergents were analysed for their effect on West Nile virus (WNV) in vitro RNA-dependent RNA polymerase activity and for their ability to release functional replication complexes from partially purified intracellular BHK-21 membrane fragments. A few previous reports indicated that flavivirus in vitro polymerase activity was sensitive to detergent treatment. The present study indicates that WNV polymerase activity is variably inhibited depending on the concentration and identity of the detergent used. Of the five detergents (Tween 20, maltoside, octylglucoside, lubrol PX and sodium deoxycholate) tested, sodium deoxycholate was the most efficient at releasing functional viral replication complexes from intracellular membranes.

A hantavirus, designated Leakey virus, was isolated from a Mus musculus captured in Real County, Texas, U.S.A. in August 1986. Virus-specific fluorescence was first detected 13 days after inoculation of Vero-E6 cells with spleen tissue from the seropositive M. musculus. Ultrastructurally, the new isolate resembled other hantaviruses. Leakey virus induced a fatal meningoencephalitis in infant Fischer rats, with viral antigen detectable in brain, lung, liver, kidney and spleen. Serum dilution, plaque reduction neutralization tests indicated that Leakey virus was antigenically distinct from Hantaan, Seoul, Puumala and Prospect Hill viruses, and therefore constitutes a new serotype.

Recovery of highly purified citrus cachexia viroid (CCaV) was accomplished by serial elution following CF-11 cellulose chromatography of a 2 m-LiCl-soluble nucleic acid preparation. The alternative herbaceous host, cucumber (Cucumis sativus cv. Suyo), yielded greater quantities of the viroid than the highest yielding citrus host, citron (Citrus medica cv. Etrog). A randomly primed cDNA probe to CCaV purified from cucumber reacted positively to extracts from citron and cucumber inoculated with the same isolate of CCaV. When tested against a broad range of other citrus viroids, the CCaV cDNA hybridized to only one, CV-IIa, which has been identified as the causal agent of a mild form of the citrus exocortis disease. Because of the apparent homology between the nucleotide sequences of CV-IIa and CCaV, and a size difference of only five to ten nucleotides, these RNAs can be considered as members of a common subgroup of citrus viroids. These two viroids have been classified by bioassay reactions as the causal agents of two distinct types of citrus disease, an ‘exocortis-like’ syndrome and cachexia. The properties of and relationships between these two members of the citrus viroid II group and the definition of the exocortis and cachexia (xyloporosis) diseases are presented.

Nucleic acid extracts from citrons (Citrus medica cv. Etrog) displaying mild and moderate symptoms associated with the exocortis disease were analysed by sequential and denaturing PAGE which revealed the presence of several viroids. A comparison was made of electrophoretic patterns displaying one or more distinct citrus viroids from field isolates of citrus with exocortis. Citrus viroids were characterized by the physical parameters of electrophoretic mobility, chromatography on CF-11 cellulose and hybridization to cDNA probes of the well characterized citrus viroids, citrus exocortis viroid, CV-Ib from the ‘citron variable viroid’ isolate, and citrus cachexia viroid. These characteristic properties combined with biological distinctions in the host range and symptom expression suggested a scheme for the organization of the citrus viroids into five major groups. The association of the symptoms induced by these citrus viroids in citron cv. Etrog, their organization into individual viroid groups and their presumed relationship to the exocortis disease of citrus are discussed.

Antisera specific for the non-structural proteins 1a and 2a of brome mosaic virus (BMV) were prepared by the gene fusion method. Plasmids were constructed which expressed parts of 1a and 2a in Escherichia coli as fusion proteins with Protein A. After induction of fusion protein synthesis in E. coli, the fusion proteins accumulated in insoluble fractions. Antisera raised against these proteins (anti-1a and anti-2a sera) reacted specifically with the respective proteins translated in vitro and in vivo. These antisera were used to investigate the involvement of the 1a and 2a proteins in the BMV replicase preparation which initiated complementary strand synthesis de novo on BMV RNA. Immunoblot analysis using these antisera revealed the presence of 1a and 2a in the BMV replicase fraction. The replicase activity was inhibited by the addition of anti-1a serum, but not anti-2a serum. Further addition of Protein A-Sepharose to remove each immunocomplex gave similar results. These results suggested the involvement of at least the 1a protein in our BMV replicase preparation.