- Volume 74, Issue 3, 1993

Inoculation of the cottontop tamarin with Epstein—Barr virus (EBV) gives rise to the development of mono- and/or oligoclonal large cell malignant lymphoma. A cDNA library was generated with the RNA extracted from an EBV-induced tamarin lymphoma biopsy in order to study the transcripts expressed in the tumour tissue. Fifteen EBV-specific cDNA clones were localized in the corresponding viral genomic fragments. Among them, two correspond to the EBNA-2 gene, and two others to the latent membrane protein gene. The majority of the cDNA clones were localized in the BamHI A fragment which has not been associated with latent expression. Furthermore, cDNAs were also found from the BamHI D and I fragments. Sequence analysis of the cDNAs localized in BamHI A showed that they correspond to a rightward transcript in the BALF-3 region, with the one clone that was sequenced containing four exons and three introns. The above results were confirmed by testing three different biopsies with the rapid amplification of cDNA ends-PCR method.

Nephropathia epidemica involves several organs including kidney, lung, liver and brain. To investigate the susceptibility of putative target cells to the agent responsible, Puumala virus, we screened established human cell lines of lung (WI-38, A-427, CCD-11Lu), kidney (A-704), liver (Hep G2), pharynx (Detroit 562), submaxillary gland (A-253) and neural (SK-N-MC, SH-SY5Y) origin as well as primary human kidney glomerular cells, endothelial cells and peripheral blood monocytes/macrophages. Propagation of the Sotkamo strain of Puumala virus was also tested in the primary kidney, spleen and lung cells of bank voles (the natural host of the virus). All of the primary cells and most of the established cell lines expressed viral protein, synthesized viral RNA and secreted infectious virus, except the neural SK-N-MC and SH-SY5Y cells. None of the tested cell types except the primary bank vole kidney cells could propagate the virus as efficiently as the Vero E6 cells. The observed host cell range is wide and consistent with a multiorgan involvement of Puumala virus. No cytopathic effects were seen in any of the infected cell cultures.

Enzootic strains of Venezuelan equine encephalitis (VEE) virus occur in the United States (Florida), Mexico, Central America and South America. Epizootic VEE first occurred in North and Central America in a widespread outbreak between 1969 and 1972. To investigate the likelihood that this epizootic VEE virus, identified as VEE antigenic subtype I-AB, evolved from enzootic viruses extant in the region, we cloned and sequenced the 26S mRNA region of the genomes of the Florida VEE subtype II virus, strain Everglades Fe3-7c, and the Middle American subtype I-E virus, strain Mena II. This region of the genome encodes the viral structural proteins. The sequences of the 26S mRNA regions of the Everglades and Mena virus genomes differed from that of the reference epizootic VEE subtype I-AB virus, Trinidad donkey strain, by 453 and 887 nucleotides and by 66 and 131 amino acids, respectively. These data confirm previous reports demonstrating significant antigenic and genetic distance between VEE I-AB virus and viruses of subtypes I-E and II. It is unlikely that the epizootic VEE I-AB virus responsible for the 1969 outbreak originated from mutation of enzootic VEE viruses in North or Middle America.

A combined qualitative and quantitative approach has been used to examine the role of virus inclusion bodies (VIBs) in the morphogenesis of bluetongue virus (BTV). VIBs were detected as early as 4 h post-infection (p.i.), and their number and profile areas increased significantly between 12 and 16 h, and 20 and 28 h p.i. respectively. Core- and virus-like particles were found within and at the periphery of the VIB matrix, respectively, and their numerical density (number per area of VIB matrix) decreased during the course of infection whereas the numerical density of virus particles in the cytoplasm increased. Virus-like particles had a diameter of 57 ± 8 nm and core-like particles appeared to fall into two size ranges, 32 ± 3 nm and 38 ± 3 nm in diameter. Both pre- and post-embedding immunoelectron microscopy procedures were used to localize BTV structural and non-structural proteins within the VIBs. The VIB matrix was labelled with antibodies to structural proteins VP5 and VP7 and non-structural proteins NS1 and NS2. Cores within VIBs contained proteins VP5, VP7 and NS1 but not VP2. Virus-like particles at the periphery of VIBs contained VP2, VP5, VP7 and NS1. The results suggest that BTV particles are synthesized, assembled and released from the perimeter of VIBs and not from within the matrix. Cores embedded in the VIBs are likely to have been trapped there during expansion of the matrix during replication.

A cDNA containing almost all of the hepatitis A virus (HAV) P3 sequences was expressed as a fusion protein with Protein A. A novel poly(U) polymerase activity was detected in the periplasmic space of Escherichia coli cells transformed with this plasmid, and this activity showed many of the expected properties of a picornavirus 3Dpol. A number of HAV-specific polypeptides were detected in these cells, and it is unclear which of these was responsible for the polymerase activity.

The functionality of the influenza virus polymerase subunits and the nucleoprotein expressed from simian virus 40 (SV40) recombinants has been tested by their ability to direct the in vivo expression of influenza virus-like RNAs. These RNAs, which contained either the chloramphenicol acetyltransferase (CAT) or haemagglutinin (HA) genes, were synthesized and reconstituted in vitro into viral ribonucleoproteins with a polymerase/nucleoprotein mixture purified from influenza virus-infected cells. Only the coinfection with SV40 recombinant viruses expressing the three polymerase subunits and the nucleoprotein allowed the expression of the transfecting CAT or HA RNAs, confirming that this set of viral genes is the minimal requirement for viral gene expression. Unexpectedly, transfection of the corresponding naked RNAs into SV40 recombinant-infected cells was as effective in directing the synthesis of CAT or HA proteins as the standard reconstituted ribonucleoprotein transfection. These results may be important for the genetic analysis of trans-acting factors involved in influenza virus transcription and replication and may open the way to rescuing influenza viruses in the absence of a helper virus.

The sequence of the 3′-terminal 1768 nucleotides of the PS and ŏo6 isolates of plum pox virus (PPV) has been determined and compared with that of the equivalent regions of other PPV isolates sequenced previously. The sequenced region is part of the PPV open reading frame encoding the last 186 amino acids of the NIb protein and the coat protein (CP, 330 amino acids), followed by a non-coding region of 220 nucleotides and a poly(A) tail. PPV-PS and PPV-ŏo6, just like PPV-El Amar, show rather high levels of nucleotide diversity in the sequence encoding the C-terminal region of the NIb protein (19.4 to 31%) and the N terminus of CP (22.8 to 41.1%) when compared with PPV-Rankovic, PPV-D and PPV-NAT, whereas the level of diversity in the rest of the CP sequence and the 3′ non-coding region is low (8 to 10.8% and 5.5 to 7.7%, respectively). However, the first 429 sequenced nucleotides of PPV-ŏo6 are very similar to those of the PPV-Rankovic, PPV-D and PPV-NAT isolates, whereas the rest of the sequence clearly resembles PPV-PS. Thus, PPV-ŏo6 seems to be the result of a natural recombination event between two wild strains of PPV. To our knowledge this is the first evidence of homologous RNA recombination (a process which could play an important role in the evolution of RNA viruses) within the potyvirus group.

The immunoreactivity of a panel of polyclonal anti-bodies and monoclonal antibodies (MAbs) raised against African isolates of potyviruses from cowpea and African yam bean was examined in ELISAs. A serological study including reference isolates followed by further characterization in differential hosts resulted in separation of the potyviruses into two distinct serogroups, one containing blackeye cowpea mosaic virus (BlCMV) and the other containing cowpea aphid-borne mosaic virus (CAMV). Using biotin-labelled MAbs, the BlCMV isolates were further subdivided into two serotypes and the CAMV isolates into five serotypes. Because both BlCMV and CAMV induce a very similar mosaic disease in cowpea, different ELISA procedures using mixed MAbs were evaluated and a single protocol was developed which allowed reliable diagnosis of both viruses.

The accumulation of two proteins, the nucleocapsid (N) protein and a non-structural (NSs) protein both encoded by the S RNA of tomato spotted wilt virus (TSWV), was followed in larvae during development and in adults of Frankliniella occidentalis after ingesting the virus for short periods on infected plants. The amounts of both proteins increased, as shown by ELISA and Western blot analysis, within 2 days above the levels ingested, indicating multiplication of TSWV in these insects. Accumulation of these proteins and of virus particles was further confirmed by in situ immunolabelling of the salivary glands and other tissues of adult thrips. The accumulation of large amounts of N and NSs protein, the occurrence of several vesicles with virus particles in the salivary glands and the massive numbers of virus particles in the salivary gland ducts demonstrate that the salivary glands are a major site of TSWV replication. The occurrence of virus particles in the salivary vesicles is indicative of the involvement of the Golgi apparatus in the maturation of the virus particles and its transport to the salivary ducts.