- Volume 79, Issue 10, 1998

The intact virion of bluetongue virus comprises ten segments of dsRNA enclosed in two concentric protein capsids. The core, which is transcriptionally active, includes three minor proteins (VP1, VP4 and VP6) which are considered to be the candidates for the core-associated enzymes that transcribe and modify full-length mRNA copies for each of the ten genome segments. Using purified recombinant VP4 protein and core-like particles containing VP4, in this report it is demonstrated that VP4 has nucleoside triphosphatase (NTPase) activity. VP4 is a nonspecific NTPase that hydrolyses four types of ribo- nucleoside triphosphate (NTP) to the corresponding nucleoside diphosphate. The substrate preference was GTP ATP UTP CTP. NTP hydrolysis by VP4 was maximal when the Mg2 or Ca2 ion concentrations were 4 mM or 6 mM, respectively. The presence of single-stranded polynucleotides poly(A), poly(U) and poly(C) had little effect on the NTPase activity. Although the enzyme exhibited a broad temperature optimum around 40 °C, the pH optimum was sharp, between pH 7 5 and 8. The K m and V max of ATP hydrolysis were calculated to be 0·25 0·05 μM ATP and 55 4 pmol ATP hydrolysed min 1 μg \ respectively. The Km was affected by the addition of poly(A) to only a small extent in contrast to the Vmax, which was increased by at least twofold.

In this study, the basis for the classification of virus isolates grouped within the genus Coltivirus, family Reoviridae, is discussed. Sequences of dsRNA segments from American (segments 9–12), European (segment 12) and Asian (segments 7– 12) isolates were characterized and polythetic criteria were defined for their taxonomic classification. These criteria (including sequence analysis) permitted the different species to be distinguished and classified into two groups. In both groups, subgroups were defined according to the degree of homology between the genomic sequences. American and European isolates are classified within group A, which includes subgroups A1 (Colorado tick fever virus species) and A2 (Eyach virus species). Asian isolates are classified in group B, which includes subgroups B1 (JKT-7075 virus species) and B2 (JKT-6423 virus species). The proteins encoded by the sequenced genomic segments were analysed. This allowed the identification of dsRNA binding domains in the proteins encoded by segment 8 of subgroup B1 isolates and segment 12 of subgroup B2 isolates. A conserved pattern of amino acids in segment 7 of group B isolates matched sequences found in the catalytic domains of protein kinases.

The polyomavirus JC (JCV) establishes a persistent infection in the kidneys, and is the virus agent that causes the demyelinating disease progressive multifocal leukoencephalopathy. PCRand DNA sequence analyses of partial JCV genomes have shown that there are at least four main JCV types, each associated with a specific geographical region. Type 1 is of European origin, Type 2 is Asian, Type 3 is found in individuals of African decent and Type 4 is a potential recombinant of Types 1 and 3, and is widely distributed throughout the population of the United States. A comprehensive phylogenetic analysis of 22 complete JCV genomes excluding part of the regulatory region was accomplished using neighbour-joining, UPGMA and maximum parsimony methods. The resulting UPGMA and parsimony phylogenies suggest that the European Type 1 strains diverged from the other types during the evolution of JCV and that each of the other genotypes (and subtypes) falls into well-supported clades. This is the first whole genome approach to phylogeny reconstruction for JCV and represents a significant improvement over earlier studies that were limited to partial JCV sequences and the neighbour-joining method.

JC polyomavirus (JCV) is ubiquitous in humans, persisting in renal tissue and excreting progeny in urine. It has been shown that the genotyping of urinary JCV offers a novel means of tracing human migrations. This approach was used to elucidate the racial composition of modern China. JCV isolates in the Old World were previously classified into nine distinct genotypes. One of them (B1) has a wide domain, encompassing part of Europe and the entirety of Asia. By constructing a neighbour-joining phylogenetic tree, all B1 isolates detected so far were classified into four distinct groups (B1-ato -d), each occupying unique domains in the world. According to this revised classification system of JCV DNAs, four genotypes (CY, SC, B1-a and -b) were found to be prevalent in China and Mongolia (Mongolia was studied instead of Inner Mongolia, which is part of China). There was a remarkable variation in the incidence of genotypes among the sites of sample collection. CY was more frequently detected in Northern China, SC was predominant in Southern China and B1-b was detected only in Mongolia. B1-a was spread throughout China. These data were statistically analysed and the observed regional differences in the incidence of genotypes were found to be significant. It is likely that these differences in JCV distribution in China reflect the intermingling of different population groups that constitute modern China.

Fowl adenovirus (FAdV) type 1, CELO strain has no homologies to mastadenovirus E1A, E1B, E3 and E4, which regulate virus gene expression, DNA replication and virus-host interaction. Similarly, the right 5 kb and left 15 kb ends of CELO virus DNA are non-homologousto mastadenoviruses. To compare CELO virus with another FAdV, 7·5 kb of the left and 17 kb of the right ends of FAdV type 8 (strain A-2A) were sequenced and nine and 17 open reading frames (ORFs), respectively, were found. This FAdV- 8 genome was similar to CELO virus in that (1) the central region contained the major structural protein genes including the fibre, pVIII, 100K, late 33K and pIVa2 genes, which were in the same order as in mastadenoviruses, (2) no homologues of mast adenovirus E1A, E1B, E3 and E4 were found in the ends, and (3) the left 6 kb and the right 13 kb ends showed no homology to mastadenoviruses. Several genomic features were unique to FAdV-8 compared to CELO virus. FAdV-8 contained one fibre gene in contrast to two in CELO virus. Three of eight unassigned ORFs in the left and five of 13 unassigned ORFs in the right ends were unique compared to CELO virus. Two sets of tandem repeats, one with five identical 33 bp repeats and the other with more than ten identical 135 bp repeats, mapped between 4·5 and 7·5 kb from the right terminus. No virus-associated RNA gene was found. Fifteen of 16 unique FAdV-8 ORFs tested were, as determined by RT-PCR, transcribed early.

The fate of herpes simplex virus 1 (HSV-1) tegument proteins during infection in Vero cells was investigated immunochemically. Input virion-associated VP13/14 and VP16 localized to the nucleus early in infection, while VP1/2 localized to the nuclear envelope of the cell and VP22 could not be detected using monoclonal antibody P43. Western blotting suggested that virion-associated VP13/14, VP16 and VP22 were stable in infected cells whereas VP1/2 appeared to be processed or modified. Further studies showed that P43 recognized a phosphorylation-sensitive epitope in VP22 and suggested that virion-associated VP22 was phos- phorylated upon entry to the cell. VP13/14 and VP16 were easily extracted from cells early in infection whereas VP22 was largely insoluble. Phosphatase treatment of soluble extracts caused a shift in the molecular mass of VP16 showing it was phosphorylated. As infection progressed VP16 was observed in discrete nuclear compartments where it co-localized with ICP8 and the capsid-associated protein VP22a. VP13/14 was also observed in the nucleus. P43 immunostaining appeared around 6 h post-infection as punctate nuclear foci which often localized to the edge of VP16-immunoreactive areas. Punctate P43 cytoplasmic staining appeared around 12 h post-infection. By 18 h the nuclear pattern had disappeared and an extensive cytoplasmic stain was observed which closely overlapped that of other tegument proteins. On the basis of these data we suggest that virion-associated VP22 is phosphorylated upon entry of the virus into the cell and that unphosphorylated VP22, which is preferentially recognized by P43, becomes available later in infection, initially in the nucleus, for packaging into virions.

A domain, previously termed RE1, exists within the herpes simplex virus type 1 genome potentially influencing expression of immediate early genes and the latency associated transcripts. This domain consists of 10 tandem copies of a CT-rich sequence. We demonstrate that this domain binds multiple host-cell factors that may allow RE1 to act either as a transcriptional regulator and/or to affect nucleo- somal and DNA structure in the latent genome.

Interactions between human cytomegalovirus (HCMV) and various carbohydrate structures were analysed using sulfated glucuronyl glycosphingolipids (SGGLs) and the structurally related glyco- sphingolipids (GLs). A thin-layer chromatography- overlay assay and a solid-phase binding assay revealed that HCMV strongly bound to sulfated glucuronyl lactosaminylparagloboside, one of the SGGLs having the repeating lactosamine structure (3Gal β 1-4GlcNAc1-)2 in addition to the 3-O- sulfated glucuronyl moiety. The virus bound less strongly to other 3-O-sulfated GLs, which included sulfated glucuronyl paragloboside and cerebroside sulfate ester, and also to (3Galβ 1-4GlcNAc1-)2- containing GLs that included nLc6Cer. Thus, a (3Galβ 1-4GlcNAc1-)2and a 3-O-sulfated saccharide seem to be important structures for the binding by HCMV. When virus particles were preincubated with these GLs, inhibitory effects were observed both on expression of the viral immediate-early gene and on plaque formation by HCMV. These effects were very well correlated with the abilities of the GLs to bind to the virus. Pretreatment of host cells with HNK-1 monoclonal antibody, which specifically recognizes SGGLs, resulted in partial inhibition of plaque formation by HCMV. These results clearly show that HCMV recognizes and binds to the sulfated carbohydrate structure in SGGL and also suggest that binding of HCMV to the specific sugar structure may play an important role in HCMV infection.

Previously, we described a highly conserved nonessential African swine fever virus (ASFV) right variable region gene, NL. Deletion of NL from the European pathogenic isolate E70 resulted in almost complete attenuation of the virus in domestic swine. To study gene function further, NL gene deletion mutants were constructed from two pathogenic African ASFV isolates, Malawi Lil-20/1 (Mal) and Pretoriuskop/96/4 (Pr4). Unexpectedly, both Mal (Mal-∆NL) and PR4 (Pr4∆NL) null mutants remained highly virulent when inoculated in swine. Mal-∆NL exhibited a disease and virulence phenotype indistinguishable from its revertant, Mal-NLR, which caused 100% mortality. Mortality among Pr4∆NL-infected animals was also high; however, a significant delay in onset of fever and viraemia and in time to death was observed. These data indicate that NL gene function is not required for ASFV virulence and that other yet-to-be identified viral determinants perform significant virulence functions in these African field isolates.

A nucleopolyhedrovirus (NPV) isolated from the looper Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) (ThorNPV) is occluded in a tetrahedral protein matrix. The ORF of the ThorNPV polyhedrin gene contains 738 nt which code for 246 amino acids of the putative polyhedrin protein with an estimated molecular mass of 28778 Da. The promoter of this gene is similar in length to the promoter of Spodoptera frugiperda NPV (SfMNPV), with a 5nt deletion before the start codon compared to those of other NPVs. When the polyhedrin gene of Autographa californica NPV (AcMNPV), whose occlusion bodies (OBs) are polyhedral, was replaced by the polyhedrin gene of ThorNPV, which produces tetrahedral OBs, tetrahedral polyhedra with properly occluded virions were produced. This work establishes the importance of the polyhedrin protein sequence in determining OB shape. Leucine at position 43 of ThorNPV polyhedrin was identified as responsible for the tetrahedral shape of ThorNPV OBs by PCR-based site-directed mutagenesis. Susceptibility to alkaline buffer of OBs formed by recombinant AcMNPV (RECAcV) carrying the polyhedrin gene of ThorNPV was slightly greater than that of native ThorNPV OBs. The LD50 of RECAcV for third-instar beet armyworm (Spodoptera exigua) was significantly lower than that of AcMNPV (253 and 31 OBs per larva, respectively).