- Volume 73, Issue 12, 1992

We have used the human myelomonocytic cell line HL-60 as a model system to determine whether human immunodeficiency virus type 1 (HIV-1) infection affects differentiation of myeloid progenitor cells. HL-60 cells were infected with three HIV-1 isolates (IIIB, NL4-3 and PM213). HIV-1 antigen expression and cytopathicity in HL-60 cells infected with each of the three isolates was delayed by approximately 15 days as compared to those in the prototypic T cell line, H9. Chronically infected HL-60 cells and clonal lines derived from them were treated with dimethyl formamide (DMF) and induced to differentiate into granulocytes. Approximately the same percentage of these cells as of DMF-treated, uninfected HL-60 cells differentiated. Superoxide production by infected and uninfected DMF-induced cells was similar. Likewise, approximately the same percentage of cells in infected and uninfected cultures became adherent and were positive for non-specific esterase when monocytic differentiation was induced. The data demonstrate that HL-60 cells infected with HIV-1 are capable of morphological and functional granulocytic and monocytic differentiation.

DNA well suited for polymerase chain reaction (PCR) amplification was purified from archival Papanicolaou smears. The detection of a wide range of human papillomavirus (HPV) types was made possible using a HPV-specific consensus primer pair, and typing was conveniently done by direct sequence analysis of the PCR product. The method could be of unique value in longitudinal and cross-sectional studies aimed at answering a number of fundamental pathological and epidemiological questions regarding HPV infection of the genital tract.

Primary cultures of normal human keratinocytes were inoculated in vitro with human papillomavirus type 1 (HPV-1), the agent responsible for deep plantar warts. Upon transfer to dead de-epidermized dermis and growth at the air-liquid interface, keratinocytes reconstituted a pseudoepidermis. Under these highly differentiating conditions, HPV-1 DNA amplification was found to take place in the reconstructed epidermis, being detectable from 7 days after the transfer and persisting for at least 10 days thereafter. The extent of keratinocyte differentiation may be insufficient to allow a complete HPV infectious cycle.

Murine gammaherpesvirus 68 (MHV-68) is able to persist in spleen cells of infected mice. To determine the cell type harbouring persistent virus, spleen cells from infected animals were separated into immunoglobulin (Ig)-positive (B cell-enriched), Ig-negative (T cell-enriched) and plastic-adherent (macrophage-enriched) fractions. These cells were co-cultivated with permissive BHK-21 cells in an infectious centre assay. The consistent recovery and enrichment of infectious centres in the Ig-positive fraction clearly demonstrates that B cells are a major site of virus persistence/latency. This observation indicates that MHV-68 is biologically similar to Epstein—Barr virus and other members of the B cell lymphotropic gammaherpesvirus 1 subgroup.

Cytomegaloviruses generally display a host range restricted to differentiated cell types from the species they infect. For human cytomegalovirus (HCMV) this has meant that with few exceptions tissue culture systems have relied on the use of primary foreskin fibroblast (HF) cells or primary human embryonic lung cells to study gene expression and virus replication functions. We have observed that primary skin fibroblast (CF) cells derived from the chimpanzee (Pan troglodytes) support the replication of a laboratory strain (Towne) of HCMV. The kinetics of gene expression of the Towne strain grown in CF or HF cells appeared to be equivalent. Titres of progeny virions grown in CF cells appeared to be reduced 10-fold relative to those of virus grown in HF cells. In contrast, replication of the Towne virus was not supported by growth in WES cells (ATCC no. CRL 1609), a chimpanzee skin fibroblast cell line transformed by an adenovirus 12-simian virus 40 hybrid. This study shows that HCMV is less parochial in its host range than previously thought.

The growth of feline enteric coronavirus strain 79-1683 in whole feline embryo cells was inhibited by the presence of 1 µg/ml of actinomycin D in the culture fluid. No virus-specific mRNAs could be detected in such cultures and yields of infectious virus were depressed by >99%. By contrast, the antigenically related feline infectious peritonitis virus strain 79-1146 was unaffected by the presence of actinomycin D, indicating a fundamental difference between the two feline coronavirus strains in their requirements for host-encoded function(s).

We have identified the binding site of monoclonal antibodies (MAbs) against the S2 subunit of the bovine coronavirus spike (S) glycoprotein. The location of this site was first investigated by using prokaryotic expression of DNA restriction fragments covering the entire S gene. The amino acid sequence containing the antibody binding site was shortened from 70 to 20 amino acids by digestion of plasmid DNA with exonuclease III, followed by sequencing of the smallest digestion product encoding an immunoreactive fusion protein. Finally we synthesized a set of nonapeptides covering the 20 amino acid sequence extending from the N-terminal residue of the S2 subunit (Ala 769 to Tyr 798). MAbs reacted mainly with six consecutive overlapping peptides with the sequence TTGYRFTN-FEPFTV. Polyclonal antibodies from hyperimmunized or convalescent animals reacted only with the recombinant proteins identified by MAbs, and the hyperimmune serum bound to the same set of peptides. This suggests that this highly conserved linear antigenic determinant corresponds to an immunodominant region. This region resembles both in location and immunodominance the linear determinant defined on the infectious bronchitis virus S2 subunit. The presence of similar regions in the N-terminal region of the S2 subunit of other coronaviruses is discussed.

The DNA sequences of the early region 3 (E3) and fibre protein genes of bovine adenovirus type 3 (BAd3) have been determined and the amino acid sequences predicted to be encoded by their open reading frames (ORFs) compared to those of the fibre and E3 proteins from other Ads. One of the BAd3-E3 proteins contains a region homologous to the 14.7K E3 protein of human Ad5 (HAd5). The putative BAd3 fibre protein contains a number of regions homologous to the HAd2 fibre protein sequence, but is predicted to be 244 amino acids longer owing to an increase in the number of repeating structural motifs of hydrophobic amino acid residues in the shaft region. Sequences to the left of the BAd3-E3 gene region contained the 3′ end of another ORF with extensive identity with the hexon-associated protein precursor (pVIII) of HAd2. Like mouse Ad1 and canine Ad1, the BAd3 E3 gene is approximately 1.5 kb, about half the size of the E3 region of HAd2 and HAd5.

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne pathogen that has caused encephalitis in equine species and humans during sporadic outbreaks in the western hemisphere. The last, and most widespread, VEE outbreak occurred in South America, Central America, Mexico and the U.S.A. (Texas) during 1969 to 1972. We have cloned and sequenced the genome of a virulent VEE subtype I-AB virus, strain 71-180, isolated in Texas in 1971. Thirty-four nucleotide differences were detected between the genome of 71-180 virus and that of the subtype I-AB Trinidad donkey (TRD) virus isolated during the 1943 VEE epizootic in Trinidad. Fifteen nucleotide changes occurred in the non-structural genes, 16 in the structural genes and three in the 3′ non-coding region. Only six of the nucleotide differences resulted in amino acid substitutions: one change in each of non-structural proteins nsP1 and nsP3, two in the E2 envelope glycoprotein, one in the 6K polypeptide and one in the E1 envelope glycoprotein. The close genetic relationship between 71-180 virus and TRD virus, commonly used for production of formalin-inactivated VEE vaccines, suggests that incompletely inactivated virulent vaccine virus may have been the source of this and other VEE outbreaks. Use of formalized virulent virus was discontinued during the 1969 to 1972 panzootic. No VEE epizootics have been reported since the introduction of the live attenuated TC-83 vaccine virus.

The antigenic sites A and C (the G-H loop and the C terminus, respectively) in VP1 of foot-and-mouth disease virus (FMDV) have been considered the immunodominant regions of the virus involved in the induction of protection. Other antigenic sites have been described but their involvement in protection has not been established. Here we report that two closely related but serologically different FMDVs (the field isolate C3 Argentina/84 and the vaccine strain C3 Resende Br/55) have identical A and C sites but differ at other antigenic sites. Such differences have been documented by reactivity with a panel of 28 monoclonal antibodies (MAbs). The two viruses reacted to the same extent with each of 13 MAbs which recognized epitopes within sites A or C, but reacted differently with six out of 15 MAbs that recognized other sites. Accordingly, sequencing of the entire region coding for the capsid proteins, for both viruses, revealed four amino acid substitutions at three antigenic sites other than A and C. The results suggest that identity of sites A and C may not be sufficient to induce cross-protection, and provide the first evidence of significant antigenic diversification of FMDV in the field mediated by amino acid substitutions outside sites A or C.

The inability to culture human parvovirus B19 in standard cell lines has rendered investigation of clinical samples for the presence of infectious virus problematic. Using haematopoietic precursors derived from first trimester foetal liver as targets for infection, and non-isotopic in situ hybridization to detect intracellular virual DNA, we have assessed infectivity in stored serum samples taken from nine volunteers at different stages following intranasal inoculation with parvovirus B19. Infectious virus was detected as early as 3 days after inoculation, the cessation of infectivity correlating with the rise in specific IgM. In all but two samples, infectivity correlated with the detection of B19 DNA by dot-blot hybridization, although in vitro culture was 10-fold more sensitive than dot-blot hybridization. B19 DNA was detected by the polymerase chain reaction in serum from one volunteer up to 36 days after inoculation, although samples containing specific antibody were non-infectious. Infection of erythroid precursors was completely inhibited by preincubation of virus with serum containing high titre B19-specific IgM and IgG. Unexpectedly, this was associated with a strong B19 DNA hybridization signal within the cytoplasm of phagocytic macrophages. This culture and detection system is a rapid and sensitive means of detecting infectious virus in serum samples, and of assessing the neutralizing ability of B19-specific antibodies.

The sequential accumulation of the protease-resistant form of the endogenous prion protein (PrP-res) was compared to levels of scrapie infectivity in the spleen and brain of scrapie-infected mice at various times after inoculation. In mouse spleen PrP-res was detected 1 week after inoculation, and increased 65-fold between 1 and 3 weeks post-inoculation and an additional 15-fold during the next 17 weeks. Infectivity in spleen reached a maximum plateau level by 3 weeks. In contrast, in mouse brain PrP-res was not detected until 8 weeks after inoculation and then increased 200-fold during the next 12 weeks. During this same time, infectivity increased approximately 10000-fold. Therefore, in both spleen and brain of scrapie-infected mice accumulation of PrP-res and infectivity appear to be associated. However, it was not possible to show quantitative correlations between PrP-res detection and infectivity, perhaps owing to the inaccuracy of the infectivity assay.

The non-structural protein NS1 of influenza A virus exhibits two modes of RNA-binding activity. One is sequence-specific binding to minus-sense virus RNA with either a 5′- or 3′-terminal common sequence as reported previously. The other was identified as binding to dsRNA and this activity did not show sequence specificity. The affinity of binding to dsRNA was much higher than that to ssRNA. A short miniature virion RNA forming a panhandle structure by pairing between the 5′- and 3′-terminal common sequences bound NS1 with higher affinity and stability than did a dsRNA of similar sequence and length.

Raspberry bushy dwarf virus (RBDV) has isometric, 33 nm diameter particles and a bipartite RNA genome. Sequencing of the larger component (RNA-1) showed that it consists of 5449 nucleotides and contains one large open reading frame encoding a putative translation product with a calculated M r of 190000. Comparisons of this polypeptide with non-structural proteins of other plant viruses revealed significant homologies with those of alfalfa mosaic virus (AlMV), brome mosaic virus (BMV), cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV). Thus RBDV belongs to the supergroup of ‘Sindbis-like’ plant viruses. The translation product of RBDV RNA-1 contains motifs characteristic of proteins with polymerase, methyltransferase and helicase activities, suggesting that this protein is involved in the replication of the viral RNA. Thus in RBDV, as in TMV, all three functional domains are combined in the single protein, whereas in AlMV, BMV and CMV these domains are distributed over the proteins encoded by RNA-1 and RNA-2. These findings support the idea that RBDV should be placed in a distinct virus genus for which the name idaeovirus has been proposed.

A ‘double-graft sandwich’ technique in which sections of potato stem from different potato cultivars were grafted between a susceptible healthy stock plant and a potato leafroll virus (PLRV)-infected scion was used to study the rate of phloem transport of PLRV in cultivars differing in resistance to PLRV infection (IR) and accumulation (AR). Resistance to phloem transport (i.e. delayed PLRV systemic movement) was found in Bismark cultivar (IR AS). This was independent of IR and AR as the rate of movement in Bismark cultivar was markedly slower than that in Omega and Spunta (IR AR), Delaware (IS AR), and Desiree and Renova (IS AS) cultivars. It operated in Bismark cultivar stems of two different ages, but did not operate against potato virus X (PVX) and was not influenced by previous infection with this virus. Aphid vector (Myzus persicae) feeding preferences and colonization rates differed between cultivars, but the cultivar characteristics responsible were unrelated to IR, AR or resistance to phloem transport. Delayed systemic movement of PLRV out of leaves inoculated with viruliferous aphids was independent of AR and resistance to phloem transport, and remained unaffected by previous infection with PVX. It was also independent of cultivar factors causing different aphid feeding preferences and colonization rates, but may be linked to IR.

A new geminivirus, tomato mottle virus (TMoV), affecting tomato production in Florida has been cloned and sequenced. Sequence analysis of the cloned replicative forms of TMoV revealed four potential coding regions for the A component [2601 nucleotides (nt)] and two for the B component (2541 nt). Comparisons of the nucleotide sequence of the TMoV genome with those of other whitefly-transmitted geminiviruses indicate that TMoV is a typical bipartite geminivirus of the New World and is closely related to but distinct from abutilon mosaic virus.