- Volume 66, Issue 7, 1985

We have used a DNA copy of segment 3 RNA of bluetongue virus serotype 17 (BTV-17) to detect sequence homology among the equivalent segments of five U.S.A. BTV serotypes (BTV-2, BTV-10, BTV-11, BTV-13 and BTV-17) as well as 14 other BTVs isolated from different endemic areas of the world. Both by in situ and Northern hybridization all the BTV serotypes were found to have RNA that reacted with the DNA probe. No homology was detected with epizootic haemorrhagic disease virus serotype 1, a related orbivirus. The BTV-17 DNA clone has also been used to detect viral RNA in infected sheep blood. This information has led us to develop a simple and sensitive procedure for the detection of viral genome-biotinylated clone DNA hybrids in vivo or in cultured cells following direct staining with either the avidin-fluorescein complex or the streptavidin-horseradish peroxidase complex.

Peripheral blood mononuclear cells (PBMC) derived from healthy individuals were stimulated with u.v.-inactivated Newcastle disease virus and the cell supernatants were assayed for both antiviral activity and alpha interferon (IFN-α) immunoreactivity. IFN-α concentrations determined by two immunoradiometric assays (IRMAs) based on monoclonal antibodies that recognize different IFN-α subtypes correlated well together (r = 0.96) and with interferon concentrations determined by the two bioassays (r = 0.82 to 0.89), but the agreement between the results of the two bioassays was not as close (r = 0.79). As judged by the agreement between determinations on duplicate inductions of the same PBMC, the IRMAs were considerably more precise than the bioassays. Despite the use of a common IFN standard there were marked differences in the absolute titres of IFN determined by the IRMAs and bioassays, highlighting the difficultires in standardizing assays for IFN-α. The IRMA results suggest that there are no major differences in the spectrum of IFN-α subtypes produced by healthy individuals under conditions of viral stimulation.

Previous work has shown that fever in influenza of ferrets occurs following release of endogenous pyrogen from virus-phagocyte interaction in the upper respiratory tract (URT), and suggested that the poor inflammatory response and correspondingly low fever elicited by A/Puerto Rico/8/34 (H1N1), compared with H3N2 reassortant clones of A/Puerto Rico/8/34-A/England/939/69, were related to its H1 and N1 surface antigens. Nasal virus levels, inflammatory and pyrexial responses produced in ferrets by clones 31 (H3N1) and 64b (H1N2) of the same reassortant system suggested a connection between the H1 antigen and low inflammatory response, but results were not conclusive. Unlike A/Puerto Rico/8/34, two recent H1N1 isolates, A/USSR/90/77 and A/Fiji/15899/83, produced a high inflammatory response yet low fever despite large amounts of virus in the URT, suggesting that either no connection exists between the acquisition of the H1 antigen and production of a low inflammatory response, or the H1 antigen of recent isolates, whilst antigenically related to that of A/Puerto Rico/8/34, is biologically different.

Injection of the protease inhibitor, aprotinin, into the allantoic cavity of embryonated eggs infected at low m.o.i. with different influenza viruses and paramyxoviruses markedly reduced multiplication by at least 100-fold. Under these conditions, most viral particles produced were non-infectious and contained uncleaved glycoproteins, presumably resulting from aprotinin suppression of protease activity.

Most definitive members of the tombusvirus group contain a satellite RNA (S-RNA); those of different tombusviruses have extensive sequence homology with each other. Replication of the S-RNA of tomato bushy stunt virus (TBSV) is supported by the genomic RNAs of other tombusviruses. Viruses regarded as possible members of the tombusvirus group do not support the replication of TBSV S-RNA and the satellite of one such virus, turnip crinkle, does not have sequence homology with that of TBSV. Passage of genomic TBSV RNA through Nicotiana benthamiana resulted in the acquisition of S-RNA whereas passage through N. clevelandii did not.

Circular molecules of potato spindle tuber viroid (PSTV) were subjected to limited digestion by various methods to produce nicked molecules. The resulting linear molecules were separated electrophoretically from circular ones, and assayed for infectivity. The linear molecules produced by treatment with RNase CI or RNase U2 were as infective as circular molecules. These linear molecules had 5′-OH and 3′-phosphate ends; almost all 3′ termini probably were in the form of 2′,3′-cyclic phosphate. However, the linear molecules produced by treatment with RNase T1 were approximately 10-fold less infective than circular molecules. The 2′,3′-cyclic phosphate at the 3′ end of these linear molecules had presumably been partially converted to 3′-phosphate. Linear molecules produced by a Mg2+-catalysed nicking reaction had a mixture of 2′- and 3′-terminal phosphates at their 3′ end and were approximately 100-fold less infective than circular ones. The infectivity of linear molecules produced by nuclease S1 digestion which lacked a 3′-phosphate was 100- to 1000-fold less than that of circular ones. These results indicate that the 3′-terminal phosphate of linear PSTV molecules is required for infectivity, and for maximum infectivity is required to be in the form of 2′,3′-cyclic phosphate.

The nucleotide sequences of cDNA clones corresponding to RNA-3 and RNA-4 of beet necrotic yellow vein virus isolates F2 and G1 have been determined. The cDNA of RNA-3 of isolate F2 is 1775 residues in length and contains a coding region of 219 codons. In isolate G1 this coding region has undergone an internal deletion of 354 nucleotides in such a way as to conserve a shortened reading frame. Otherwise, the RNA-3 sequences of the two isolates were closely similar. RNA-4 of isolate F2 has an extrapolated length of 1431 residues and contains an open reading frame of 282 codons. This open reading frame has undergone an internal deletion of 324 nucleotides in one cDNA clone of RNA-4(G1) with conservation of a shortened reading frame. Sequence analysis of other RNA-4(G1) cDNA clones revealed, however, that the exact boundaries of the deletion are not always the same. RNA-3 and RNA-4 of each isolate are more than 90% homologous for the 3′-terminal 200 nucleotides. Short homologous sequences are also present in RNA-3 and RNA-4 of isolate F2 flanking the regions deleted in each of these RNAs in the G1 isolate. These homologous sequences probably play a role in the deletion process.

A topoisomerase activity is associated with herpes simplex virus type 1. The enzyme was recovered from purified virions which were disrupted with 6 m-guanidine-HC1 followed by renaturation of extracted proteins. Based upon the following observations, the virion activity is classified as a type I topoisomerase: (i) the linking number of a unique DNA topoisomer is altered in steps of one; (ii) ATP and MgCl2 are not required for activity; (iii) the enzyme can be trapped in a covalent complex with DNA; (iv) the covalent linkage to DNA is through a 3′ phosphoryl bond. A number of lines of evidence strongly indicate that the topoisomerase is external to the nucleocapsid. For example, the activity was released by treatment of intact virions with NP40, and subsequent washing steps extracted most residual activity. When guanidine extracts were prepared from nucleocapsids, topoisomerase activity was not detectable. Finally, DNA within the virion did not appear to contain covalently attached proteins with properties similar to topoisomerases. Thus, the enzyme appears to be a component of the envelope or tegument structure of the virion.

Particles of carrot red leaf virus (CRLV; luteovirus group) purified from chervil (Anthriscus cerefolium) contain a single ssRNA species of mol. wt. about 1.8 × 106 and a major protein of mol. wt. about 25000. CRLV acts as a helper for aphid transmission of carrot mottle virus (CMotV; ungrouped) from mixedly infected plants. Virus preparations purified from such plants possess the infectivity of both viruses but contain particles indistinguishable from those of CRLV; some of the particles are therefore thought to consist of CMotV RNA packaged in CRLV coat protein. When RNA from such preparations was electrophoresed in agarose/polyacrylamide gels, CMotV infectivity was associated with an RNA band that migrated ahead of the CRLV RNA band and had an estimated mol. wt. of about 1.5 × 106, similar to that previously found for the infective ssRNA extracted directly from Nicotiana clevelandii leaves infected with CMotV alone. Preparations of dsRNA from CMotV-infected N. clevelandii leaves contained two species: one of mol. wt. about 3.2 × 106, presumably the replicative form of the infective ssRNA, and the other, mol. wt. about 0.9 × 106, of unknown origin and function. The infective agent in buffer extracts of CMotV-infected N. clevelandii was resistant to RNase (although the enzyme acted as a reversible inhibitor of infection at high concentrations) and is therefore not unprotected RNA. It may be protected within the approximately 52 nm enveloped structures previously reported.