- Volume 69, Issue 3, 1988

We have recently reported the isolation of two group A swine rotaviruses each lacking normal genomic RNA segment 11 and showing instead one extra segment that migrated abnormally on gel electrophoresis. Hybridization studies performed with segment-specific probes and with a purified abnormal RNA segment showed that the extra bands had sequence homology to normal segment 11. Analysis of protein profiles of normal and rearranged strains showed that the gene product of segment 11 had no apparent change in its relative electrophoretic migration, suggesting that the rearranged genes remained functional.

High molecular weight human cytomegalovirus (CMV) DNA was isolated from agarose-embedded infected human diploid cells by employing field inversion gel electrophoresis. A high yield of CMV DNA molecules was obtained within 1 week of infecting the cell culture. Labelling of the viral DNA with biotin by nick translation enabled the detection of CMV-infected cells in sections of paraffin-embedded human adrenal gland by in situ hybridization.

Organs of naturally infected mink were examined for the presence of Aleutian disease virus (ADV) DNA by in situ hybridization. Spleen, lymph nodes, thymus, bone marrow, kidney, liver, lung and small intestine were found to be positive for ADV to differing extents. Infected lymphoid organs showed a focal distribution of pospositive cells. Southern blot analysis of DNA extracted from infected organs revealed replicative forms of viral DNA in spleen and bone marrow samples only. These findings are consistent with a lymphotropism of ADV in vivo. Compared to the situation after experimental infection of mink these results indicate additional sites of virus replication and/or persistence of the naturally occurring disease.

Prion protein (PrP) forms the fibrils or prion rods isolated from scrapie-infected brain and has been proposed as the major component of the infectious agent of this slowly progressive spongiform encephalopathy. In previous Northern blot analyses PrP-specific mRNAs have been found in both normal and scrapie-infected brains but not in spleen, an organ which harbours large titres of infectivity. In the present study, mouse PrP DNA was used to probe for PrP mRNA in assorted tissues and cells. A reexamination of mouse and hamster spleens revealed that they contained low levels of PrP mRNA (approx. 0.8% of that in brain mRNA). No consistent differences were observed between normal and scrapie-infected tissues. Also positive for PrP mRNA under stringent hybridization conditions were mouse epithelial, neuroblastoma, erythroid, B-lymphocytic and embryo fibroblast tissue culture cell lines, a hamster ovary cell line, a rat glioma cell line, and human T lymphocytic and neutroblastoma cell lines. In contrast, no PrP mRNA was detected in two mouse myeloid cell lines and one T cell lymphoma. These results provide evidence that PrP is a protein common to numerous, but not all, cell types besides those of the brain.

The capacity of lectins to inhibit viral haemolysis of chicken erythrocytes was tested, to evaluate the role of carbohydrate in the fusion reaction. Pretreatment of cells with pea lectin provided a 70% to 85% haemolysis inhibition with WSN influenza virus, but only 10% to 14% with PR8 influenza virus. Pea lectin did not detectably bind to virus, nor did it inhibit virus binding to cells, but it did inhibit WSN influenza virus elution. Additionally, pea lectin was active against Sendai virus and B/Lee influenza virus, but inactive against Newcastle disease virus. Haemolysis by WSN and PR8 influenza viruses was unaffected in cells pretreated with concanavalin A, peanut, wheatgerm or soybean lectins. A possible role of cellular carbohydrate in virus-cell fusion is discussed.

The development of autoimmunity was investigated in BALB/c and C58 mice infected with lactate dehydrogenase-elevating virus (LDV). Autoantibodies reactive by ELISA with syngeneic central nervous system antigens appeared early during LDV infection of both strains of mice, and were maintained for many months. Western blot analysis indicated that the LDV-induced autoantibodies reacted with a variety of different brain antigens, and mouse strain differences in the pattern of autoreactivity were observed. LDV infection of C58 and BALB/c mice also stimulated antibodies reactive with syngeneic liver-, kidney- and spleen-derived antigens, and in Swiss outbred mice heart-reactive antibodies were observed following LDV infection. These results show that autoimmunity is a feature of the deregulation of the immune system which occurs during LDV infection.

Virus-like particles (VLPs) with a mean diameter of 32 nm were recovered from the stools of three acute phase cases of enterically transmitted non-A, non-B hepatitis (ET-NANBH) occurring in the Soviet Union, North Africa and North America. VLPs from two of these cases were studied in detail and were shown to react specifically with antibody in acute phase sera obtained from other cases of ET-NANBH in Asia, the Soviet Union, North Africa and North America. Partially purified VLPs were found to sediment at 183S in sucrose gradients and to cross-react with antibody in acute phase sera from geographically isolated cases of ET-NANBH. The latter virus preparations were also used to document the seroconversion of experimentally ET-NANBH-infected cynomolgus macaques to 32 nm VLPs. Our findings indicate that one virus or class of viruses is responsible for the majority of ET-NANBH.

Comparison by HPLC of tryptic digests of coat proteins from six biologically and serologically distinct potyviruses, namely bean yellow mosaic virus, Johnson grass mosaic virus, passion-fruit woodiness virus (PWV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and water-melon mosaic virus II, demonstrated that each potyvirus can be distinguished from the others. HPLC of tryptic peptides from coat proteins of four strains of PVY, two strains of PWV and three strains of SCMV showed that peptide patterns of strains from the same potyvirus were very similar. These findings were supported by amino-terminal amino acid sequence analysis of the peptides. The use of enzymes from different sources and variation in the temperature (35 °C to 40 °C) and time (16 to 20 h) of digestion caused small variations in the profiles but did not change the main features of the peptide patterns of each potyvirus. The results suggest that HPLC profiles of tryptic digests of the coat proteins of potyviruses could be useful criteria for the identification and classification of potyviruses.

Insertion mutations introduced in vitro into cloned DNA of tomato golden mosaic virus that considerably shortened the length of the open reading frames (ORFs) AL1, AL2/AL3, BL1 or BR1, abolished the ability of the DNA to infect Nicotiana benthamiana seedlings. Mutants in which ORF AR1 was similarly shortened by an insertion or a 28 bp deletion were infectious, showing that the formation of coat protein or virions is not required for replication and systemic spread of virus DNA, although the appearance of symptoms was delayed in infections with the deletion mutant. Mutants with larger deletions (178 bp to 603 bp) in ORF AR1 were not infectious. Infections could be initiated with mixtures of AL1 and AL2/AL3 mutants, or BL1 and BR1 mutants, primarily as a result of complementation, although a low proportion of wild-type DNA A molecules regenerated by recombination or reversion was detected in the progeny of infection with the DNA A mutants.

Double-stranded cDNA copies of RNA-1 and RNA-2 of tomato ringspot virus were cloned into pUC9. Comparison of restriction enzyme maps of eight clones indicated that 1.9 kb at the 3′ termini of RNA-1 and RNA-2 were similar. Five of these clones were used in Northern hybridization analyses and found to contain sequences either unique to RNA-1 or RNA-2, or common to RNA-1 and RNA-2. Southern blot analyses using clones derived from RNA-1 and RNA-2 confirmed that there is a 1.9 kb nucleotide sequence homology at the 3′ termini. A sequence homology of this magnitude has not been reported previously for other plant viruses with multipartite ssRNA genomes.

Infection of cowpea (Vigna unguiculata) cells or protoplasts with cowpea mosaic virus is accompanied by the appearance of characteristic cytopathic structures. A major constituent of these cytopathic structures, represented in thin sections by electrondense material, was shown to contain viral non-structural proteins by its reaction with antisera to non-structural proteins and their detection with colloidal gold-labelled Protein A. With this same technique virus particles were found to accumulate mainly in the cytoplasm.

In reticulocyte lysates maize rayado fino virus RNA directed the synthesis of a large number of polypeptides ranging in M r from 15000 to 165000. Partial proteolysis showed that many of the larger polypeptides contained the same peptides. When tRNA was added to the lysate proportionally more of the large M r products were produced. No polypeptides were detected that comigrated with coat proteins and no coat protein was detected in translation products by ELISA or by immunoprecipitation. We found little evidence of polyprotein degradation among the translation products and no evidence for encapsidated subgenomic mRNAs for the capsid polypeptides.