- Volume 70, Issue 11, 1989

DNA from infectious laryngotracheitis virus (ILTV) was randomly sheared and cloned into the M13 bacteriophage. Clones containing ILTV DNA were sequenced and the predicted amino acid sequences were compared to the known sequences of other herpesviruses using computer analysis. Twenty-one ILTV genes were identified, 20 by comparison to varicella-zoster virus and 19 by comparison to herpes simplex virus type 1; only 12 genes, giving consistently lower homology scores, were found by comparison with the gammaherpesvirus Epstein-Barr virus, indicating that ILTV sequences bear greater similarity to other alpha- than to gammaherpesvirus sequences.

A 1·4 kb fragment (nucleotides 2430 to 3901) encoding portions of the human parvovirus B19 structural proteins was inserted into the pRIT2 plasmid expression vector containing the gene encoding staphylococcal Protein A under the control of the phage λ promoter PR. The fusion protein was used to raise antibodies in rabbits. The sera were shown by immune electron microscopy to agglutinate B19 particles and were also shown to recognize the VP2 B19 capsid protein, by Western blot analysis. The B19 antigenicity of the fusion protein was confirmed by immunoblot and enzyme immunoassay with IgG and IgM anti-B19-positive reference human sera.

A new human papillomavirus type (HPV-56) was identified by low stringency Southern blot analysis with an HPV-31 DNA probe, in a cervical intraepithelial neoplasm (CIN). The DNA of this virus was molecularly cloned and shown to be a new HPV type based on the absence of cross-reactivity to HPV types 1 to 55 under high-stringency hybridization conditions. At low stringency, HPV-56 was most related to HPV types 30 and 45. The deduced organization of the open reading frames of HPV-56, from hybridization and partial nucleotide sequence analyses, reveals a typical HPV genome. HPV-56 was detected in two of 464 normal cervical tissues, in five of 227 cervical condylomas and CIN, and in two of 84 invasive cancers of the cervix.

A panel of eight neutralizing monoclonal antibodies (MAbs) against the fusion (F) protein of Newcastle disease virus (NDV) has been shown to locate a major antigenic site on the basis of competitive binding assay and additivity index studies. Five epitopes (A1 to A5) have been located within this site on the F protein of the Beaudette C strain of NDV on the basis of cross-resistance plaque assays of MAb-resistant mutants raised against these MAbs. Epitopes A1, A4 and A5 are distinct; epitope A2 partially overlaps epitope A3. Nucleotide sequence analysis of the F genes of MAb-resistant mutants showed that each predicted single amino acid substitutions ranging from amino acid residues 157 to 171 for epitope A4 and at residues 72, 78, 79 and 343 for epitopes A1, A2, A3 and A5 respectively. These locations indicate that both the F1 and F2 fragments are involved in the formation of a single antigenic site and suggest the involvement of extensive protein folding in the active form of this F protein.

Unambiguous assignment of restriction enzyme patterns to six individual serotypes of human rhinovirus was accomplished after amplification of a 380 bp DNA fragment derived from the 5′ non-coding region. This was possible even though serotypes 1A and 1B and serotypes 2 and 49 differed only at 10 and 15 positions respectively. The method utilizes the conserved and variable components of this part of the genome and provides the basis for a simple and rapid method for typing of human rhinoviruses.

The sequence of the 3′-terminal 1370 nucleotides of barley yellow mosaic virus (BaYMV) RNA 1 was determined. The sequence contains a long open reading frame (ORF) of 1137 nucleotides and a non-coding region of 231 nucleotides upstream of the poly(A) tail. Mapping of the partial amino acid sequences of the capsid protein onto the putative translational product of the ORF indicates that the 3′-proximal region of RNA 1 encodes the capsid protein which consists of 297 amino acids with an M r of 32334; the capsid protein is produced by proteolytic processing from a precursor polypeptide at a glutamine-alanine dipeptide. The removal of the N- and C-terminal regions of the capsid protein by mild proteolysis of intact virus particles indicates that both terminal regions are exposed on the external surfaces of virus particles. Alignment of the BaYMV capsid protein sequence with those of some potyviruses showed only small blocks of homology which contrast with the extensive matches among potyviruses. This fact together with the genome organization and the vector specificity clearly distinguishes BaYMV from potyviruses.

The nucleotide sequences of the 5′ and 3′ non-coding regions of pepper mild mottle virus strain S (PMMV-S) RNA were determined; they are more like corresponding sequences of tomato mosaic virus (ToMV) RNA than those of any other tobamovirus reported so far. The 5′ leader contains a 68 nucleotide guanosine-free sequence which differs in several nucleotides from the corresponding sequences in genomic RNA of tobacco mosaic virus (TMV) and ToMV. The messenger activity of PMMV-S RNA in vitro and the polypeptide translation products made were similar to those of TMV RNA. It therefore seems unlikely that qualitative or quantitative differences in translation in vivo account for the milder symptoms induced by PMMV-S, and its lesser replication, than TMV. The 3′ non-coding region of PMMV-S RNA is 199 nucleotides long and can be folded into the same secondary structure as the RNA of other tobamoviruses.

Complementary DNA copies of the genomic RNA of melon necrotic spot virus (MNSV) have been cloned and the region deduced to encode the coat protein has been sequenced. The putative coat protein coding region, located near the 3′ end of the genome, consists of 1170 nucleotides and has the potential to encode a 390 amino acid protein of M r 41840. Our data show that although MNSV is a carmovirus, its coat protein more closely resembles those of the tombusviruses than those of the carmoviruses sequenced to date, in both the extent of sequence similarity and in the length of the random/arm and protruding domains of the coat protein. Furthermore, dot matrix comparisons revealed sequence similarity between the coat protein protruding domains of MNSV and the cucumber necrosis tombusvirus. This similarity may be involved in one or more of the biological properties these two viruses share, such as the ability to infect cucumbers naturally and to be transmitted by the soil-inhabiting fungus Olpidium radicale.

Cowpea mosaic virus (CPMV) M RNA is translated in vitro into two polyproteins of M r values 105 000 (105K) and 95K. Using antiserum against the small capsid protein VP23, these proteins have now been detected in cowpea protoplasts, a few hours after inoculation with CPMV. These proteins could also be detected at later stages of infection, but only when proteolytic processing was inhibited by the addition of ZnCl2. Using antiserum against a synthetic peptide, corresponding to a part of the overlapping C-terminal ends of the 58K and 48K proteins, the 58K protein, which is the amino-terminal cleavage product of the 105K protein, was found in the cytoplasmic fraction of infected protoplasts. The 48K protein, derived from the 95K protein, was detected in both the cytoplasmic and membrane fractions of protoplasts. The presence of the 105K, 95K, 58K and 48K proteins in CPMV-infected protoplasts indicates that separate initiation codons on the M RNA are used in vivo to produce the 105K and 95K polyproteins, as demonstrated in vitro.

Apple chlorotic leaf spot virus (ACLSV) RNA was translated in a rabbit reticulocyte lysate. Two major polypeptides of M r 105000 (105K) and 51 K, and some less prominent polypeptides (88K, 66K, 28K and 23K) were produced. The 23K polypeptide was identified as the coat protein on the basis of its electrophoretic mobility and serological reaction to virus antiserum. Time course and immuno-precipitation experiments suggested that the 23K polypeptide might be generated from the 28K polypeptide by post-translational cleavage.