Entire Genomic Sequence of Novel Canine Papillomavirus Type 13

Papillomaviruses are associated with benign and malignant neoplasias of the skin and mucous membranes. The sequence of a novel canine papillomavirus was determined from DNA detected in the oral cavity of a dog. The sequence of the novel virus canine papillomavirus type 13 (CPV13) shares the highest levels of similarity with the Tau papillomaviruses CPV2 and CPV7.

Identification and genomic characterization of a novel human torque teno virus of 3.2 kb

In the process of searching for the recently described small anelloviruses 1 and 2 (SAVs) with the genomic DNA length of 2.2 or 2.6 kb in human sera, we isolated a novel virus with its genomic organization resembling those of torque teno virus (TTV) of 3.8–3.9 kb and torque teno mini virus (TTMV) of 2.8–2.9 kb. The entire genomic sequence of three isolates (MD1-032, MD1-073 and MD2-013), which comprised 3242–3253 bases and exhibited 76–99 % identities with the SAVs within the overlapping sequence, was determined. Although the MD1-032, MD1-073 and MD2-013 isolates differed by 10–28 % from each other over the entire genome, they segregated into the same cluster and were phylogenetically distinguishable from all reported TTVs and TTMVs. These results suggest that SAVs are deletion mutants of the novel virus with intermediate genomic length between those of TTV and TTMV and that the novel virus can be classified into a third group of the genus Anellovirus.

High Throughput Methods for Gene Identification, Cloning and Functional Genomics Using the GeneTAC™ G3 Robotics Workstation

Using a single robotic platform, the GeneTAC™ G3, we have automated most of the processes involved in the cloning and characterisation of novel disease causing genes by addressing the following; firstly, identifying the BACs of interest and making shotgun libraries. Secondly, automating the set up of sequencing reactions using methodology that eliminates the need for DNA preparation of 384 clones. Thirdly, generating sub-libraries using selective re-arraying of library clones to enable the determination of the entire genomic sequence of the gene. Fourthly, determining gene function by combination of differential screening and mini Northerns using microarrays printed using the GeneTAC™ G3 system and hybridised using the GeneTAC™ HybStation (Genomics Solutions, Ann Arbor, USA).

Genomic and evolutionary characterization of TT virus (TTV) in tupaias and comparison with species-specific TTVs in humans and non-human primates

TT virus (TTV) was recovered from the sera of tupaias (Tupaia belangeri chinensis) by PCR using primers derived from the noncoding region of the human TTV genome, and its entire genomic sequence was determined. One tupaia TTV isolate (Tbc-TTV14) consisted of only 2199 nucleotides (nt) and had three open reading frames (ORFs), spanning 1506 nt (ORF1), 177 nt (ORF2) and 642 nt (ORF3), which were in the same orientation as the ORFs of the human prototype TTV (TA278). ORF3 was presumed to arise from a splicing of TTV mRNA, similar to reported human TTVs whose spliced mRNAs have been identified, and encoded a joint protein of 214 amino acids with a Ser-, Lys- and Arg-rich sequence at the C terminus. Tbc-TTV14 was less than 50% similar to previously reported TTVs of 3·4–3·9 kb and TTV-like mini viruses (TLMVs) of 2·8–3·0 kb isolated from humans and non-human primates, and known animal circoviruses. Although Tbc-TTV14 has a genomic length similar to animal circoviruses (1·8–2·3 kb), Tbc-TTV14 resembled TTVs and TLMVs with regard to putative genomic organization and transcription profile. Conserved motifs were commonly observed in the coding and noncoding regions of the Tbc-TTV14 genome and in all TTV and TLMV genomes. Phylogenetic analysis revealed that Tbc-TTV14 is the closest to TLMVs, and is closer to TTVs isolated from tamarin and douroucouli than to TTVs isolated from humans and chimpanzees. These results indicate that tupaias are naturally infected with a new TTV species that has not been identified among primates.

Nucleotide Sequence and RT-PCR Detection of a Virus Associated with Grapevine Rupestris Stem-Pitting Disease

Grapevine rupestris stem pitting (RSP) is a graft-transmissible disease of unknown etiology. We have characterized a virus associated with this disease. The entire genomic sequence (GenBank accession number AF026278) consisted of 8,725 nucleotides excluding a poly(A) tail. Six open reading frames (ORF) were found. ORF1 potentially encodes a polypeptide with a methyltransferase domain, a papain-like proteinase domain, a helicase domain, and a RNA-dependent RNA polymerase domain; ORF2, ORF3, and ORF4 compose a triple-gene block; ORF5 encodes a coat protein; and ORF6 is located near the 3′ end with unknown function. Sequence analysis indicated that the virus is most similar to apple stem-pitting virus and may be allied with the carla- and potexviruses and grouped with other viruses that infect woody hosts. A specific reverse-transcription polymerase chain reaction (RT-PCR)-based detection method was developed. Among 62 grapevine sources known to be infected with rupestris stem-pitting disease, 60 sources tested positive by RT-PCR. Among 43 healthy vines tested, all were negative. The name grapevine rupestris stem-pitting-associated virus is proposed.