Molecular cloning and characterisation of the griffon vulture (Gyps fulvus) toll-like receptor 1

The toll-like receptor (TLR) family is an ancient pattern recognition receptor family, conserved from insects to mammals. Members of the TLR family are vital to immune function through the sensing of pathogenic agents and initiation of an appropriate immune response. In this study, we cloned a cDNA encoding for a griffon vulture (Gyps fulvus) orthologue of mammalian TLR1 (CD281). The predicted 650 amino acid sequence comprised an extracellular domain with five leucine-rich repeats (LRR) and an LRR-C-terminal (LRR-CT) motif, followed by a 23 amino acid transmembrane segment, and a 190 amino acid intracytoplasmic region containing the Toll/IL-1R (TIR) domain. Vulture TLR1 and TIR domain showed 64% and 86% amino acid sequence similarity with chicken sequences. The tissue and cell expression pattern of vulture TLR1 were analysed by real time-PCR (RT-PCR) and correlated with the ability to respond to various pathogenic challenges. Despite the similarities in the overall structure and expression pattern of vulture TLR1 with other vertebrate TLRs, the length of the vulture TLR ectodomain, number and position of LRRs and N-glycosylation sites suggest structural differences that may have functional implications.     

Identification of a novel cofilin-related molecule (Der f 31) as an allergen from Dermatophagoides farinae

House dust mite (HDM) allergen is a major cause of allergic disease. In this study, two-dimensional immunoblot and Matrix-Assisted Laser Desorption Ionization tandem Time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify Der f 31. After Der f 31 was cloned, expressed and purified, skin prick test (SPT), Immune inhibitory assays, Western blot, ELISA and asthmatic mouse model were employed to examine the allergenicity of recombinant Der f 31. The gene of Der f 31 includes 447 bps, and encoded 148 amino acids. Positive responses of SPT to r-Der f 31 were 32.5% in 43 HDM-allergic patients. r-Der f 31 can induce allergic pulmonary inflammation in the mouse model. In conclusion, Der f 31 is a novel subtype of dust mite allergens.     

Identification and purification of a novel fish allergen from largemouth bass (Micropterus salmoides)

The prevalence of fish allergies has become a serious health problem and has increased alarmingly over the past few years. To contain this problem, we would need to identify all commonly consumed fish allergens. To date, however, no report has identified largemouth bass allergens. This study attempted to identify and purify the major allergen implicated in the allergic response to largemouth bass (Micropterus salmoides), a freshwater fish widely consumed in China. Fifteen patients with a positive history of type I allergy to fish were recruited from skin-prick test and the allergy screen. Total protein extracts and purified allergenic protein from bass were tested for their immunoglobulin E–binding properties. Immunoblot assay resulted in strong reactivity with the 17-kDa protein in all patients. In summary, nucleoside diphosphate kinase B was identified as a novel fish allergen in largemouth bass. This finding is important for allergy diagnoses and the treatment of freshwater fish–allergic disorders.     

Identification of a novel HLA-B*55 variant (B*5513) from a Korean family

The novel allele, B*5513, was identified from three siblings of a Korean family. Direct DNA sequencing analysis revealed that B*5513 differed from B*5502 by a single-nucleotide substitution at codon 116 (TTA-->TTC) resulting in an amino acid change from leucine to phenylalanine. This substitution altered serologic reactivity of the B55 molecule to B22-specific alloantisera.     

Identification of a novel Babesia sp. from a sable antelope (Hippotragus niger Harris, 1838)

Babesiosis in a sable antelope (Hippotragus niger Harris, 1838) was first reported in 1930; the parasite was named Babesia irvinesmithi. Recently, specimens from an adult sable that presented with a sudden onset of disease and that subsequently died during immobilization were submitted for molecular characterization. Microscopic examination of thin blood smears revealed the presence of small piroplasms. DNA was extracted from blood samples; the V4 variable region of the 18S rRNA gene was amplified and analyzed using the reverse line blot (RLB) assay. Amplicons did not hybridize with any of the Babesia or Theileria species-specific probes present on the blot and hybridized only with a Babesia or Theileria genus-specific probe, suggesting the presence of a novel species. The full-length 18S rRNA gene sequence was obtained and aligned with published sequences of related genera, and phylogenetic trees were constructed. Sequence similarity analyses indicated that a Babesia species, designated Babesia sp. (sable), was present. The sequence showed its highest similarity to B. orientalis and to an unnamed Babesia species previously detected in bovine samples. The latter was later established to be Babesia occultans. A Babesia sp. (sable)-specific RLB oligonucleotide probe was designed and used to screen 200 South African sable samples, but so far, no other sample has been found to be positive for the presence of Babesia sp. (sable) DNA. In summary, we identified a novel piroplasm parasite from a sable antelope that died from an unknown illness. While the parasite was observed in blood smears, there is no direct evidence that it was the cause of death.     

Identification and characterization of a novel tospovirus species using a new RT-PCR approach

Summary.  A novel tospovirus serologically distinct from all established tospo- virus species was found in Thailand in Physalis minima L. The S RNA of this virus was cloned by a new RT-PCR approach revealing a nucleotide sequence of 3257 nucleotides. The ambisense RNA segment encoded a nonstructural protein (NSs) of 469 amino acids, with a predicted Mr of 53.2 kDa, and a nucleoprotein (N) of 279 amino acids and a Mr of 31.0 kDa, so far the largest N protein known for any tospovirus species. N protein sequence comparisons revealed closest relationship to the species Watermelon bud necrosis virus (58% identity), Watermelon silver mottle virus and Peanut bud necrosis virus (57%) and a distant relationship to Peanut yellow spot virus (23%) and Peanut chlorotic fanspot virus (22%). Received March 27, 2000 Accepted July 27, 2000     

Identification of a novel NOG gene mutation (P35S) in an Italian family with symphalangism

Symphalangism (SYM or SYM1) is an autosomal dominant disorder characterized by multiple joint fusions. The disease is caused by mutations of the NOG gene, that maps to chromosome 17q22. So far, only six independent NOG mutations have been identified. We have analysed an Italian family in which father and son had bilateral symphalangism and detected a novel NOG mutation (P35S), originated in the father from a c.914C>T transition. A different mutation in the same codon (P35R) has been previously described. Comparison between different noggin gene hortologs shows that codon 35 is conserved. Therefore, this codon should play an important role in NOG gene function. This is the first mutation described for NOG after the initial report of NOG mutations being causative of SYM.     

Identification of a novel Brevibacterium species isolated from humans and description of Brevibacterium sanguinis sp. nov

Six coryneforms isolated from blood and dialysate fluid were phenotypically similar to Brevibacterium casei, but 16S rRNA gene sequencing and DNA-DNA hybridization indicate that they belong to a new species for which the name Brevibacterium sanguinis is proposed.     

A novel species of mastrevirus (family Geminiviridae) isolated from Digitaria didactyla grass from Australia

Mastreviruses (family Geminiviridae) that infect monocotyledonous plants occur throughout the temperate and tropical regions of Asia, Africa, Europe and Australia. Despite the identification of a very diverse array of mastrevirus species whose members infect African monocots, few such species have been discovered in other parts of the world. For example, the sequence of only a single monocot-infecting mastrevirus, Chloris striate mosaic virus (CSMV), has been reported so far from Australia, even though earlier biological and serological studies suggested that other distinct mastreviruses were present. Here, we have obtained the complete nucleotide sequence of a virus from the grass Digitaria didactyla originating from Australia. Analysis of the sequence shows the virus to be a typical mastrevirus, with four open reading frames, two in each orientation, separated by two non-coding intergenic regions. Although it showed the highest levels of sequence identity to CSMV (68.7%), their sequences are sufficiently diverse for the virus to be considered a member of a new species in the genus Mastrevirus, based on the present species demarcation criteria. We propose that the name first used during the 1980s be used for this species, Digitaria didactyla striate mosaic virus (DDSMV).     

Identification of a novel subtype of South River virus (family Bunyaviridae)

We previously reported the isolation of South River virus (SORV) from a pool of mosquitoes collected in the Yucatan Peninsula of Mexico (Farfan-Ale et al. in Vector Borne Zoonotic Dis 10:777-783, 5). The isolate (designated SORV-252) was identified as SORV after a 197-nucleotide region of its small RNA genome segment was sequenced. In the present study, the complete small and medium RNA genome segments and part of the large RNA genome segment of SORV-252 were sequenced and shown to have 92%, 85% and 90% nucleotide sequence identity, respectively, to the homologous regions of the prototype SORV isolate (NJO-94F). To determine the antigenic relationship between SORV-252 and NJO-94F, cross-plaque reduction neutralization tests (PRNTs) were performed using sera from mice inoculated with these viruses. SORV-252 and NJO-94F were distinguishable in the cross-neutralization assays; there was a twofold difference in the PRNT titers in one direction and a fourfold difference in the other direction, suggesting that SORV-252 represents a novel subtype of SORV. Additionally, SORV-252 and NJO-94F have distinct plaque morphologies in African green monkey kidney (Vero) cells. In conclusion, we provide evidence that a novel subtype of SORV is present in the Yucatan Peninsula of Mexico.     

Identification of two novel membrane proteins from the Tiger frog virus (TFV)

The Tiger frog virus (TFV) belongs to the genus Ranavirus in the family Iridoviridae, and its genome was completely sequenced in 2002. In order to better understand the viral structure and functional genes involved in infection and virus-host interactions, two candidate genes, ORF001L and ORF020R, were selected for our study. ORF001L and ORF020R were analyzed by genomic comparison and by using the TMHMM software. Both genes were conserved in the genus Ranavirus, may encode putative membrane proteins, and were determined as late genes by temporal analysis. In order to identify whether these two proteins were structural proteins or not, ORF001L and ORF020R were cloned and expressed in the pET32a (+) vector. Antisera against the two proteins were prepared by immunization of mice with purified proteins. Western blot analysis suggested that both ORF001L and ORF020R were structural proteins. Indirect immunofluorescence assay (IFA) revealed that the subcellular location of the two proteins was confined to the cytoplasm, especially at the viral assembly site (AS). Immunogold electron microscopy (IEM) further localized these two proteins, showing that they were envelope proteins.     

Identification and in silico analysis of novel von Hippel-Lindau (VHL) gene variants from a large population

Mutational inactivation of the VHL gene is the cause of von Hippel-Lindau (VHL) disease, an autosomal dominant hereditary cancer syndrome predisposing to haemangioblastomas, pheochromocytomas and clear-cell renal carcinomas. The gene product (pVHL) functions as an adapter in cellular processes including cell growth and apoptosis. VHL mutation analysis was carried out in 426 unrelated subjects with phenotypes ranging from VHL syndrome, to isolated VHL-related tumours that could represent the first manifestation of the disease. A total of 111 individuals were found to carry alterations, with large deletions representing 40% of the variants. Eighteen of the 95 detected variants were novel, seemingly disease-causing mutations; their pathogenic role has been evaluated in silico for effects on protein folding and interactions. Putative regions of interaction between pVHL and proteins involved in common pathways have been identified, assessing possible implications for the presence of mutations in these regions. All new variants predicted to truncate or cause complete pVHL loss of structure were associated with phenotypes consistent with VHL type 1. Seven of the new amino acid substitutions are disease-causing mutations, one is a neutral variant, whereas the results for two remain ambiguous. Our combined molecular and in silico approach for the evaluation of putative disease-causing mutations contributes to the interpretation of the potential pathogenicity of these novel variants.     

Isolation of mycoplasma spp. from racing pigeons (Columba livia)

Live and dead racing pigeons (Columba livia) from five lofts in Norfolk and Suffolk were examined clinically and cultured for Mycoplasma spp. Both clinically healthy birds and those showing signs of mild respiratory disease were included. The oropharynx was the culture site for 130 live birds, the nasal sinuses and other tissues for 58 carcases. Mycoplasma columbinum, M. columborale and M. columbinasale were isolated from the oropharynges and nasal sinuses; M. columbinum and M. columbinasale from the brain and M. columbinum and M. columborale from lungs and air sacs. One or more of these three Mycoplasma spp. was isolated at necropsy from 28% of 58 pigeons. Only 11% of 37 pigeons reacted sero-logically by the metabolism inhibition test to M. columbinum and none to M. columborale. Twenty-five birds examined for M. gallisepticum antibody by the haemagglutination-inhibition test were negative. No sex or age predilection to infection with Mycoplasma was apparent. About 10% of pigeons in all five lofts showed clinical signs of the respiratory disease sometimes described as 'mycoplasmosis catarrh', but most dead birds from which Mycoplasma spp. were isolated also had concomitant infections of various kinds. Although suggestive, the results of these investigations provide no clear evidence that Mycoplasma spp. are aetiologically involved in natural respiratory disease of pigeons. No conclusive satisfactory treatment was found for the elimination of mycoplasmas.     

Molecular cloning of a novel human papillomarvirus (type 58) from an invasive cervical carcinoma

A novel human papillomavirus type (HPV) was cloned from an invasive cervical carcinoma. The viral clone showed no homology with other known prototypes of HPV (HPV-1 through HPV-57), except HPV-33 by Southern blot analysis under stringent conditions. It showed less than 20% homology to HPV-33 by reassociation kinetic analysis. The restriction endonuclease map of the clone was different from those of other HPV types and its predicted genome organization surmised by hybridization with subgenomic fragment probes of HPV-33 DNA showed the typical HPV genome organization. The results indicate that this clone is a new type of HPV, designated as HPV-58, distinct from the other known types of HPV. HPV-58 was detected in none of 6 specimens of cervical condylomata acuminata, in 7 of 58 specimens of cervical intraepithelial neoplasia, and in 4 of 50 specimens of invasive cervical carcinoma studied in Nagano prefecture, Japan.