Comparative restriction endonuclease analysis of varicella-zoster virus clinical isolates

The DNAs of 67 isolates of varicella-zoster virus (VZV) obtained from 31 individuals were compared by restriction endonuclease analysis using BamHI, EcoRI, PstI and SmaI. All of the epidemiologically unrelated 26 isolates could be differentiated using SmaI and another one or two enzymes. However, the DNA cleavage profiles of multiple VZV isolates from the same patient and the isolates from a group of patients who were infected with VZV from the same source were found to be identical to each other, as reported previously. No patients were found who were simultaneously infected with different VZV strains. Moreover, VZV showed no change in DNA fragment profiles after serial passages not only through human embryonic lung cells but also through patients.     

Comparative complete genome analysis of Indian type A foot-and-mouth disease virus field isolates

Comparative complete genome analysis of 17 serotype A Indian field isolates representing different genotypes and sub-lineages is presented in this report. Overall 79% of amino acids were invariant in the coding region. Chunk deletion of nucleotide was observed in S and L fragment of 5′-UTR. More variability which is comparable to that of capsid coding region was found in L and 3A region. Functional motifs and residues critical for virus biology were conserved most. Polyprotein cleavage sites accepted few changes. Many sites were detected to be under positive selection in L, P1, 2C, 3A, 3C, and 3D region and of which some are functionally important and antigenically critical. Genotype/lineage specific signature residues could be identified which implies evolution under different selection pressure. Transmembrane domain could be predicted in 2B, 2C, 3A, and 3C proteins in agreement with their membrane binding properties. Phylogenetic analysis at complete coding region placed the isolates in genotype IV, VI, and VII and two broad clusters comprising VP3⁵⁹-deletion and non-deletion group within genotypes VII. The VP3⁵⁹-deletion group has diversified genetically with time giving rise to three lineages. Incongruence in tree topology observed for different non structural protein coding region and UTRs-based phylogeny indicate suspected recombination.     

Species identification and subtyping of Ureaplasma parvum and Ureaplasma urealyticum using PCR-based assays

There is good evidence that the organism currently known as Ureaplasma urealyticum should be divided into two species-U. parvum (previously U. urealyticum biovar 1) and U. urealyticum (previously U. urealyticum biovar 2). In this study, we designed a series of primers, targeting the 16S rRNA gene and 16S rRNA-23S rRNA intergenic spacer regions, the urease gene subunits, and the 5' ends of the multiple-banded antigen (MBA) genes, to identify and subtype these Ureaplasma species. All of the species-specific primer pairs could distinguish the two species, but only subtype-specific primer pairs targeting the MBA genes could distinguish subtypes within each species. U. parvum was separated into three subtypes, represented by serovars 1, 3/14, and 6. U. urealyticum was also separated into three subtypes by PCR and/or direct sequencing. Subtype 1 consisted of serovars 2, 5, 8, and 9; subtype 2 contained serovars 4, 10, 12, and 13; and subtype 3 contained serovars 7 and 11. A selection of primer pairs was used to identify and subtype 78 clinical ureaplasma isolates from vaginal swabs of pregnant women and to identify and subtype ureaplasmas directly in 185 vaginal swabs in which they had been previously detected. U. parvum was identified in 228 (87%) of 263 isolates or specimens, and U. urealyticum was identified in 50 (19%) (both were present in 6%). Serovars 3/14 (48%) and 1 (43%) were most common among U. parvum isolates, and subtypes 2 (62%) and 1 (34%) were most common among U. urealyticum isolates. This new PCR-based typing system will facilitate future studies of the relationship between individual Ureaplasma species or subtypes and human disease.     

Phase variation of the multiple banded protein in Ureaplasma urealyticum and Ureaplasma parvum

Ureaplasma urealyticum and U. parvum are common commensals and, possibly, pathogens of the human urogenital tract. Like other Mycoplasmatales they possess variable surface proteins. The multiple banded (MB) protein shows a striking variability of its molecular weight. This is caused by changes of the number of C-terminal repeating units. In this study, selective pressure was imposed against cytadherence of U. urealyticum and U. parvum. Ureaplasmas were co-incubated with either erythrocytes or HeLa cells and the cell-bound fraction was removed. Additionally, U. urealyticum populations were transferred serially through broth containing specific polyclonal antibodies. Both approaches led to the emergence of escape variants in which no MB protein was detectable. PCR studies with several primers on different parts of the mba gene indicated major differences between wild-type strains and MB-negative escape variants. In experiments with clonal lineages, however, the loss of the MB protein was shown to be reversible. Therefore, it is proposed that the multiple banded proteins of U. urealyticum and U. parvum are subjected to a phase-switching mechanism as it has already been described for several other Mycoplasmatales.     

Ureaplasma parvum Prosthetic Joint Infection Detected by PCR

We describe the first reported case of Ureaplasma parvum prosthetic joint infection (PJI) detected by PCR. Ureaplasma species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material). U. parvum is a relatively new species name for certain serovars of Ureaplasma urealyticum, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient''s failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected U. parvum in all samples. U. parvum-specific PCR testing was performed on all synovial fluid samples to confirm the U. parvum detection.     

Comparative analysis of genome sequences of three isolates of Orf virus reveals unexpected sequence variation

Orf virus (ORFV) is the type species of the Parapoxvirus genus. Here, we present the genomic sequence of the most well studied ORFV isolate, strain NZ2. The NZ2 genome is 138 kbp and contains 132 putative genes, 88 of which are present in all analyzed chordopoxviruses. Comparison of the NZ2 genome with the genomes of 2 other fully sequenced isolates of ORFV revealed that all 3 genomes carry each of the 132 genes, but there are substantial sequence variations between isolates in a significant number of genes, including 9 with inter-isolate amino acid sequence identity of only 38-79%. Each genome has an average of 64% G+C but each has a distinctive pattern of substantial deviation from the average within particular regions of the genome. The same pattern of variation was also seen in the genome of another parapoxvirus species and was clearly unlike the uniform patterns of G+C content seen in all other genera of chordopoxviruses. The availability of genomic sequences of three orf virus isolates allowed us to more accurately assess likely coding regions and thereby revise published data for 24 genes and to predict two previously unrecognized genes.     

Ureaplasma parvum as a Cause of Sternal Wound Infection

Few reports in the literature have documented the isolation of Ureaplasma species from sternal wounds. A case of sternal wound infection likely due to Ureaplasma parvum is described. When routine bacterial cultures from a sternal wound infection fail to yield a pathogen, diagnostic testing for mycoplasmas and ureaplasmas should be considered.     

Simple method for determining biovar and serovar types of Ureaplasma urealyticum clinical isolates using PCR-single-strand conformation polymorphism analysis

Ureaplasma urealyticum has been associated with urethritis in men, obstetric problems in women, and respiratory distress syndrome in preterm infants. U. urealyticum can be divided into two biovars comprising 14 serovars. Partial sequences of genes encoding the multiple-banded antigens of the cell surface are known. Using a commercially available precast DNA mutation detection gel system, we have developed a simple and reproducible PCR-single-strand conformation polymorphism analysis method for differentiating the biovars of this species that reveals five patterns among the 14 serovars and enables clinical isolates to be typed directly from broth cultures.     

Fluoroquinolone resistance in Ureaplasma parvum in the United States

We report the first case of naturally occurring fluoroquinolone resistance in Ureaplasma spp. from the United States. Resistance in this case probably developed as a result of mutations in the gyrA and parC genes of the DNA gyrase/topoisomerase IV complex that occurred in the presence of antimicrobial selective pressure.     

Occurrence of Ureaplasma parvum and Ureaplasma urealyticum in Women with Cervical Dysplasia in Katowice, Poland

The aim of this study was to evaluate the occurrence of genital mycoplasmas, especially Ureaplasma parvum and Ureaplasma urealyticum, in women with atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade squamous intraepithelial lesions (HSIL), compared to women with normal cytology living in Katowice, Poland. Two sterile swabs were used to obtain material from the posterior vaginal fornix of 143 women with squamous intraepithelial lesions and 39 healthy women: first for general bacteriology, second for detection of urogenital mycoplasmas using Mycoplasma IST2 kit. From each positive Mycoplasma IST2 culture DNA was isolated and PCR was performed for identification of U. parvum and U. urealyticum. Mycoplasma IST was positive in 34.1% cases. Urogenital mycoplasmas were demonstrated in women with HSIL significantly more often compared to women with LSIL, ASCUS, and with normal cytology. DNA of U. parvum was demonstrated in majority of Mycoplasma IST2-positive cases, U. urealyticum DNA-only in 9 (4.9%). Predominance of 3/14 serovars of U. parvum was demonstrated. U. urealyticum biovar 2 was present more often in women with squamous intraepithelial lesions.     

A quantitative analysis of Ureaplasma urealyticum and Ureaplasma parvum compared with host immune response in preterm neonates at risk of developing bronchopulmonary dysplasia

Multiplex, real-time PCR for the identification of Ureaplasma urealyticum and Ureaplasma parvum was performed on nucleic acids extracted from sequential endotracheal aspirates obtained from preterm neonates born at <29 weeks of gestation and ventilated for more than 48 h admitted to two level 3 neonatal intensive care units. Specimens were obtained shortly after birth and sequentially up until extubation. One hundred fifty-two specimens (93.8%) contained material suitable for analysis. Ureaplasma spp. were identified in 5 of 13 neonates studied. In most cases, the DNA load of the detected Ureaplasma species was low and decreased over time. In addition, changes in detectable Ureaplasma species DNA did not relate to changes in the inflammatory marker C-reactive protein (CRP) or respiratory status. All but two blood samples obtained at times of suspected sepsis were culture positive for other microorganisms; the species cultured were typically coagulase-negative staphylococci and were associated with increased levels of CRP (>10 mg/liter). This study was limited by the small number of patients examined and does not have the power to support or contradict the hypothesis that postnatal lung infection with Ureaplasma parvum is causally related to bronchopulmonary dysplasia (BPD) or adverse respiratory outcomes after preterm birth. However, in this study, increases in CRP levels were not associated with patients in whom Ureaplasma parvum was detected, in contrast to the detection of other bacterial species.     

Modified indirect immunofluorescence test for serotyping large numbers of Ureaplasma urealyticum clinical isolates.

A technical modification of the indirect immunofluorescence test for serotyping Ureaplasma urealyticum clinical isolates is described. The use of a tissue culture plate filled with ureaplasma agar made the serotyping easier to perform and proved to be very handy, especially for performance of large series.     

Preliminary profile of the Cryptosporidium parvum genome: an expressed sequence tag and genome survey sequence analysis

Cryptosporidium parvum is a protozoan enteropathogen that infects humans and animals and causes a pronounced diarrheal disease that can be life-threatening in immunocompromised hosts. No specific chemo- or immunotherapies exist to treat cryptosporidiosis and little molecular information is available to guide development of such therapies. To accelerate gene discovery and identify genes encoding potential drug and vaccine targets we constructed sporozoite cDNA and genomic DNA sequencing libraries from the Iowa isolate of C. parvum and determined approximately 2000 sequence tags by single-pass sequencing of random clones. Together, the 567 expressed sequence tags (ESTs) and 1507 genome survey sequences (GSSs) totaled one megabase (1 mb) of unique genomic sequence indicating that approximately 10% of the 10.4 mb C. parvum genome has been sequence tagged in this gene discovery expedition. The tags were used to search the public nucleic acid and protein databases via BLAST analyses, and 180 ESTs (32%) and 277 GSSs (18%) exhibited similarity with database sequences at smallest sum probabilities P(N)< or =10(-8). Some tags encoded proteins with clear therapeutic potential including S-adenosylhomocysteine hydrolase, histone deacetylase, polyketide/fatty-acid synthases, various cyclophilins, thrombospondin-related cysteine-rich protein and ATP-binding-cassette transporters. Several anonymous ESTs encoded proteins predicted to contain signal peptides or multiple transmembrane spanning segments suggesting they were destined for membrane-bound compartments, the cell surface or extracellular secretion. One-hundred four simple sequence repeats were identified within the nonredundant sequence tag collection with (TAA)(> or =6)/(TTA)(> or =6) and (TA)(> or = 10)/(AT)(> or =10 ) being the most prevalent, occurring 40 and 15 times, respectively. Various cellular RNAs and their genes were also identified including the small and large ribosomal RNAs, five tRNAs, the U2 small nuclear RNA, and the small and large virus-like, double-stranded RNAs. This investigation has demonstrated that survey sequencing is an efficient procedure for gene discovery and genome characterization and has identified and sequence tagged many C. parvum genes encoding potential therapeutic targets.     

Development of monoclonal antibodies against Ureaplasma urealyticum serotypes and their use for serotyping clinical isolates

Monoclonal antibodies (MAbs) against Ureaplasma urealyticum serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of U. urealyticum strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.     

Chlamydia trachomatis: genome sequence analysis of lymphogranuloma venereum isolates

Chlamydia trachomatis is the most common cause of sexually transmitted infections in the UK, a statistic that is also reflected globally. There are three biovariants of C. trachomatis: trachoma (serotypes A-C) and two sexually transmitted pathovars; serotypes D-K and lymphogranuloma venereum (LGV). Trachoma isolates and the sexually transmitted serotypes D-K are noninvasive, whereas the LGV strains are invasive, causing a disseminating infection of the local draining lymph nodes. Genome sequences are available for single isolates from the trachoma (serotype A) and sexually transmitted (serotype D) biotypes. We sequenced two isolates from the remaining biotype, LGV, a long-term laboratory passaged strain and the recent "epidemic" LGV isolate-causing proctitis. Although the genome of the LGV strain shows no additional genes that could account for the differences in disease outcome, we found evidence of functional gene loss and identified regions of heightened sequence variation that have previously been shown to be important sites for interstrain recombination. We have used new sequencing technologies to show that the recent clinical LGV isolate causing proctitis is unlikely to be a newly emerged strain but is most probably an old strain with relatively new clinical manifestations.     

Comparative full genome analysis revealed E1: A226V shift in 2007 Indian Chikungunya virus isolates

The resurgence of Chikungunya virus (CHIKV) in the form of unprecedented explosive epidemic after a gap of 32 years in India is a point of major public health concern. In 2007 again there was outbreak in Kerala, India, affecting more than 25,000 cases with many reported mortalities. To understand the molecular basis of this high virulence and its implication in large-scale epidemic, a detailed systematic serological, virological and molecular investigation was undertaken with the epidemic samples of Kerala-2007. The comparative analysis of full genome sequence of Chikungunya virus isolate of 2007 with 2006 revealed three unique substitutions in structural and non-structural genes of 2007 isolate [two in E1 region (V14A and A226V) and one in Nsp1 (M184T)]. Our finding further substantiates the association of A226V shift in E1 gene with evolutionary success possibly due to adaptation in the mosquito vector with progression of epidemic, as observed in Reunion Island. This A226V shift which was absent in all 2006 Indian isolates, is found to be present in the four 2007 isolates, analysed in this study. These unique molecular features of the 2007 isolates with the progression of the epidemic from 2005 to 2007 demonstrate their high evolutionary and epidemic potential and thereby suggesting possible implication in higher magnitude and virulence of this outbreak.     

Multilocus analysis of Cryptosporidium hominis and Cryptosporidium parvum isolates from sporadic and outbreak-related human cases and C. parvum isolates from sporadic livestock cases in the United Kingdom

Cryptosporidium parvum and Cryptosporidium hominis isolates from sporadic, drinking water-associated, and intrafamilial human cases together with C. parvum isolates from sporadic cases in livestock were collected in the United Kingdom between 1995 and 1999. The isolates were characterized by analysis of three microsatellite markers (ML1, GP15, and MS5) using PCR amplification. Within C. hominis, four alleles were detected within the GP15 and MS5 loci, and a single type was detected with ML1. C. parvum was more polymorphic; 12 alleles were detected with GP15, 6 were detected with MS5, and 3 were detected with ML1. Multilocus analysis of polymorphisms within the three microsatellite loci was combined with those reported previously for an extrachromosomal small double-stranded RNA. Forty multilocus types were detected within these two species: 9 were detected in C. hominis, and 31 were detected in C. parvum. In C. hominis, heterogeneity was almost exclusively found in samples from sporadic cases. Similarity analysis identified three main groups within C. parvum, and the group that predominated in human infection was also found in livestock. Multilocus types of C. parvum previously identified only in humans were not detected in livestock. Isolates of both C. hominis and C. parvum from separate waterborne outbreaks were genetically homogeneous, suggesting preferential or point source transmission of certain types of these two species of parasites.     

Comparative analysis of trichomonad genome sizes and karyotypes

In parasitic protists, the genome sizes range from 2.9Mb in Encephalitozoon cuniculi to about 160Mb in Trichomonas vaginalis. The suprisingly large genome size of the former human parasite resulted from the expansion of various repetitive elements, specific gene families, and possibly from large-scale genome duplication. The reason for this phenomenon, as well as whether other trichomonad species have undergone a similar genome expansion, is not known. In this work we studied the genomes of nine selected species of the Trichomonadea group. We found that each species has a characteristic karyotype with a stable and haploid number of chromosomes. Relatively large genome sizes were found in all the tested species, although over a rather broad range (86-177Mb). The largest genomes were typically observed in the Trichomonas and Tritrichomonas genera (133-177Mb), while Tetratrichomonas gallinarum contains the smallest genome (86Mb). The genome size correlated with the cell volume, however, no relationship between genome size and the site of infection or trichomonad phagocytic ability was observed. The data presented here provide primary information towards selecting a trichomonad species for future large-scale sequencing to elucidate the evolution of unusual parabasalid genomes.     

The first complete genome sequences of clinical isolates of human coronavirus 229E

Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to insufficient pressure to maintain gene integrity of non-essential genes. We present here the first full-length genome sequence of two clinical isolates. Each encoded gene was compared to Inf-1. In general, little sequence changes were noted, most could be attributed to genetic drift, since the clinical isolates originate from 2009 to 2010 and VR740 from 1962. Hot spots of substitutions were situated in the S1 region of the Spike, the nucleocapsid gene, and the non-structural protein 3 gene, whereas several deletions were detected in the 3??UTR. Most notable was the difference in genome organization: instead of an ORF4A and ORF4B, an intact ORF4 was present in clinical isolates.