Genomic characterization of a poxvirus isolated from a child

A poxvirus was isolated from a six-year-old girl. The comparative analyses of the genome of this isolate (H-CP-LSax) which were carried out using the restriction endonucleases BamHI, HindIII, KpnI, MluI, NcoI, SacI, and SmaI revealed that this isolate is a member of the genus orthopoxvirus. Since the girl had never been vaccinated against smallpox, and had close contact to domestic animals, including cats, rabbits and guinea pigs, the genome of H-CP-LSax virus was genetically analysed in comparison with other known orthopoxviruses. The analysis demonstrates clearly that the HindIII clevage pattern of H-CP-LSax DNA is different from the HindIII DNA cleavage patterns of vaccinia virus, cowpox virus, rabbit poxvirus, cat poxvirus, ectromelia virus, and okapi poxvirus. Surprisingly, it was found that the HindIII and SmaI cleavage patterns of the DNA of one out of six elephant poxviruses which were analysed under the same conditions were virtually identical to the HindIII and SmaI cleavage patterns of H-CP-LSax DNA. Although SmaI and HindIII digestion of both virus genomes gave the same fragment patterns, the viral DNAs can be distinguished from each other by the restriction endonucleases SacI, BamHI, and KpnI, which also show high similarities in the fragmentation patterns of both viruses. The results obtained in this study indicate three possibilities concerning the origin of H-CP-LSax virus. Firstly that the H-CP-LSax virus originated from an unknown animal species. Secondly, that this virus is a variant of elephant poxvirus in which the HindIII and SmaI sites are extremely conserved, and finally that H-CP-LSax can be a recombinant virus of unknown origin.     

Genomic characterization of pestiviruses isolated from lambs and kids in southern Italy

A nested polymerase chain reaction was used to identify 13 pestivirus strains isolated from small ruminants in several mixed (sheep and goats) flocks of Southern Italy, and for classification as bovine viral diarrhoea virus (BVDV) type 1, BVDV type 2, and Border disease virus (BDV) genotypes. Of the nine ovine isolates, two were characterized as BVDV type 1, and seven as BVDV type 2. The four pestiviruses isolated from kids belong to BVDV type 1. None of the pestivirus strains tested could be classified as 'true' BDV (genotype 3). Although BVDV type 2 has been described in Europe rarely, the characterization of BD/90-1M strain as BVDV type 2, isolated in Italy in 1990, demonstrates that this genotype has been circulating in Italy since the 1990s.     

Molecular characterization of Bacillus thuringiensis isolated from diverse habitats of India

Bacillus thuringiensis (Bt) strains were isolated from 94 samples from different geographical regions. Novel types of crystalline inclusion bodies were observed from some of the isolates. Crystalline inclusions of bipyramidal, spherical and cuboidal morphology were found produced by most of the isolates. Isolate GS12 showed crystal on one side of spore while isolate GM108 formed crystals on both termini of spore. Isolate GN31 produced large sized bipyramidal crystals. SDS-PAGE analysis of the spore crystal suspension showed major protein bands in the range of 29 and 140 kDa. Two new serovars of Bt viz. GS4 and GN24 having H3abce and H3ab serotype respectively were isolated. Toxicity comparable to the reference strain Bacillus thuringiensis subs. kurstaki (Btk) HD-1 was observed for the isolates GM20, GM17 and MP3 against larvae of Helicoverpa armigera. Some of the isolates harboring cry genes like cry1Ac and cry2 did not show any toxicity towards H. armigera while most of the isolates were harboring cry1, cry1Ac and cry2 gene.     

Biological and genomic characterization of two newly isolated Elizabethkingia anophelis bacteriophages

BackgroundElizabethkingia anophelis is an opportunistic pathogen that infects newborns and immunocompromised patients. Because the infection is associated with high mortality as a result of its intrinsic resistance to antibiotics, alternative treatment methods are needed. Our previous study successfully isolated the world's first E. anophelis phage, TCUEAP1, which showed beneficial protection to E. anophelis-infected mice. More new bacteriophages are needed in order to provide sufficient choices to combat E. anophelis infections.MethodsIn the current study, two new phages infecting E. anophelis were isolated from wastewater and were designated as TCUEAP2 and TCUEAP3. Further experiments, namely, transmission electron microscopy (TEM), infection assay, host-range analysis, and sequencing were performed to determine their biological and genomic characteristics.ResultsTEM analysis revealed that both TCUEAP2 and TCUEAP3 possess an icosahedral head with a non-contractile tail, and belong to the Siphoviridae family. Further experiments revealed that TCUEAP3 has a longer latent period and higher burst size compared to TCUEAP2. Host range analysis showed that both TCUEAP2 and TCUEAP3 have a narrow host range, infecting only their respective hosts. The genomic size of phage TCUEAP2 was 42,403 bps containing 61 predicted open reading frames (ORFs), whereas the genome size of TCUEAP3 was 37,073 bps containing 40 predicted ORFs.ConclusionDue to the distinct biological characteristics of TCUEAP2 and TCUEAP3, they may be satisfactory for clinical uses such as preparation of phage cocktails or decontamination in clinical settings.     

Salmonella typhimurium isolated from bacteraemia.

During a two year period, a total of 15 strains of S. typhimurium were isolated and analysed by phage typing. Of these, 13 were found untypable, while two strains belonged to phage 76 and 22. All the strains were sensitive to Gentamicin and Cephaloridine. All but one showed multiple drug resistance.     

Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China

The complete genomic sequence of Newcastle disease virus (NDV) strain NDV08-004, isolated from domestic ducks in China, was determined in this study. The genome is 15198 nucleotides (nt) in length, follows the “rule of six” and contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. Compared with the full genome sequences of Class II NDV strains, the NDV08-004 isolate has a 12-nt insertion (TGGGAGACGGGG) in the phosphoprotein gene between nucleotides 2381 and 2382 of the genome (numbered according to the genomic sequence of the La Sota strain, which consists of 15186 nt). Strain NDV08-004 has the motif ¹¹²E-Q-Q-E-R-L¹¹⁷ at the cleavage site of the fusion protein, which is typical of lentogenic NDV strains, and this is in agreement with the results of pathogenic tests based on the mean death time (MDT) and the intracerebral pathogenicity index (ICPI). Phylogenetic analysis based on the full genome revealed that all the NDV strains studied could be divided into two distinct clades, namely class I and class II, and the NDV08-004 isolate characterized in this study was grouped in class I. Further phylogenetic analysis based on a 374-bp fragment of the F gene in class I strains of NDV demonstrated that NDV08-004 belongs to genotype 3, and should be therefore similar to strains obtained from live bird markets in Hong Kong in recent years.     

Antibiotic sensitivity of E. coli and Salmonella isolated from different water sources in Kashmir, India

The objective of the present study was to determine the prevalence of Salmonella and Escherichia coli, the serotypes involved and the antibiogram of the various isolates obtained from different sources of water supply (streams, Dal Lake, tube wells and community supply water) in Kashmir, India, during the years 2009–2010. A total of 100 samples, 25 from each source, were taken for the present study. All samples were evaluated for the presence of Salmonella and E. coli, stereotyped and tested for antimicrobial susceptibility. A total of 60 isolates of E. coli and 12 isolates of Salmonella were obtained. The antimicrobial susceptibilities of different isolates of E. coli and Salmonella from different water sources were examined by disc diffusion method. The high in vitro sensitivity was shown by 100 % of E. coli isolates for ciprofloxacin, 95 % for amoxycillin/clavulanic acid, 93.33 % for each of cephotaxime and amikacin and 91.67 % for lavofloxacin and gentamicin. The isolates registered an intermediate response to rifampicin (85.00 %), lomefloxacin (83.33 %) and norfloxacin (83.33 %) and showed resistance to erythromycin (98.33 %). The isolates of Salmonella were 100 % sensitive for gentamicin, 91.67 % for each of cefixime, ciprofloxacin, amikacin and lavofloxacin; 83.33 % for each of cephotaxime and amoxycillin/clavulanic acid and showed resistance to erythromycin (100 %), nalidixic acid (100 %) and rifampicin (91.67 %). The isolates, however, showed an intermediate resistance to lomefloxacin (83.34 %) and norfloxacin (66.67). The high incidence of multi-drug-resistant strains of E. coli and Salmonella is due to injudicious use of antibiotics and exchange of antibiotic resistance among bacterial populations.     

Prevalence and characterization of plasmid-mediated quinolone resistance genes in Salmonella isolated from poultry in Korea

The purpose of this study was to investigate the prevalence and characteristics of plasmid-mediated quinolone resistance (PMQR) genes qnr, aac(6′)-Ib-cr, and qepA in a total of 185 non-duplicate Salmonella spp. isolated from hatcheries, poultry farms, and poultry slaughterhouses during the period 2001 to 2010 in Korea. Additionally, mutation analysis of quinolone resistance determining regions (QRDRs), conjugation experiments, and plasmid analysis were performed in the PMQR-positive isolates. Among the 185 isolates, six (3.2%) contained qnr genes (two qnrB4 and four qnrS1) but none carried the aac(6′)-Ib-cr or qepA genes. Among the six PMQR-positive isolates, one showed a single mutation (Ser83-Phe substitution) in the QRDRs of gyrA. Among them, three were non-susceptible (intermediate or resistant) to nalidixic acid (minimum inhibitory concentration [MIC] ≥256 µg/ml), ciprofloxacin (MIC 2 µg/ml), and levofloxacin (MIC 4 µg/ml), but others were susceptible to all of the three fluoroquinolones. They were resistant to six or more antimicrobial agents tested and were able to transfer quinolone resistance to recipient Escherichia coli J53 by conjugation. By performing a hybridization test, plasmids harbouring qnrB4 and qnrS1 genes were less than 8 kb and about 70 kb in size, respectively. The horizontal dissemination of qnrS1 gene was mediated by IncN plasmid. Compared with the recipient strain, MICs of the transconjugants increased two-fold to four-fold for nalidixic acid, and eight-fold to 16-fold for ciprofloxacin and levofloxacin. This report is the first to describe the detection of qnr genes in Salmonella spp. isolated from poultry in Korea. Widespread horizontal transfer of these genes among bacteria may be a serious public health concern because these can rapidly increase fluoroquinolone resistance. To ensure the public health, it is essential to continuously survey and carefully monitor the spread of PMQR genes in Salmonella from poultry.     

Genomic characterization of an enterovirus 97 strain isolated in Shandong,China

The genomic characterization of human enterovirus 97 (EV97) strain isolated from an acute flaccid paralysis case in Shandong province, China in 1999, is described. The strain, designated as 99188/SD/CHN/1999/EV97 (abbreviated as 99188), had a genome of 7394 nucleotides. Compared with other EV97 strains, it had 81.3–83.3% nucleotide similarity and 94.0–95.4% amino acid similarity in VP1 coding region, and it had 81.4% complete genomic similarity with prototype strain BAN99-10355. The most striking feature was the deletion of 18 nucleotides in the 3′ end of VP1 coding region, combined with two deletions and one insertion in 5′ and 3′ untranslated regions. All these findings demonstrated the strain 99188 had a distant genetic relationship with other EV97 strains. In the phylogenetic trees generated from VP1 and 3D sequences of human enterovirus species B (HEV-B), the lineages of strain 99188 were not congruent, suggesting the event of recombination. Similarity plot analysis further provided the evidence of recombination with other strains of HEV-B in P2 and P3 coding region. This is the first finding of EV97 in China and the third genomic sequence of EV97 reported.     

Isolation and characterization of Thermus bacteriophages

Summary. One-hundred-fifteen bacteriophage strains were isolated from alkaline hot springs in Iceland, New Zealand, Russia (Kamchatka), and the U.S.A. The phages belonged to the Myoviridae, Siphoviridae, Tectiviridae, and Inoviridae families. Over 50% of isolates were isometric or filamentous. One type of siphovirus had giant tails of over 800 nm in length. Phages were further characterized by host range, genome size, DNA restriction endonuclease digestion patterns, and temperature and pH sensitivity. Myoviruses and tectiviruses had a worldwide distribution. Most phages were narrowly host-specific and all were highly resistant against heating and alkaline and acidic pH. This is the first time that tectiviruses and filamentous phages are reported for bacteria of the Thermus-Deinococcus phylum. The presence of tectiviruses, inoviruses, and myoviruses is attributed to acquisition from ancestral γ-proteobacteria by horizontal gene transfer.     

Genomic characterization and integrative properties of phiSMA6 and phiSMA7, two novel filamentous bacteriophages of Stenotrophomonas maltophilia

Two novel filamentous phages, phiSMA6 and phiSMA7, were isolated from Stenotrophomonas maltophilia environmental strain Khak84. We identified and annotated 11 potential open reading frames in each phage. While the overall layout of the functional gene groups of both phages was similar to that of the known filamentous phages, they differed from them in their molecular structure. The genome of phiSMA6 is a mosaic that evolved by acquiring genes from at least three different filamentous S. maltophilia phages and one Xanthomonas campestris phage related to Cf1. In the phiSMA6 genome, a gene similar to the bacterial gene encoding the mating pair formation protein trbP was also found. We showed that phiSMA6 possesses lysogenic properties and upon induction produces high-titer lysates. The genome of phiSMA7 possesses a unique structure and was found to be closely related to a prophage present in the chromosome of the completely sequenced S. maltophilia clinical strain D457. We suggest that the other three filamentous phages of S. maltophilia described previously also have the capacity to integrate into the genome of their bacterial host.     

Genomic and proteinic characterization of strain S, a rickettsia isolated from Rhipicephalus sanguineus ticks in Armenia.

Strain S, a spotted fever group (SFG) rickettsia isolated from Rhipicephalus sanguineus ticks collected in Armenia, was identified. Microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis and Western immunoblotting, PCR and then restriction fragment length polymorphism analysis, pulsed-field gel electrophoresis, and 16S rRNA gene sequencing were used to compare strain S with reference isolates. Strain S was found to possess proteinic, antigenic, and genomic patterns which were unique among SFG rickettsiae. Strain S is characterized by its high degree of pathogenicity for experimental animals, but its role as a potential human pathogen should be determined. The role of R. sanguineus ticks in the epidemiology of SFG rickettsiae is discussed.     

Genomic sequence analysis of Helicoverpa armigera nucleopolyhedrovirus isolated from Australia

The complete genomic sequence of Helicoverpa armigera nucleopolyhedrovirus from Australia, HearNPV-Au, was determined and analyzed. The HearNPV-Au genome was 130,992 bp in size with a G + C content of 39 mol% and contained 134 predicted open reading frames (ORFs) consisting of more than 150 nucleotides. HearNPV-Au shared 94 ORFs with AcMNPV, HearSNPV-G4 and SeMNPV, and was most closely related to HearSNPV-G4. The nucleotide sequence identity between HearNPV-Au and HearSNPV-G4 genome was 99 %. The major differences were found in homologous regions (hrs) and baculovirus repeat ORFs (bro) genes. Five hrs and two bro genes were identified in the HearNPV-Au genome. All of the 134 ORFs identified in HearNPV-Au were also found in HearSNPV-G4, except the homologue of ORF59 (bro) in HearSNPV-G4. The sequence data strongly suggested that HearNPV-Au and HearSNPV-G4 belong to the same virus species.     

Erratum to: Genomic characterization of the first class I Newcastle disease virus isolated from the mainland of China

The complete genomic sequence of Newcastle disease virus (NDV) strain NDV08-004, isolated from domestic ducks in China, was determined in this study. The genome is 15198 nucleotides (nt) in length, follows the “rule of six” and contains a 55-nt leader sequence at the 3′ end and a 114-nt trailer sequence at the 5′ end. Compared with the full genome sequences of Class II NDV strains, the NDV08-004 isolate has a 12-nt insertion (TGGGAGACGGGG) in the phosphoprotein gene between nucleotides 2381 and 2382 of the genome (numbered according to the genomic sequence of the La Sota strain, which consists of 15186 nt). Strain NDV08-004 has the motif ¹¹²E-Q-Q-E-R-L¹¹⁷ at the cleavage site of the fusion protein, which is typical of lentogenic NDV strains, and this is in agreement with the results of pathogenic tests based on the mean death time (MDT) and the intracerebral pathogenicity index (ICPI). Phylogenetic analysis based on the full genome revealed that all the NDV strains studied could be divided into two distinct clades, namely class I and class II, and the NDV08-004 isolate characterized in this study was grouped in class I. Further phylogenetic analysis based on a 374-bp fragment of the F gene in class I strains of NDV demonstrated that NDV08-004 belongs to genotype 3, and should be therefore similar to strains obtained from live bird markets in Hong Kong in recent years.     

Genomic characterization of hepatitis C virus isolates from Argentina

Thirty-three Argentinian patients infected with hepatitis C virus (HCV) were studied for viral genotyping. The patients included 10 hemophiliac and 4 polytransfused children and 19 adults: 3 polytransfused, 7 dialyzed and 9 sporadic cases. Core-based genotyping permitted the classification of 31 samples. Genotypes II, I and V were the most frequent: 21 (63.6%), 16 (48.4%) and 10 (30.3%) of the 33 patients, respectively. Only one polytransfused patient carried genotype IV. Genotype II was detected in 7 out of 9 sporadic cases. Thirteen patients (39.3%) were coinfected with two genotypes, and 2 others were coinfected with three genotypes. The remaining 2 samples which could not be typed were characterized following the restriction fragment length polymorphism (RFLP) method, and were classified as type 1. One of these had two consecutive transitional mutations in the 5′ untranslated region (5′ UTR). © 1995 Wiley-Liss, Inc.     

Molecular characterization of an enterotoxin from Salmonella typhimurium

In this study, we describe the molecular and antigenic characteristics of a cloned enterotoxin from Salmonella typhimurium strain Q1. The full length Salmonella enterotoxin gene (stn), localized on a 2.8 kb Cla I/PstI DNA fragment, was cloned from a genomic library of Salmonella . Based on nucleotide sequence analysis, the stn gene contained 749 bp that would encode a protein having a molecular size of 29 073. The most unusual feature of the stn gene was the presence of a rare initiation codon (TTG) in lieu of the typical ATG codon, which required site-directed mutagenesis to confirm the precise initiation site. The expression of the stn gene in a bacteriophage T7 RNA polymerase/promoter system was enhanced by introducing a typical ATG start codon and an optimal Shine-Dalgarno sequence upstream of the stn gene by site-directed mutagenesis. The stn gene was located opposite the hydHG operon that regulates labile hydrogenase activity in Salmonella species and Escherichia coli . The overall amino acid sequence of the enterotoxin was quite dissimilar to any other published sequence, including cholera toxin or other adenylate cyclase-activating proteins. However, an intriguing similarity in a small region of the amino acid sequence of Stn was observed with portions of the amino acid sequences from several other protein toxins known to ADP-ribosylate host cell proteins. This region of homology may indicate a conserved motif, within the active site, that is involved in the stimulation of adenylate cyclase activity.     

Genomic characterization of a novel group A lamb rotavirus isolated in Zaragoza, Spain

An ovine rotavirus (OVR) strain, 762, was isolated from a 30-day-old lamb affected with severe gastroenteritis, in Zaragoza, Spain, and the VP4, VP7, VP6, NSP4, and NSP5/NSP6 genes were subsequently characterized molecularly. Strain OVR762 was classified as a P[14] rotavirus, as the VP4 and VP8* trypsin-cleavage product of the VP4 protein revealed the highest amino acid (aa) identity (94% and 97%, respectively) with that of the P11[14] human rotavirus (HRV) strain PA169, isolated in Italy. Analysis of the VP7 gene product revealed that OVR762 possessed G8 serotype specificity, a type common in ruminants, with the highest degree of aa identity (95-98%) shared with serotype G8 HRV, bovine rotavirus, and guanaco (Lama guanicoe) rotavirus strains. Moreover, strain OVR762 displayed a bovine-like NSP4 (genotype E2) and NSP5/NSP6 (genotype H3), and a VP6 genotype I2, as well as a long electropherotype pattern. This is the first report of a lamb rotavirus with P[14] and G8 specificities, providing additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.