Genetic variability in the coat protein gene of Potato virus X and the current relationship between phylogenetic placement and resistance groupings

The complete coat protein nucleotide sequences of 11 Potato virus X isolates from Australia and two from Britain were compared to those of 72 others. On phylogenetic analysis, clade I contained all 11 Australian sequences, and sub-clade II-1 contained the two new British sequences. Clade I isolates were from six different continents, but those in sub-clades II-1 and II-2 were only from Europe and the Americas, respectively. Clade I contained isolates in strain groups 1, 3 and 4, and sub-clades II-1 and II-2, isolates in strain groups 2 and 4. Thus, strain group 4 now occurs within both clades.     

Molecular characterization of domestic and exotic potato virus S isolates and a global analysis of genomic sequences

Five potato virus S (PVS) isolates from the USA and three isolates from Chile were characterized based on biological and molecular properties to delineate these PVS isolates into either ordinary (PVS^O) or Andean (PVS^A) strains. Five isolates – 41956, Cosimar, Galaxy, ND2492-2R, and Q1 – were considered ordinary strains, as they induced local lesions on the inoculated leaves of Chenopodium quinoa, whereas the remaining three (FL206-1D, Q3, and Q5) failed to induce symptoms. Considerable variability of symptom expression and severity was observed among these isolates when tested on additional indicator plants and potato cv. Defender. Additionally, all eight isolates were characterized by determining the nucleotide sequences of their coat protein (CP) genes. Based on their biological and genetic properties, the 41956, Cosimar, Galaxy, ND2492-2R, and Q1 isolates were identified as PVS^O. PVS-FL206-1D and the two Chilean isolates (PVS-Q3 and PVS-Q5) could not be identified based on phenotype alone; however, based on sequence comparisons, PVS-FL206-1D was identified as PVS^O, while Q3 and Q5 clustered with known PVS^A strains. C. quinoa may not be a reliable indicator for distinguishing PVS strains. Sequences of the CP gene should be used as an additional criterion for delineating PVS strains. A global genetic analysis of known PVS sequences from GenBank was carried out to investigate nucleotide substitution, population selection, and genetic recombination and to assess the genetic diversity and evolution of PVS. A higher degree of nucleotide diversity (π value) of the CP gene compared to that of the 11K gene suggested greater variation in the CP gene. When comparing PVS^A and PVS^O strains, a higher π value was found for PVS^A. Statistical tests of the neutrality hypothesis indicated a negative selection pressure on both the CP and 11K proteins of PVS^O, whereas a balancing selection pressure was found on PVS^A.     

Analysis of Iranian <Emphasis Type="Italic">Potato virus S</Emphasis> isolates

Two hundred forty potato samples with one or more symptoms of leaf mosaic, distortion, mottling and yellowing were collected between 2005 and 2008 from seven Iranian provinces. Forty-four of these samples tested positive with double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) using a Potato virus S (PVS) polyclonal antibody. Of these 12 isolates of PVS were selected based on the geographical location for biological and molecular characterization. The full coat protein (CP) and 11K genes from 12 PVS isolates were PCR amplified, cloned and sequenced. All 12 PVS isolates showed mosaic symptoms on Nicotiana debneyii and N. tabacum cv. Whiteburly and local lesion on Chenopodium amaranticolor, C. quinoa and C. album. The Iranian isolates share between 93 and 100% pairwise nucleotide identity with other PVS^O isolates. Based on maximum likelihood phylogenetic analysis coupled with pairwise identity analysis, we propose 15 genotypes for the PVS^O strain and 3 genotypes for the PVS^A strain.     

A comparison of the nucleotide sequence homologies between isolates of the Andean and ordinary strains of potato virus S and their relationship to other carlaviruses

Sequence homologies between the RNAs of five isolates of the ordinary strain of potato virus S (PVS^o), four isolates of the Andean strain (PVS^A), potato virus M(PVM), and the type member of the carlavirus group, carnation latent virus (CLV), were compared using cDNA solution hybridization. A high degree of homology (90–100%) was detected between the majority of PVS^o and PVS^A isolates, but lower levels of homology (<10%) were observed between PVS, PVM, and CLV.     

Complete genome sequence of a novel potato virus S strain infecting Solanum phureja in Colombia

Potato virus S (PVS) (genus Carlavirus, family Betaflexiviridae) is one of the most prevalent viruses in potato crops (Solanum tuberosum and S. phureja) around the world, causing reductions in crop yields between 10 and 20 %. Symptoms of PVS infection may include leaf mottling, rugosity of leaves, deepening of the veins and reductions in crop yields between 10 and 20 %. Virions are flexuous rods of 610-710 nm with a positive-sense ssRNA genome of approximately 8500 nt comprising six ORFs, a 5′CAP and a 3′poly-A tail. PVS has been classified into two groups: PVS^O (Ordinary) and PVS^A (Andean). PVSA induces severe symptoms in infected plants, such as premature senescence and defoliation, and is more efficiently transmitted by aphids than PVS^O. To date, only five PVS genomes have been completely sequenced, including those of three PVS^O and two PVS^A strains. Currently, there are no reports of complete PVS genome sequences from Andean South America. In this work, we present the complete genomic sequence of a novel PVS strain infecting S. phureja that is clearly distinct from currently known PVS isolates.     

Variation in the coat protein sequence of British isolates of <Emphasis Type="Italic">Turnip yellow mosaic virus</Emphasis> and comparison with previously published isolates

Summary. Isolates of Turnip yellow mosaic virus (TYMV) were collected from wild cabbage (Brassica oleracea) on a 400 m stretch of Dorset coastline. The coat protein genes of four isolates showed high homology in nucleotide sequence (0.970–1.000, mean 0.987). Lower levels of homology where found to previously published sequences of Australian isolates [10] (0.725–0.775, mean 0.741). The amino acid composition of the Dorset isolates showed high levels of homology (0.964–1.000, mean 0.986). Numerous amino acid substitutions occurred between the Dorset and Australian isolates (0.705–0.819, mean 0.742). Comparison with other isolates showed large genetic distances between the Dorset isolates and both European and Australian isolates.     

Genetic variability of the coat protein sequence of pea seed-borne mosaic virus isolates and the current relationship between phylogenetic placement and resistance groups

Nucleotide sequences of complete or partial coat protein (CP) genes were determined for 11 isolates of pea seed-borne mosaic virus (PSbMV) from Australia and one from China, and compared with known sequences of 20 other isolates. On phylogenetic analysis, the isolates from Australia and China grouped into 2 of 3 clades. Clade A contained three sub-clades (Ai, Aii and Aiii), Australian isolates were in Ai or Aiii, and the Chinese isolate in Aii. Clade A contained isolates in pathotypes P-1, P-2 and U-2; clade B, one isolate in P-2; and clade C, only isolates in P-4.     

Comparison of the coat protein genes of Mirafiori lettuce big-vein virus isolates from Australia with those of isolates from other continents

The complete coat protein nucleotide encoding sequences of 13 Mirafiori lettuce big-vein virus isolates from Australia were compared to those of 23 other isolates, including one from Australia. On phylogenetic analysis, sub-clade A1 contained isolates from Australia (13), Europe and Japan, A2 contained isolates from Australia (1), Europe and South America, and B1 and B2 contained only European isolates. In the amino acid sequences deduced, the N-terminus and central regions varied considerably between clades A and B. Mean Dn/Ds ratios were 0.112, 0.076, 0.187 and 0.063 for all isolates, Australian isolates, clade A isolates and clade B isolates, respectively.     

Incidence of inducible beta-lactamases in gram-negative septicemia isolates from twenty-nine european laboratories

In 1984 the European Study Group on Antibiotic Resistance (ESCAR), which is made up of 29 laboratories in 12 European countries, consecutively collected gram-negative bacilli and staphylococci isolates from blood and using the microdilution method performed susceptibility testing with 11 beta-lactam antibiotics. A total of 2,578 isolates were collected; 68% were gram-negatives and 32% staphylococci.Pseudomonas spp. accounted for 12% of the strains,Enterobacter spp. 7%,Serratia spp. 3%, indole-positiveProteus spp. 1%,Citrobacter spp. andMorganella spp. 0.9% each. Strains with inducible beta-lactamases were detected by the cefoxitin disc diffusion method in 11% of all gram-negatives and in 67% of the relevant species. The production of inducible beta-lactamase was confirmed by elevated MICs to and decreased killing by piperacillin, cefotaxime and ceftazidime after induction of enzyme production with low concentrations of cefoxitin. This phenomenon was not observed with mecillinam or the new penem Sch 34343.     

Zucchini yellow mosaic virus: biological properties, detection procedures and comparison of coat protein gene sequences

Between 2006 and 2010, 5324 samples from at least 34 weed, two cultivated legume and 11 native species were collected from three cucurbit-growing areas in tropical or subtropical Western Australia. Two new alternative hosts of zucchini yellow mosaic virus (ZYMV) were identified, the Australian native cucurbit Cucumis maderaspatanus, and the naturalised legume species Rhyncosia minima. Low-level (0.7%) seed transmission of ZYMV was found in seedlings grown from seed collected from zucchini (Cucurbita pepo) fruit infected with isolate Cvn-1. Seed transmission was absent in >9500 pumpkin (C. maxima and C. moschata) seedlings from fruit infected with isolate Knx-1. Leaf samples from symptomatic cucurbit plants collected from fields in five cucurbit-growing areas in four Australian states were tested for the presence of ZYMV. When 42 complete coat protein (CP) nucleotide (nt) sequences from the new ZYMV isolates obtained were compared to those of 101 complete CP nt sequences from five other continents, phylogenetic analysis of the 143 ZYMV sequences revealed three distinct groups (A, B and C), with four subgroups in A (I-IV) and two in B (I-II). The new Australian sequences grouped according to collection location, fitting within A-I, A-II and B-II. The 16 new sequences from one isolated location in tropical northern Western Australia all grouped into subgroup B-II, which contained no other isolates. In contrast, the three sequences from the Northern Territory fitted into A-II with 94.6-99.0% nt identities with isolates from the United States, Iran, China and Japan. The 23 new sequences from the central west coast and two east coast locations all fitted into A-I, with 95.9-98.9% nt identities to sequences from Europe and Japan. These findings suggest that (i) there have been at least three separate ZYMV introductions into Australia and (ii) there are few changes to local isolate CP sequences following their establishment in remote growing areas. Isolates from A-I and B-II induced chlorotic symptoms in inoculated leaves of Chenopodium quinoa, but an isolate from A-II caused symptomless infection. One of three commercial ZYMV-specific antibodies did not detect all Australian isolates reliably by ELISA. A multiplex real-time PCR using dual-labelled probes was developed, which distinguished between Australian ZYMV isolates belonging to phylogenetic groups A-I, A-II and B-II.     

Genetic characterization of the causative agent of besnoitiosis in goats in Iran on the basis of internal transcribed spacer rDNA and its comparison with <Emphasis Type="Italic">Besnoitia</Emphasis> species of other hosts

This study was conducted to identify genetic characteristics of Besnoitia spp. isolated from goat in Iran. Molecular analysis of two portions of nuclear ribosomal DNA (ITS1 and ITS2) was used for the genetic characterization of the Besnoitia species. Comparison of the sequencing data of the Iranian Besnoitia samples obtained in the present study (GenBank accession number HM008988) with those previously reported for other Besnoitia spp. in the GenBank database revealed a particularly close relationship between the present goat Besnoitia samples and the Besnoitia samples from the cattle, caribou, and equids (Besnoitia besnoiti, Besnoitia tarandi, and Besnoitia bennetti). This is the first use of a genetic approach to interrogate the identity of the species of Besnoitia infecting Iranian goats. Also, the results of the present study showed the occurrence of a similar sequence polymorphism for ITS1 and ITS2 in all Iranian isolates, which exhibit 100% identity in these ribosomal sequences, to those of B. besnoiti previously reported from Israel. Although the ITS1 sequence of Iranian goat isolates is identical to European cow isolates, the ITS2 sequences derived from present Besnoitia genotype differed in one nucleotide position compared with other European B. besnoiti. Further studies should be employed based on this molecular data to identify the natural definitive host in order to complete the life cycle of this distinct genotype of parasite.     

Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.     

Genomovar Diversity Amongst<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis> Complex Isolates From an Australian Adult Cystic Fibrosis Unit

In this study, a combination of recA-based PCR assays and 16S rDNA restriction fragment length polymorphism (RFLP) analysis was used to determine the genomovar diversity of clinical Burkholderia cepacia complex isolates. Twenty-eight isolates were prospectively collected from patients attending a large Australian adult cystic fibrosis (CF) unit, 22 isolates were referred from other Australian CF units and a further eight isolates originated from patients without CF. The 28 prospectively collected isolates were distributed amongst the following genomovars: Burkholderia cepacia genomovar I (28.6%), Burkholderia multivorans (21.4%), Burkholderia cepacia genomovar III (39.3%), Burkholderia vietnamiensis (3.6%) and Burkholderia ambifaria (7.1%). The results of this study highlight the usefulness of 16S rDNA RFLP typing for the identification of other Burkholderia spp. and non-fermenting gram-negative bacteria.     

Indigenous and introduced potyviruses of legumes and <Emphasis Type="Italic">Passiflora</Emphasis> spp. from Australia: biological properties and comparison of coat protein nucleotide sequences

Five Australian potyviruses, passion fruit woodiness virus (PWV), passiflora mosaic virus (PaMV), passiflora virus Y, clitoria chlorosis virus (ClCV) and hardenbergia mosaic virus (HarMV), and two introduced potyviruses, bean common mosaic virus (BCMV) and cowpea aphid-borne mosaic virus (CAbMV), were detected in nine wild or cultivated Passiflora and legume species growing in tropical, subtropical or Mediterranean climatic regions of Western Australia. When ClCV (1), PaMV (1), PaVY (8) and PWV (5) isolates were inoculated to 15 plant species, PWV and two PaVY P. foetida isolates infected P. edulis and P. caerulea readily but legumes only occasionally. Another PaVY P. foetida isolate resembled five PaVY legume isolates in infecting legumes readily but not infecting P. edulis. PaMV resembled PaVY legume isolates in legumes but also infected P. edulis. ClCV did not infect P. edulis or P. caerulea and behaved differently from PaVY legume isolates and PaMV when inoculated to two legume species. When complete coat protein (CP) nucleotide (nt) sequences of 33 new isolates were compared with 41 others, PWV (8), HarMV (4), PaMV (1) and ClCV (1) were within a large group of Australian isolates, while PaVY (14), CAbMV (1) and BCMV (3) isolates were in three other groups. Variation among PWV and PaVY isolates was sufficient for division into four clades each (I-IV). A variable block of 56 amino acid residues at the N-terminal region of the CPs of PaMV and ClCV distinguished them from PWV. Comparison of PWV, PaMV and ClCV CP sequences showed that nt identities were both above and below the 76-77% potyvirus species threshold level. This research gives insights into invasion of new hosts by potyviruses at the natural vegetation and cultivated area interface, and illustrates the potential of indigenous viruses to emerge to infect introduced plants.     

Pathotyping of Australian isolates of Marek's disease virus and association of pathogenicity with meq gene polymorphism

We report the pathotyping of six Australian isolates of Marek's disease virus-1 (MDV1) isolated between 1992 and 2004 and association of virulence with meq gene polymorphism. Unvaccinated and herpesvirus of turkeys (HVT)-vaccinated specific pathogen free chickens were challenged at day 5 with 500 plaque forming units of Marek's disease virus. The isolates induced gross Marek's disease lesions in 53 to 94% of unvaccinated chickens, and HVT induced a protective index ranging from 38 to 100% by 56 days post challenge. This experiment provides evidence that current Australian isolates of MDV1 vary significantly in pathogenicity. However, there was no clear evidence that the most virulent recent isolates were more pathogenic than isolates from the 1980s or that any of the isolates belong to the highest pathotype category of very virulent plus. Evidence is presented that virulence can be predicted by measurements taken as early as 13 days post challenge. The meq gene sequences of five of the isolates used in the experiment were determined. When compared with the very virulent US isolate Md5, there was a 177 base-pair insertion and distinct point mutations in each of the five isolates. There were no individual mutations in the meq sequences that correlated with levels of virulence. However, amino acid alignment of the five Australian and 14 international isolates revealed that the number of repeat sequences of four prolines (PPPP repeats) in the meq gene (overall range 2 to 8) was strongly associated with virulence across all isolates, with the most pathogenic isolates having the fewest number of repeats. The results suggest that the presence of the 177 base-pair insertion alone is not an indicator of attenuation. Rather, the number of PPPP repeats, independent of the presence of the insertion, is a better indicator of pathogenicity.     

Genetic variability in the coat protein genes of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus

Summary. Available data suggests that lettuce big-vein disease is caused by the ophiovirus Mirafiori lettuce big-vein virus (MLBVV) but not by the varicosavirus Lettuce big-vein-associated virus (LBVaV), although the latter is frequently associated with the disease. Since the disease occurs worldwide, the putative coat protein (CP) open reading frames of geographically distinct isolates of MLBVV and LBVaV were sequenced. Comparison of both nucleotide and amino acid sequences showed a high level of sequence similarity among LBVaV isolates. Phylogenetic analysis of LBVaV CP nucleotide sequences showed that most of the Spanish isolates clustered in a phylogenetic group whereas English isolates were more similar to the USA isolate. An Australian isolate was closely related to the Dutch isolate. Genetic diversity among MLBVV CP nucleotide sequences was higher ranging from 0.2% to 12%. Phylogenetic analysis of MLBVV CP nucleotide sequences revealed two distinct subgroups. However, this grouping was not correlated with symptom development on lettuce or the geographic origin of the MLBVV isolates. Finally, a quick method based on RFLP analysis of RT-PCR amplicons was developed for assigning MLBVV isolates to the two subgroups.     

Prevalence and antibiotic sensitivity of Danish versus other European bacterial isolates from intensive care and hematology/oncology units

The prevalence and antibiotic sensitivity patterns of bacteria collected consecutively from medical and surgical intensive care units (ICUs) and from hematology/oncology units in nine hospitals in Denmark were determined and compared to data collected simultaneously in 12 other European countries. Bacterial isolates from 794 Danish patients were tested and compared to 8,625 isolates from European patients. The minimal inhibitory concentrations of eight different antibiotics were determined using a microdilution plate. Similar to findings in European countries, the most common source of bacterial isolates in Danish units was the respiratory tract (49 %), followed by blood (18 %), urinary tract (14 %) and surgical wounds (10 %).Staphylococcus aureus was the most prevalent respiratory organism in Danish units, whereasEnterobacteriaceae andPseudomonas aeruginosa dominated in other countries. In blood,Escherichia coli was most prevalent in Denmark while coagulase-negative staphylococci were predominant in other countries. Urinary tract isolates were dominated byEscherichia coli in both Denmark and the other countries, butEnterococcus faecalis andPseudomonas aeruginosa were more frequently isolated in the other countries.Staphylococcus aureus was the most frequent wound isolate in Denmark, whileEnterobacteriaceae other thanEscherichia coli dominated in other European countries. Thus, in DenmarkEscherichia coli andStaphylococcus aureus, followed byPseudomonas aeruginosa andKlebsiella spp. (from ICUs) orEnterococcus spp. andKlebsiella spp. (from hematology/oncology units), are the most prominent pathogens in these units today. Indicator organisms of antibotic consumption (Pseudomonas aeruginosa and methicillin-resistant coagulase-negative staphylococci andStaphylococcus aureus) were more frequent in other European countries than Denmark. In general, the Danish isolates were more sensitive to antibiotics than the European isolates. Thus, the sensitivity of all organisms to third-generation cephalosporins was about 10 % higher in Denmark as compared to Europe, and for many frequent pathogens, the sensitivity matched the fourth-generation cephalosporin, cefpirome. A lower total antibiotic consumption and the infrequent use of broad-spectrum cephalosporins in Denmark may contribute to these differences.     

Identification of new isolates of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> that cluster with less common viral strains

Summary Turnip mosaic virus (TuMV) was found infecting cultivated brassicas and wild and cultivated ornamental Brassicaceae plants in different regions of Spain. Five new TuMV isolates, originating from different host plant species (Brassica cretica, Brassica juncea, Brassica napus, Eruca vesicaria subsp. sativa and Sisymbrium orientale), have been identified. The nucleotide sequences of the coat protein (CP) genes of the five isolates were determined. Phylogenetic analysis of the CP sequences showed that the five isolates grouped into two different clusters. The three isolates from the central region of Spain clustered with a previously reported Pisum sativum isolate from southeastern Spain, whereas the other two isolates from the eastern region clustered with two Italian and two Greek isolates. Both clusters were genetically distinct and belonged to the multi-lineage group OBR. The OBR group contains mainly TuMV isolates from hosts other than Brassica spp. and Raphanus sativus and mostly originating from Mediterranean countries. These new sequences provide further phylogenetic resolution of the OBR group. Although new TuMV isolates have been found in Spain, they were not associated with any serious disease outbreaks.     

Multilocus phylogenetic analysis of <Emphasis Type="Italic">Cryptosporidium andersoni</Emphasis> (Apicomplexa) isolated from a bactrian camel (<Emphasis Type="Italic">Camelus bactrianus</Emphasis>) in China

This is the first report of cryptosporidiosis in a bactrian camel (Camelus bactrianus) in China. Two Cryptosporidium isolates derived from the same bactrian camel (3-year-old) in November 2005 and April 2006 were characterized using sequence and phylogenetic analysis of the small-subunit rRNA (18S rRNA), 70-kDa heat shock protein (HSP70), actin and Cryptosporidium oocyst wall protein (COWP) genes. The sequences of the 18S rRNA and COWP were identical to all other Cryptosporidium andersoni isolates although minor differences were noticed between the isolates and the USA isolate at the actin locus (99.2% of similarity). The sequence of the HSP70 was identical to the Japanese C. andersoni isolate, with a minor difference from the Australian C. andersoni isolate (99.7% of similarity). Cross-transmission studies demonstrated that the C. andersoni isolates did not infect immunosuppressed or immunocompetent Kun-ming mice, severe combined immunodeficiency mice, and immunosuppressed or immunocompetent calves. Among the C. andersoni isolates reported so far, only isolates from Japan could infect SCID mice. Thus, the C. andersoni isolates from the bactrian camel were biologically similar to most bovine C. andersoni isolates characterized so far, but are different from bovine isolates from Japan.     

Comparison of the coat protein genes of Lettuce big-vein associated virus isolates from Australia with those of isolates from other continents

The complete coat protein (CP) nucleotide sequences of seven Lettuce big-vein associated virus (LBVaV) isolates from Australia were compared to those of 22 other LBVaV and five tobacco stunt virus (TStV) isolates. On phylogenetic analysis, clade I contained only LBVaV isolates from Europe, sub-clade IIa only Australian LBVaV isolates, IIb only Japanese LBVaV isolates, and IIc only TStV isolates from Japan. In the amino acid sequences deduced, the central region of the gene was most divergent. Mean Dn/Ds ratios were 0.283 and 0.124 for clades I and II, respectively. The suggestion that TStV is a strain of LBVaV was supported.