A2 Isolates Research Articles

Expansion and Evolution of Streptococcus pneumoniae Serotype 19A ST320 Clone as Compared to Its Ancestral Clone, Taiwan19F-14 (ST236)

The Streptococcus pneumoniae serotype 19A sequence type (ST) 320 clone, derived from an international Taiwan(19F)-14 (ST236) clone, has become prevalent in many countries. The dynamics of invasive pneumococcal disease (IPD) were determined using the database of the National Notifiable Disease Surveillance System in Taiwan. The virulence of 19A ST320 and Taiwan(19F)-14 (ST236) were assessed in mice. By constructing an isogenic serotype 19F variant of the 19A ST320 strain (19F ST320), we analyzed the role of capsular type and genetic background on the difference in virulence between 19A ST320 and Taiwan(19F)-14 (ST236). Between 2008 and 2011, IPD due to serotype 19A increased from 2.1 to 10.2 cases per 100 000 population (P < .001); IPD due to any serotype also significantly increased (P = .01). Most serotype 19A isolates belonged to ST320. Using competition experiments in a murine model of colonization, we demonstrated that 19A ST320 outcompeted Taiwan(19F)-14 (ST236; competitive index, 20.3; P = .001). 19F ST320 was 2-fold less competitive than the 19A ST320 parent (competitive index, 0.47; P = .04) but remained 14-fold more competitive than Taiwan(19F)-14 (ST236; competitive index, 14.7; P < .001). Genetic evolution of pneumococcal clones from Taiwan(19F)-14 (ST236) to 19A ST320 has made this pneumococcus better able to colonize of the nasopharynx. This evolution reflects not only a switch in capsular serotype but also changes in other loci.

HOST RANGE VARIATION AND GENETIC DIVERSITY OF IRANIAN ISOLATES OF RALSTONIA SOLANACEARUM FROM POTATO AND TOMATO WITH RAPD AND (GTG)5-PCR

Of 170 Iranian isolates of Ralstonia solanacearum from wilting potato and tomato plants, 130 were ana- lyzed for genetic and pathogenicity diversity. Specific PCR assays identified all the isolates as R. solanacearum biovar N2 (2T) and 2A. Based on pathogenicity and PCR tests, the isolates were divided into two pathogenic groups. Group I, corresponding to race 3, was patho- genic to potato and tomato, whereas group II, corre- sponding to race 1, was pathogenic to tomato, potato, eggplant and tobacco. Molecular analysis was done with random amplified polymorphic DNA (RAPD) and rep- PCR using (GTG)5 primer. RAPD and (GTG)5-PCR analysis divided the 130 Iranian isolates of R. solanacearum into eight haplotypes that were grouped in three clusters. Cluster I comprised all biovar 2A isolates with four haplotypes, cluster II and III contained biovar N2 isolates with four haplotypes. Both RAPD and (GTG)5-PCR enabled discrimination of R. solanacearum strains according to biovar and the host of origin.

First Report on the Occurrence of A2 Mating Type of the Cucurbit Downy Mildew Agent Pseudoperonospora cubensis in China.

Pseudoperonospora cubensis is a foliar pathogen of cucurbits. In cucumber, it produces chlorotic, angular lesions with dark sporangia on their under-surface. The distribution of pathotypes and mating types of P. cubensis were investigated in seven provinces in China. Twenty-nine isolates were obtained from Guangdong, Anhui, Hubei, Sichuan, Shandong, Beijing, and Harbin, one to six isolates from each province. They were collected from cucumber during summer 2011, except those from Harbin, which were collected from cucumber in summer 2012. Isolates were tested for pathogenicity and mating type. Pathogenicity was tested by inoculation of detached leaves of: cucumber (Cucumis sativum cvs. Bet-Alpha, SMR18), melon (Cucumis melo reticulatus cv. Ananas-Yokneam), pumpkin (Cucurbita maxima cv. Tripoli), squash (Cucurbita pepo, cv. Beruti), butternut gourd (Cucurbita moschata cv. Waltham), watermelon (Citrullus lanatus, cv. Malali), and sponge gourd (Luffa cylindrica, cultivar unknown). Mating type was determined by oospore production in melon leaf discs inoculated with mixed sporangia of a test isolate and A1 or A2 tester isolates, as recently described (2). The results showed that 22 isolates belonged to pathotype 3, sporulating on cucumber and melon; five isolates belonged to pathotype 6, sporulating on cucumber, melon, pumpkin, squash, and butternut gourd; one isolate belonged to pathotype 5, sporulating on the above five species and watermelon (1), and one isolate was capable of sporulating on the mentioned five species and sponge gourd, herein called pathotype 7. Of the 29 isolates tested, 22 belonged to the A1 mating type, six to the A2 mating type, and one did not produce oospores with either testers. Of the six A2 isolates, two originated from Sichuan, one from Beijing, and three from Harbin. All 22 isolates belonging to pathotype 3 were A1, whereas the other six isolates (four pathotype 6, one pathotype 5, and one pathotype 7) were A2. Crosses made between Chinese isolates belonging to opposite mating types resulted in the formation of abundant oospores in detached melon or cucumber leaves. The results prove that the A2 mating type of P. cubensis occurs in China. This explains the abundant occurrence of oospores of P. cubensis in nature in China (4). The data corroborate with Runge et al. (3), who suggested that the recent changes in the population structure of P. cubensis around the world resulted from the migration of a new genotype of P. cubensis from the Far East to Europe and the U.S.A. In Israel, a new pathotype attacking both Cucumis and Cucurbita appeared in 2002 (1), and A2 mating type first appeared in 2010 (2).

Pneumococcal Serotypes Causing Acute Otitis Media Among Children in Barcelona (1992–2011)

There is scarce information about changes in serotypes and clonal types of Streptococcus pneumoniae causing acute otitis media (AOM) in recent years, particularly in European countries. Pneumococcal serotypes and clones from S. pneumoniae strains isolated from children with AOM who were attended at Hospital Sant Joan de Déu, Barcelona (1992 to 2011), were studied. Heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in June 2001. We defined 3 periods: prevaccine period 1992 to 2001, early vaccine period 2002 to 2006 and late vaccine period 2007 to 2011. There were 376 pneumococcal strains causing AOM, and 373 (99.2%) of them were serotyped. AOM caused by PCV7 serotypes declined significantly: 161 of 245 (65.7%) episodes in 1992 to 2001 versus 22 of 67 (32.8%) in 2002 to 2006 versus 8 of 61 (13.1%) in 2007 to 2011 P < 0.001. In the last period (2007 to 2011), the potential serotype coverage for the PCV10 was 16.4% and for the PCV13 was 68.9% (P < 0.001). Serotype 19A increased from 5.7% in 1992 to 2001 to 42.6% in 2007 to 2011 (P < 0.001). Among strains with penicillin minimal inhibitory concentration ≥0.12 μg/mL (n = 241), serotype 19A rose from 2.3% in the first period to 57.9 % in the last period (P < 0.001). The clonal-type ST320 was initially detected in 2005, and in the period 2007 to 2011, the ST320 was found in 72.7% of nonsusceptible serotype 19A isolates. Among children with AOM, a rapid expansion of the multiresistant clone ST320 expressing serotype 19A has been observed in Barcelona. The implementation of PCV13, which includes this serotype, may decrease the prevalence of AOM and reduce antimicrobial resistance.

Prevalence, characterization and antimicrobial susceptibility of Salmonella enterica and Yersinia enterocolitica in pigs at slaughter in Italy

In 2005–2008, 1152 samples (451 faecal samples, 451 carcass swabs and 250 tonsils) were collected from 451 finishing pigs slaughtered in three abattoirs of northern Italy. In two abattoirs, 34 scalding water samples were collected. The aim of this study was to investigate the faecal and palatine tonsil carriage rate of Salmonella enterica and Yersinia enterocolitica in pigs at slaughter and the degree of carcass contamination by these bacteria. Typing of the isolates, virulence characterization and antimicrobial testing were also performed.S. enterica was isolated from 21.5% of the faecal samples, 10.9% of the carcasses and 10.4% of the tonsils, but not from scalding water. Nineteen different serovars were identified among 172 S. enterica isolates. The prevalent serovars were Derby (41.3%), Rissen (12.2%), Typhimurium (11%), 4,[5],12:i:— (8.7%) and Give (4.1%). S. enterica ser. Typhimurium and S. enterica ser. 4,[5],12:i:— isolates were phage-typed and PT DT120 was the most common (23.5%). Y. enterocolitica was detected in 17.1% of the faecal samples, 2.4% of the carcasses, 10.8% of the tonsils and 11.8% of the scalding water samples. A total of 119 isolates were found, four of them in water. Of the 115 Y. enterocolitica isolates of pig origin, 24 (20.9%) were 4/O:3 and 4 (3.5%) were 2/O:9. Y. enterocolitica 4/O:3 represented 85.7% of the pathogenic isolates found in all types of samples and 100% of those found in tonsils. In 4/O:3 isolates the most common virulence-associated genes were ystA (100%), inv (95.8%), ail (87.5%) and yadA (54.2%). In 2/O:9 isolates the prevalent genes were ail (100%), inv (100%) and ystA (100%), followed by ystB (25.0%). The majority (75.7%) of Y. enterocolitica isolates was biotype 1A, belonging to 13 serotypes (O:3; O:5; O:4,32-4,33; O:6,30-6,31; O:7,8-8; O:7,8-8-8,19; O:7,13; O:8; O:9; O:13; O:16-16,29; O:41,42-41,43; O:52). The most common virulence genes in 1A isolates were inv (95.4%) and ystB (72.4%).The antimicrobial resistance test showed that all Salmonella isolates were susceptible to cefotaxime, ciprofloxacin, cefalothin, gentamicin and enrofloxacin. Resistances to tetracycline (56%), sulphonamide compounds (42%) and streptomycin (34%) were the most common. All Y. enterocolitica isolates were susceptible to ciprofloxacin, ceftazidime, cefotaxime, chloramphenicol, enrofloxacin, gentamicin, kanamicin and neomycin. Most isolates were resistant to cefalothin (92%) and ampicillin (89%).Apparently, carcass contamination by S. enterica and Y. enterocolitica was more likely attributable to cross-contamination than to self-contamination, suggesting that good hygienic measures and slaughtering procedures can control transmission of these pathogens to pork meat.

The presence of resistance mutations to protease and polymerase inhibitors in Hepatitis C virus sequences from the Los Alamos databank

Several new direct-acting antiviral (DAA) drugs are in development for chronic hepatitis C viral (HCV) infection, and NS3-NS4A serine protease and the NS5B RNA-dependent RNA polymerase have been the major targets. HCV variants displaying drug-resistant phenotypes have been observed both in vitro and during clinical trials. Our aim was to characterize amino acid changes at positions previously associated with resistance in protease (NS3) and polymerase (NS5B) regions from treatment-naïve HCV patients infected with genotypes 1a, 1b and 3a. All 1383 NS3 protease sequences (genotype 1a=680, 1b=498 and 3a=205) and 806 NS5B polymerase sequences (genotypes 1a=471, 1b=329, 3a=6) were collected from Los Alamos databank. Genotype 3a protease sequences showed the typical low-level resistance mutation V36L. NS3 sequences from other genotypes presented mutations on positions 36, 39, 41, 43, 54, 80, 109, 155 and 168 in a frequency lower than 2%, except for the mutation Q80R found in 35% of genotype 1a isolates. Polymerase sequences from genotype 3a patients showed five typical mutations: L419I, I424V, I482L, V499A and S556G. Two positions presented high polymorphism in the NS5B region from genotype 1a (V499A) and genotype 1b (C316N) subjects. Our results demonstrated a natural profile of genotype 3a that can be associated with the pre-existence of HCV variants resistant to first-generation protease inhibitors and to non-nucleoside polymerase inhibitors. Likewise, genotype 1b isolates and genotype 1a sequences exhibited pre-existing mutations associated with resistance to Palm II and Thumb I polymerase inhibitors, respectively.

Epidemiology of serotype 19A isolates from invasive pneumococcal disease in German children

BackgroundThis study presents an analysis of 159 serotype 19A isolates from IPD in children before and after the general recommendation for childhood pneumococcal conjugate vaccination in Germany in July 2006. Vaccination formulations used were PCV7, PCV10 (from April 2009) and PCV13 (from Dec. 2009, replacing PCV7).MethodsIsolates from invasive pneumococcal disease in children were serotyped using the Quellung reaction, tested for antibiotic susceptibility and analysed for their multi locus sequence type.ResultsIn an analysis of 3328 isolates from invasive pneumococcal disease (IPD) in children that were sent to the German National Reference Center for Streptococci between July 1997 and June 2011, we show that the proportion of 19A isolates ranged between 1.7 and 4.2% in the period 1997 to 2006. After the recommendation for pneumococcal conjugate childhood vaccination, which was issued in July 2006, the proportion of 19A isolates increased significantly to 15.0% in 2010/11. Eight clonal complexes (CC) and groups accounted for 77.2% and 65.3% of all serotype 19A isolates before and after vaccination, respectively. While three CCs and several STs were not detected after vaccine introduction, four CCs and several STs first appeared after vaccination, including three ST320 isolates that could be traced to recent imports from the US, UK and India. The proportion of penicillin-nonsusceptible and of multidrug-resistant 19A isolates moderately increased after vaccine introduction. A significant increase in the use of cephalosporins and azithromycin was noted post-vaccination (p=0.00001 and p=0.0013 respectively).ConclusionsThe prevalence of serotype 19A in Germany has increased significantly between July 2007 and June 2011. Possible reasons for this are the introduction of pneumococcal conjugate vaccination, increased use of cephalosporins and azithromycin, import of multidrug-resistant isolates and increased reporting.

Host Preference of Mating Type in Pseudoperonospora cubensis, the Downy Mildew Causal Agent of Cucurbits.

The A2 mating type of Pseudoperonospora cubensis was first discovered in Israel in May 2010 on butternut gourd (Cucurbita moschata) (1). We monitored the occurrence of the A2 mating type of P. cubensis in isolates collected during May 2010 through September 2012 from downy mildew-infected cucurbit crops growing along the coastal plain of Israel. Mating type was determined by oospore production in melon leaf discs co-inoculated with sporangia of a test isolate mixed with sporangia of A1 or A2 tester isolates (2). The A1 and A2 tester isolates were maintained at 14°C (14 h light/day) by repeated inoculation of detached leaves of cucumber and pumpkin, respectively. The 29 isolates that were collected from cucumber (Cucumis sativum) were all A1. Of the 33 isolates collected from pumpkin (Cucurbita maxima), squash (C. pepo), or butternut gourd (C. moschata), 88% were A2 and 12% were A1. The host preference of mating type in P. cubensis was monitored at Bar-Ilan University farm during April to July 2012, among about 800 plants of eight cucurbit species (~100 plants per species) that were grown side-by-side in three adjacent net-houses (two 6 × 50 m and one 6 × 100 m) and exposed to natural infection. Downy mildew developed on cucumber, melon, pumpkin, squash, and butternut gourd, but not on watermelon, sponge gourd (Luffa cylindrica), or Momordica balsamina. Three-hundred and three isolates of P. cubensis were collected and tested for mating type: 123 from cucumber, 53 from melon, 30 from pumpkin, 48 from butternut gourd, and 41 from squash. The cucumber isolates expressed A1, A2, and A1A2 at a ratio of 94.3%, 3.3%, and 2.4%, respectively; the melon isolates 58.5%, 26.4%, and 15.1%; the pumpkin isolates 0%, 96.7%, and 3.3%; the butternut isolate 7.3%, 87.3%, and 5.5%; and the squash isolates 2.4%, 97.6%, and 0%, respectively. A1A2 isolates produce oospores when crossed with either A1 or A2 tester isolates. This is the first evidence suggesting a preference of A1 isolates to Cucumis spp. and of A2 isolates to Cucurbita spp. similar preference was recently observed among Chinese isolates of this pathogen (unpublished data). The mechanism(s) controlling this preference is not known. Classical genetics is currently employed to P. cubensis in order to understand if it derives from true linkage. The practical implication for downy mildew management is that growing cucumber/melon in close proximity to pumpkin/squash/butternut gourd should be avoided as it may enhance oospore production in nature. Oospores in soil were recently shown to serve as a primary source of downy mildew infection in cucumber (3).

Imported Hepatitis E Virus, Central African Republic, 2011

Imported Hepatitis E Virus, Central African Republic, 2011

Dynamics of Streptococcus pneumoniae Serotypes Causing Acute Otitis Media Isolated from Children with Spontaneous Middle-Ear Drainage over a 12-Year Period (1999–2010) in a Region of Northern Spain

The aim of this study was to determine the serotype and clonal distribution of pneumococci causing acute otitis media (AOM) and their relationship with recurrences and mixed infections with other microorganisms under the influence of the 7-valent pneumococcal conjugate vaccine (PCV7). To do this, all pneumococcal isolates collected from the spontaneous middle-ear drainage of children <5 years old diagnosed of AOM by their pediatrician or their general practitioner from 1999 to 2010 were phenotypically characterized and the most frequent serotypes were genotyped. In the 12-year study, 818 episodes of pneumococcal AOM were detected, mostly (70.5%) in children younger than 2 years old. In 262 episodes (32%), the pneumococci were isolated with another bacterium, mainly (n = 214) Haemophilus influenzae. Mixed infections were similar in children under or over 2 years old. The most frequent serotypes were 19A (n = 227, 27.8%), 3 (n = 92, 11.2%) and 19F (n = 74, 9%). Serotypes included in the PCV7 sharply decreased from 62.4% in the pre-vaccination (1999–2001) to 2.2% in the late post-vaccination period (2008–2010). Serotype diversity steadily increased after the introduction of the PCV7 but decreased from 2008–2010 due to the predominant role of serotype 19A isolates, mostly ST276 and ST320. The prevalence of serotype 3 doubled from 6.1% (20/326) in 1999–2004 to 14.6% (72/492) in 2005–2010. Relapses mainly occurred in male infants infected with isolates with diminished antimicrobial susceptibility. Reinfections caused by isolates with the same serotype but different genotype were frequent, highlighting the need for genetic studies to differentiate among similar strains. In conclusion, the main change in pneumococcal AOM observed after the introduction of the PCV7 was the sharp decrease in vaccine serotypes. Also notable was the high burden of serotype 19A in total pneumococcal AOM before and especially after the introduction of the PCV7, as well as in relapses and reinfections.

Population Genetic Structure of Phytophthora cinnamomi Associated with Avocado in California and the Discovery of a Potentially Recent Introduction of a New Clonal Lineage

Phytophthora root rot (PRR) of avocado (Persea americana), caused by Phytophthora cinnamomi, is the most serious disease of avocado worldwide. Previous studies have determined that this pathogen exhibits a primarily clonal reproductive mode but no population level studies have been conducted in the avocado-growing regions of California. Therefore, we used amplified fragment length polymorphism based on 22 polymorphic loci and mating type to investigate pathogen diversity from 138 isolates collected in 2009 to 2010 from 15 groves from the Northern and Southern avocado-growing regions. Additional isolates collected from avocado from 1966 to 2007 as well as isolates from other countries and hosts were also used for comparative purposes. Two distinct clades of A2 mating-type isolates from avocado were found based on neighbor joining analysis; one clade contained both newer and older collections from Northern and Southern California, whereas the other clade only contained isolates collected in 2009 and 2010 from Southern California. A third clade was also found that only contained A1 isolates from various hosts. Within the California population, a total of 16 genotypes were found with only one to four genotypes identified from any one location. The results indicate significant population structure in the California avocado P. cinnamomi population, low genotypic diversity consistent with asexual reproduction, potential evidence for the movement of clonal genotypes between the two growing regions, and a potential introduction of a new clonal lineage into Southern California.

Characterization of Yersinia enterocolitica Biotype 1A Strains Isolated from Swine Slaughterhouses and Markets

Yersinia enterocolitica is an important foodborne pathogen that causes illness in humans and animals. Y. enterocolitica is also the most heterogeneous species of the genus and is divided into distinct serotypes and over six biotypes. Y. enterocolitica biotype 1A strains are classically considered as nonpathogenic; however, some biotype 1A isolates have been considered as causative of gastrointestinal disease, yielding symptoms indistinguishable from those produced by pathogenic biotypes. Even after decades of isolation of clinical strains, the pathogenic mechanisms of these isolates are still not fully understood. In the present study, 122 Yersinia enterocolitica biotype 1A strains isolated from swine slaughterhouses and meat markets in Sao Paulo, Brazil, were characterized according to the presence of the virulence genes ail, virF, and ystA. A total of 94 strains were positive to at least one virulence gene (77.05%), and 67 were positive to all of them (54.92%). Twenty-two strains were submitted to PFGE genotyping resulting in 22 distinct pulsotypes, varying from 50% to 84% of genetic similarity. Any clustering tendency among pulsotypes related to origin, isolation site, or even virulence profile was not observed. The present study reports an important contamination of the environment in swine slaughterhouses, meat markets, and pork, by potentially virulent Y. enterocolitica biotype 1A.

Histopathological Investigations of the Infection Process and Propagule Development of &lt;em&gt;Phytophthora ramorum&lt;/em&gt; on Rhododendron Leaves

In Europe, cultivated rhododendron is one of the most important hosts for Phytophthora ramorum . To better understand leaf infection and leaf capacity for sporulation, infection studies were carried out. Detached leaves of Rhododendron ‘Catawbiense Grandiflorum ́ and `Brigitte ́ were inoculated with zoospore suspensions of P. ramorum isolates of mating type A1 and A2. ‘Catawbiense Grandiflorum’ developed much more leaf necrosis than ‘Brigitte’ (average necrotic leaf area after 55 days 106.5 mm 2 versus 0.12 mm 2 ). The trichomes on the ‘Brigitte’ leaves seemed to prevent the germ tubes from detecting the stomata. P. ramorum germ tubes from encysted zoospores invaded the leaf tissue via the stomata. Appressoria-like structures were observed. On the infected leaves new hyphae grew out of the stomata. Sporangia and chlamydospores developed on the mycelium from germinated zoospore cysts growing on the leaf surface as well as on the hyphae growing out of the stomata after infection. They could be observed mainly on the necrotic leaf areas. Single healthy-looking oospores developed within 12 days on the ‘Brigitte’ leaves. Observation of the infection process on leaves of Rhododendron ‘Cunningham’s White’ with the scanning electron microscope confirmed the various stages of the infection process.

Phytophthora infestans Field Isolates from Gansu Province, China are Genetically Highly Diverse and Show a High Frequency of Self Fertility

The genetic diversity of 85 isolates of Phytophthora infestans collected in 2007 from Gansu province in China was determined and compared with 21 isolates collected before 2004. Among them, 70 belonged to the A1 mating type and 15 were self-fertile (SF). The mitochondrial DNA haplotypes revealed both Ia (25%) and IIa (75%) haplotypes. Metalaxyl resistance occurred with high frequency (54%) in Gansu. Simple sequence repeat (SSR) genotyping revealed 26 genotypes (13 from the Tianshui region) among the 85 isolates, and 18 genotypes among the 21 isolates collected before 2004, without overlap in genotypes detected in the two groups. Cluster analysis showed clear subdivisions within the different mating type isolates. Among Gansu's isolates, Nei's and Shannon's diversity indices were highest in isolates collected in Tianshui where both A1 and SF isolates were found. Analysis of molecular variance of isolates from Gansu indicated that 51% and 49% of the variance was explained by within-area and among-area variance, respectively. The results suggest that the occurrence of SF isolates increases the risk of sexual reproduction, the formation of oospore as initial inocula in the field, and affects the genotypic diversity in the population.

Cucumber vein yellowing virus isolate-specific expression of symptoms and viral RNA accumulation in susceptible and resistant cucumber cultivars

Since its outbreak in Spain during the fall of 2000, Cucumber vein yellowing virus (CVYV) has become into a limiting factor in cucurbit cultivation in this country, particularly in cucumber. The impact of the virus has been limited after the introduction of resistant cucumber cultivars and the implementation of cultural practices to control whitefly, the virus vector. In Spain, CVYV isolates are genetically similar and cucumber resistance breaking isolates have not been reported. A CVYV isolate from Jordan (Jor) was previously reported as being more virulent in cucumber cv. Beit Alfa than an isolate from Israel. To study the reactions of different cucumber cultivars to the virulent Jor isolate in comparison with a Spanish isolate of (Alm 1A), we have inoculated both isolates in 10 commercial cucumber cultivars with resistance to the virus. Also, we have used real-time RT-PCR to estimate virus accumulation of Jor and Alm 1A isolates in three of these cultivars, one described as susceptible and two as resistant to CVYV. The Spanish Alm 1A isolate caused mild or no symptoms in resistant cultivars whereas Jor isolate caused intense symptoms in all cultivars. Alm1 A genomic RNA accumulated similarly in the three cultivars, whereas the accumulation of Jor genomic RNA was lower in the resistant cultivars than in the susceptible cultivar. Also the Jor isolate and accumulated earlier and at a higher level than the Alm 1A isolate in the three cultivars.

Genome Analyses of an Aggressive and Invasive Lineage of the Irish Potato Famine Pathogen

Pest and pathogen losses jeopardise global food security and ever since the 19th century Irish famine, potato late blight has exemplified this threat. The causal oomycete pathogen, Phytophthora infestans, undergoes major population shifts in agricultural systems via the successive emergence and migration of asexual lineages. The phenotypic and genotypic bases of these selective sweeps are largely unknown but management strategies need to adapt to reflect the changing pathogen population. Here, we used molecular markers to document the emergence of a lineage, termed 13_A2, in the European P. infestans population, and its rapid displacement of other lineages to exceed 75% of the pathogen population across Great Britain in less than three years. We show that isolates of the 13_A2 lineage are among the most aggressive on cultivated potatoes, outcompete other aggressive lineages in the field, and overcome previously effective forms of plant host resistance. Genome analyses of a 13_A2 isolate revealed extensive genetic and expression polymorphisms particularly in effector genes. Copy number variations, gene gains and losses, amino-acid replacements and changes in expression patterns of disease effector genes within the 13_A2 isolate likely contribute to enhanced virulence and aggressiveness to drive this population displacement. Importantly, 13_A2 isolates carry intact and in planta induced Avrblb1, Avrblb2 and Avrvnt1 effector genes that trigger resistance in potato lines carrying the corresponding R immune receptor genes Rpi-blb1, Rpi-blb2, and Rpi-vnt1.1. These findings point towards a strategy for deploying genetic resistance to mitigate the impact of the 13_A2 lineage and illustrate how pathogen population monitoring, combined with genome analysis, informs the management of devastating disease epidemics.

Genotyping and Molecular Characterization of Hepatitis B Virus from Human Immunodeficiency Virus-Infected Individuals in Southern Africa

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) are hyperendemic in sub-Saharan Africa. The HBV genotypes prevailing in HIV-infected Africans are unknown. Our aim was to determine the HBV genotypes in HIV-infected participants and to identify clinically significant HBV mutations. From 71 HBV DNA+ve HIV-infected participants, 49 basic core promoter/precore (BCP/PC) and 29 complete S regions were successfully sequenced. Following phylogenetic analysis of 29 specimens in the complete S region, 28 belonged to subgenotype A1 and one to D3. Mutations affecting HBeAg expression at the transcriptional (1762T1764A), translational (Kozak 1809–1812, initiation 1814–1816, G1896A with C1858T), or post translational levels (G1862T), were responsible for the high HBeAg-negativity observed. The G1862T mutation occurred only in subgenotype A1 isolates, which were found in one third (7/21) of HBsAg−ve participants, but in none of the 18 HBsAg+ve participants (p<0.05). Pre-S deletion mutants were detected in four HBsAg+ve and one HBsAg−ve participant/s. The following mutations occurred significantly more frequently in HBV isolated in this study than in strains of the same cluster of the phylogenetic tree: ps1F25L, ps1V88L/A; ps2Q10R, ps2 R48K/T, ps2A53V and sQ129R/H, sQ164A/V/G/D, sV168A and sS174N (p<0.05). ps1I48V/T occurred more frequently in females than males (p<0.05). Isolates with sV168A occurred more frequently in participants with viral loads >200 IU per ml (p<0.05) and only sS174N occurred more frequently in HBsAg−ve than in HBsAg+ve individuals (p<0.05). Prior to initiation of ART, ten percent, 3 of 29 isolates sequenced, had drug resistance mutations rtV173L, rtL180M+rtM204V and rtV214A, respectively. This study has provided important information on the molecular characteristics of HBV in HIV-infected southern Africans prior to ART initiation, which has important clinical relevance in the management of HBV/HIV co-infection in our unique setting.

Re-emergence of the Ib mitochondrial haplotype within the British and Irish Phytophthora infestans populations

Since its arrival in the British Isles in 1845 Phytophthora infestans has remained the most destructive pathogen of potato. In the ensuing period, the British and Irish P. infestans populations have undergone major displacements following the immigration of novel strains. Here we report the re-emergence of the Ib mitochondrial DNA haplotype in the British and Irish P. infestans populations associated with the 6_A1 genotype. Historically associated with the previously panglobally distributed clonal lineage US-1, the Ib haplotype has not been detected (with the exception of a single isolate in the mid 1990s) in the British or Irish P. infestans populations since the early 1980s. The 6_A1 isolates analysed possessed mtDNA Ib, but were otherwise quite unlike US-1, having the Pep allozyme genotype 96/96 and novel RG57 and SSR fingerprints. These genetic characteristics strongly suggest that the appearance of the 6_A1 genotype in these populations has resulted from migration (possibly after a recombination event elsewhere). This study highlights the advantages of utilising a range of different markers in pathogen monitoring.

Replication of Hepatitis C Virus Genotype 3a in Cultured Cells

Hepatitis C virus (HCV) genotype 3a is widespread worldwide, but no replication system exists for its study. We describe a subgenomic replicon system for HCV genotype 3a. We determined the consensus sequence of an HCV genome isolated from a patient, and constructed a subgenomic replicon using this clone. The replicon was transfected into HuH-7 cells and RNA replication was confirmed. We identified cell culture-adaptive mutations that increased colony formation multiple-fold. We have therefore established a genotype 3a replicon system that can be used to study this HCV genotype.

Clinical isolates of Yersinia enterocolitica Biotype 1A represent two phylogenetic lineages with differing pathogenicity-related properties

BackgroundY. enterocolitica biotype (BT) 1A strains are often isolated from human clinical samples but their contribution to disease has remained a controversial topic. Variation and the population structure among the clinical Y. enterocolitica BT 1A isolates have been poorly characterized. We used multi-locus sequence typing (MLST), 16S rRNA gene sequencing, PCR for ystA and ystB, lipopolysaccharide analysis, phage typing, human serum complement killing assay and analysis of the symptoms of the patients to characterize 298 clinical Y. enterocolitica BT 1A isolates in order to evaluate their relatedness and pathogenic potential.ResultsA subset of 71 BT 1A strains, selected based on their varying LPS patterns, were subjected to detailed genetic analyses. The MLST on seven house-keeping genes (adk, argA, aroA, glnA, gyrB, thrA, trpE) conducted on 43 of the strains discriminated them into 39 MLST-types. By Bayesian analysis of the population structure (BAPS) the strains clustered conclusively into two distinct lineages, i.e. Genetic groups 1 and 2. The strains of Genetic group 1 were more closely related (97% similarity) to the pathogenic bio/serotype 4/O:3 strains than Genetic group 2 strains (95% similarity). Further comparison of the 16S rRNA genes of the BT 1A strains indicated that altogether 17 of the 71 strains belong to Genetic group 2. On the 16S rRNA analysis, these 17 strains were only 98% similar to the previously identified subspecies of Y. enterocolitica. The strains of Genetic group 2 were uniform in their pathogenecity-related properties: they lacked the ystB gene, belonged to the same LPS subtype or were of rough type, were all resistant to the five tested yersiniophages, were largely resistant to serum complement and did not ferment fucose. The 54 strains in Genetic group 1 showed much more variation in these properties. The most commonly detected LPS types were similar to the LPS types of reference strains with serotypes O:6,30 and O:6,31 (37%), O:7,8 (19%) and O:5 (15%).ConclusionsThe results of the present study strengthen the assertion that strains classified as Y. enterocolitica BT 1A represent more than one subspecies. Especially the BT 1A strains in our Genetic group 2 commonly showed resistance to human serum complement killing, which may indicate pathogenic potential for these strains. However, their virulence mechanisms remain unknown.