Genetic characterization of a new insect flavivirus isolated from Culex pipiens mosquito in Japan

We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.     

Characterization of Pseudomonas aeruginosa strains isolated from cystic fibrosis patients by different typing methods

Pseudomonas (P.) aeruginosa strains isolated from the sputum of cystic fibrosis patients (CFP) are frequently difficult to type by conventional typing methods. The purpose of this study was to develop a random amplified polymorphic DNA (RAPD) analysis for the routine typing of these strains. Sixty P. aeruginosa non-repetitive strains recovered from CFP in a teaching hospital were typed. Thirty-five non-serotyped strains were studied by RAPD-PCR analysis with primers 272 and 208. RAPD data were performed to establish the relatedness between bioptype, antibiotic susceptibility and genotype. Fifty-five percent of strains are multiresistant, and no relation was found between antibiotype and biotype. A possible correlation between various phenotypes belonging to a single genotype was observed. RAPD typing characterized 30 distinct genotypes and two small clusters of strains were observed among isolates with each primer. Strains belonging to one cluster were present in two (6%) of the 35 strains. Strains belonging to the other cluster were present in three (8%) of the 35 strains. The occurrence of these clusters indicates that cross-infection may occur. The results indicate also that only the RAPD method can establish a clonal relation whereas the other methods may only reflect phenotypical differences, and thus are inadequate to type these strains.     

Population variation of West Nile virus confers a host-specific fitness benefit in mosquitoes

West Nile virus is similar to most other RNA viruses in that it exists in nature as a genetically diverse population. However, the role of this genetic diversity within natural transmission cycles and its importance to virus perpetuation remain poorly understood. Therefore, we determined whether highly genetically diverse populations are more fit compared to less genetically diverse WNV populations. Specifically, we generated three WNV populations that varied in their genetic diversity and evaluated their fitness relative to genetically marked control WNV in vivo in Culex quinquefasciatus mosquitoes and chickens. Our results demonstrate that high genetic diversity leads to fitness gains in vector mosquitoes, but not chickens.     

Virulence profiles and antibiotic susceptibility patterns of Klebsiella pneumoniae strains isolated from different clinical specimens

Aim of the study

To detect virulence factors in 54 Klebsiella pneumoniae isolates from different clinical specimens: urine (26), blood (11), pus (11), lung (four), cerebrospinal fluid (one) and ascitic fluid (one).

Material and methods

PCR was used to investigate virulence genes encoding adhesins (fimH-1, mrkD, kpn, ycfM), siderophores (entB: enterobactin, iutA: aerobactin, irp-1, irp-2, ybtS, fyuA: yersiniabactin, iroN: catechols receptor), protectines or invasins (rmpA, magA, traT) and toxins (hlyA, cnf-1). The serum resistance, capsule and hypermucoviscosity, and ability to form biofilm and produce siderophores were sought by phenotypic assays. The in vivo virulence was assessed in mice infected by intraperitoneal way. Antimicrobial susceptibility was tested by diffusion method.

Results

The most common virulence genes were fimH-1 (100%), mrkD (96.3%), ycfM (96.3%), and entB (100%). kpn and yersiniabactin genes were found at medium rates of 63% and 46.3% and at lower prevalence, were genes traT (1.8%), iroN (3.7%), iutA (5.5%) and rmpA (3.7%). magA, hlyA and cnf-1 genes were not detected. The capsule, serum resistance, biofilm formation, mannose-sensitive or -resistant haemagglutination and hypermucoviscosity were observed in 100%, 92.6%, 88.8%, 94.4%, 68.5% and 9.2% of isolates, respectively. The prevalence of siderophores was consistent with that of genotypic detection. The LD₅₀ in mice was very low (< 10² CFUs) for isolates with the most virulence factors. A rate of 74.1% of isolates showed a multidrug resistance (MDR) pattern.

Conclusions

The distribution of virulence profiles according to the clinical origin suggests a role of enterobactin in urinary infections and yersiniabactin in the invasiveness. The fimbriae F1 and F3, capsule, enterobactin, serum resistance and biofilm formation, were commonly found in isolates, they seem to be at the basis of classic pathogenicity of K. pneumoniae. The invasiveness enhancers, aerobactin, yersiniabactin, catechols receptor, mucoid factor and hypermucoviscosity, detected concomitantly in some isolates, constitute a threat for vulnerable populations, even more if they are in combination with antibiotic resistance.     

Lack of evidence for an interaction between Buchnera GroEL and Banana bunchy top virus (Nanoviridae)

Circulative plant viruses such as luteovirids and geminiviruses have been shown to bind to GroEL proteins produced by endosymbiotic bacteria harboured within hemipteran vectors. These interactions seem to prevent the degradation of the viral particles in the aphid's haemocoel. Similarly to luteovirids and geminiviruses, Banana bunchy top virus (BBTV), a member of the Nanoviridae family, is transmitted in a persistent, circulative manner and can be detected in the haemolymph of the aphid vector, Pentalonia nigronervosa. To date, it is not known if BBTV can interact with GroEL. In this study, we localised and inferred the phylogeny of a Buchnera aphidicola endosymbiont inhabiting P. nigronervosa. Furthermore, we predicted the 3D structure of Buchnera GroEL and detected the protein in the haemolymph of P. nigronervosa. Interactions were tested using 3 different assays: immunocapture PCR, dot blot, and far-western blot assays; however, none of them showed evidence of a BBTV–GroEL interaction. We concluded that it was unlikely that BBTV interacted with Buchnera GroEL either in vitro or in vivo and we discuss possible alternatives by which BBTV viral particles are able to avoid the process of degradation in the aphid haemocoel.     

Peruvian horse sickness virus and Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia

During 1997, two new viruses were isolated from outbreaks of disease that occurred in horses, donkeys, cattle and sheep in Peru. Genome characterization showed that the virus isolated from horses (with neurological disorders, 78% fatality) belongs to a new species the Peruvian horse sickness virus (PHSV), within the genus Orbivirus, family Reoviridae. This represents the first isolation of PHSV, which was subsequently also isolated during 1999, from diseased horses in the Northern Territory of Australia (Elsey virus, ELSV). Serological and molecular studies showed that PHSV and ELSV are very similar in the serotype-determining protein (99%, same serotype). The second virus (Rioja virus, RIOV) was associated with neurological signs in donkeys, cattle, sheep and dogs and was shown to be a member of the species Yunnan orbivirus (YUOV). RIOV and YUOV are also almost identical (97% amino acid identity) in the serotype-determining protein. YUOV was originally isolated from mosquitoes in China.     

Staphylococcal cassette chromosome mec characterization of methicillin-resistant Staphylococcus aureus strains isolated at the military hospital of Constantine/Algeria

Staphylococcal cassette chromosome mec is a genetic mobile element that carries the gene mecA mediating the methicillin resistance in staphylococci. The aim of this study is to type the Staphylococcal cassette chromosome mec (SCCmec) in 64 non-redundant methicillin-resistant Staphylococcus aureus (MRSA) strains recovered at the military hospital of Constantine (Algeria) between 2005 and 2007. Methicillin resistance was detected by oxacillin and cefoxitin discs and PBP2a test, and then confirmed by mecA PCR. The SCCmec complex types were determined by real time PCR. The analysis showed that 50 isolates were hospital acquired (HA-MRSA) and 14 were community-acquired (CA-MRSA). SCCmec type IV and V (traditionally attributed to CA-MRSA) were harbored by both HA-MRSA and CA-MRSA, while SCCmec type I, II and III were not recorded. These findings motivate more investigations to be carried on HA-MRSA in our hospital and other national health care centers.     

Unequal distribution of resistance-conferring mutations among Mycobacterium tuberculosis and Mycobacterium africanum strains from Ghana

Isoniazid (INH) and rifampicin (RMP) resistance in Mycobacterium tuberculosis complex (MTC) isolates are mainly based on mutations in a limited number of genes. However, mutation frequencies vary in different mycobacterial populations. In this work, we analyzed the distribution of resistance-associated mutations in M. tuberculosis and M. africanum strains from Ghana, West Africa. The distribution of mutations in katG, fabG1-inhA, ahpC, and rpoB was determined by DNA sequencing in 217 INH-resistant (INH^r) and 45 multidrug-resistant (MDR) MTC strains isolated in Ghana from 2001 to 2004. A total of 247 out of 262 strains investigated (94.3%) carried a mutation in katG (72.5%), fabG1-inhA (25.1%), or ahpC (6.5%), respectively. M. tuberculosis strains mainly had katG 315 mutations (80.1%), whereas this proportion was significantly lower in M. africanum West-African 1 (WA1) strains (43.1%; p < 0.05). In contrast, WA1 strains showed more mutations in the fabG1-inhA region (39.2%, p < 0.05) compared to M. tuberculosis strains (20.9%). In 44 of 45 MDR strains (97.8%) mutations in the 81-bp core region of the rpoB gene could be verified. Additionally, DNA sequencing revealed that 5 RMP-susceptible strains also showed mutations in the rpoB hotspot region. In conclusion, although principally the same genes were affected in INH^rM. tuberculosis and M. africanum strains, disequilibrium in the distribution of mutations conferring resistance was verified that might influence the efficiency of molecular tests for determination of resistance.     

Characterization of biofilms formed by Candida parapsilosis, C. metapsilosis, and C. orthopsilosis

Infections due to Candida parapsilosis have been associated with the ability of this fungus to form biofilms on indwelling medical devices. Recently, C. parapsilosis isolates were reclassified into 3 genetically non-identical classes: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Little information is available regarding the ability of these newly reclassified species to form biofilms on biomedical substrates. In this study, we characterized biofilm formation by 10 clinical isolates each of C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Biofilms were allowed to form on silicone elastomer discs to early (6 h) or mature (48 h) phases and quantified by tetrazolium (XTT) and dry weight assays. Surface topography and three-dimensional architecture of the biofilms were visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), respectively. Metabolic activity assay revealed strain-dependent biofilm forming ability of the 3 species tested, while biomass determination revealed that all 3 species formed equivalent biofilms (P>0.05 for all comparisons). SEM analyses of representative isolates of these species showed biofilms with clusters of yeast cells adherent to the catheter surface. Additionally, confocal microscopy analyses showed the presence of cells embedded in biofilms ranging in thickness between 62 and 85 μm. These results demonstrate that similar to C. parapsilosis, the 2 newly identified Candida species (C. orthopsilosis and C. metapsilosis) were able to form biofilms.     

Characterization of the circulating hemocytes in mud crab (Scylla olivacea) revealed phenoloxidase activity

This study focused on an isolation and characterization of the circulating hemocytes in mud crab, Scylla olivacea. Isolation of specific cell types of hemocytes from crab hemolymph was accomplished by using 60% Percoll density gradient centrifugation. Four separated bands of the hemocytes were successfully obtained. Characterization of these isolated hemocytes by light microscope using trypan blue-rose bengal staining, rose bengal–hematoxilin staining, and phase contrast revealed four distinct types of hemocyte cells. Using their specific morphology and granularity, they were identified as hyaline cell (HC), small granular cell (SGC), large granular cell (LGC) and mixed granular cell (MGC). Transmission electron microscopy (TEM) revealed more details on specific cell size, size of cytoplasmic granule, and nuclear to cytoplasmic ratio, and confirmed the classification. Relative abundance of these cells types in the hemolymph of an adult crab were 15.50 ± 8.22% for HC, 55.50 ± 7.15% for SGC, 13.50 ± 5.28% for LGC, and 15.50 ± 3.50% for MGC. Proteomic analysis of protein expression for each specific cell types by two-dimensional electrophoresis identified two highly abundant proteins, prophenoloxidase (ProPO) and peroxinectin in LGC. Determination of phenoloxidase (PO) activity in each isolated cell types using in vitro and in situ chemical assays confirmed the presence of PO activity only in LGC. Based on an increased PO activity of crab hemolymph during the course of White Spot Syndrome Virus (WSSV) infection, these results suggest that prophenoloxidase pathway was employed for host defense mechanism against WSSV and it may link to the role of large granular hemocyte.     

Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line

We previously demonstrated that Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication is restricted in permissive BmN-4 cells upon coinfection with Hyphantria cunea NPV (HycuNPV). Here, we show that HycuNPV-encoded hycu-ep32 gene is responsible for the restricted BmNPV multiplication in HycuNPV-coinfected BmN-4 cells. The only homologue for hycu-ep32 is in Orgyia pseudotsugata NPV. hycu-ep32 could encode a polypeptide of 312 amino acids, and it contains no characteristic domains or motifs to suggest its possible functions. hycu-ep32 is an early gene, and Hycu-EP32 expression reaches a maximum by 6 h postinfection. hycu-ep32-defective HycuNPV, vHycuΔep32, was generated, indicating that hycu-ep32 is nonessential in permissive SpIm cells. In BmN-4 cells, HycuNPV infection resulted in a severe global protein synthesis shutdown, while vHycuΔep32 did not cause any specific protein synthesis shutdown. These results indicate that the restriction of BmNPV multiplication by HycuNPV is caused by a global protein synthesis shutdown induced by hycu-ep32 upon coinfection with HycuNPV.     

Virulence of Mycobacterium avium complex strains isolated from immunocompetent patients

Mycobacterium avium complex (MAC) disease has been increasing worldwide not only in immunocompromised but also in immunocompetent humans. However, the relationship between mycobacterial strain virulence and disease progression in immunocompetent humans is unclear. In this study, we isolated 6 strains from patients with pulmonary MAC disease. To explore the virulence, we examined the growth in human THP-1 macrophages and pathogenicity in C57BL/6 mice. We found that one strain, designated 198, which was isolated from a patient showing the most progressive disease, persisted in THP-1 cells. In addition, strain 198 grew to a high bacterial load with strong inflammation in mouse lungs and spleens 16 weeks after infection. To our knowledge, strain 198 is the first isolated MAC strain that exhibits hypervirulence consistently for the human patient, human macrophages in vitro, and even for immunocompetent mice. Other strains showed limited survival and weak virulence both in macrophages and in mice, uncorrelated to disease progression in human patients. We demonstrated that there is a hypervirulent clinical MAC strain whose experimental virulence corresponds to the serious disease progression in the patients. The existence of such strain suggests the involvement of bacterial virulence in the pathogenesis of pulmonary MAC disease in immunocompetent status.     

Characterization of a small plaque variant of West Nile virus isolated in New York in 2000

A small-plaque variant (SP) of West Nile virus (WNV) was isolated in Vero cell culture from kidney tissue of an American crow collected in New York in 2000. The in vitro growth of the SP and parental (WT) strains was characterized in mammalian (Vero), avian (DF-1 and PDE), and mosquito (C6/36) cells. The SP variant replicated less efficiently than did the WT in Vero cells. In avian cells, SP growth was severely restricted at high temperatures, suggesting that the variant is temperature sensitive. In mosquito cells, growth of SP and WT was similar, but in vivo in Culex pipiens (L.) there were substantial differences. Relative to WT, SP exhibited reduced replication following intrathoracic inoculation and lower infection, dissemination, and transmission rates following oral infection. Analysis of the full length sequence of the SP variant identified sequence differences which led to only two amino acid substitutions relative to WT, prM P54S and NS2A V61A.     

Isolation and phylogenetic analysis of Mucambo virus (Venezuelan equine encephalitis complex subtype IIIA) in Trinidad

In the 1950s and 1960s, alphaviruses in the Venezuelan equine encephalitis (VEE) antigenic complex were the most frequently isolated arboviruses in Trinidad. Since then, there has been very little research performed with these viruses. Herein, we report on the isolation, sequencing, and phylogenetic analyses of Mucambo virus (MUCV; VEE complex subtype IIIA), including 6 recently isolated from Culex (Melanoconion) portesi mosquitoes and 11 previously isolated in Trinidad and Brazil. Results show that nucleotide and amino acid identities across the complete structural polyprotein for the MUCV isolates were 96.6-100% and 98.7-100%, respectively, and the phylogenetic tree inferred for MUCV was highly geographically- and temporally-structured. Bayesian analyses suggest that the sampled MUCV lineages have a recent common ancestry of approximately 198 years (with a 95% highest posterior density (HPD) interval of 63-448 years) prior to 2007, and an overall rate of evolution of 1.28 × 10^− 4 substitutions/site/yr.     

Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei

Trypanosomatids are unicellular parasites living in a wide range of host environments, which to large extent shaped their mitochondrial energy metabolism, resulting in quite large differences even among closely related flagellates. In a comparative manner, we analyzed the activities and composition of mitochondrial respiratory complexes in four species (Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and Trypanosoma brucei), which represent the main model trypanosomatids. Moreover, we measured the activity of mitochondrial glycerol-3-phosphate dehydrogenase, the overall oxygen consumption and the mitochondrial membrane potential in each species. The comparative analysis suggests an inverse relationship between the activities of respiratory complexes I and II, as well as the overall activity of the canonical complexes and glycerol-3-phosphate dehydrogenase. Our comparative analysis shows that mitochondrial functions are highly variable in these versatile parasites     

Gene disruption reveals a dispensable role for Plasmepsin VII in the Plasmodium berghei life cycle

Plasmepsins (PM), aspartic proteases of Plasmodium, comprises a family of ten proteins that perform critical functions in Plasmodium life cycle. Except VII and VIII, functions of the remaining plasmepsin members have been well characterized. Here, we have generated a mutant parasite lacking PM VII in Plasmodium berghei using reverse genetics approach. Systematic comparison of growth kinetics and infection in both mosquito and vertebrate host revealed that PM VII depleted mutants exhibited no defects in development and progressed normally throughout the parasite life cycle. These studies suggest a dispensable role for PM VII in Plasmodium berghei life cycle.     

Compartmentalized expression of light-induced clock genes in the suprachiasmatic nucleus of the diurnal grass rat (Arvicanthis niloticus)

Photic responses of the circadian system are mediated through light-induced clock gene expression in the suprachiasmatic nucleus (SCN). In nocturnal rodents, depending on the timing of light exposure, Per1 and Per2 gene expression shows distinct compartmentalized patterns that correspond to the behavioral responses. Whether the gene- and region-specific induction patterns are unique to nocturnal animals, or are also present in diurnal species is unknown. We explored this question by examining the light-induced Per1 and Per2 gene expression in functionally distinct SCN subregions, using diurnal grass rats Arvicanthis niloticus. Light exposure during nighttime induced Per1 and Per2 expression in the SCN, showing unique spatiotemporal profiles depending on the phase of the light exposure. After a phase delaying light pulse (LP) in the early night, strong Per1 induction was observed in the retinorecipient core region of the SCN, while strong Per2 induction was observed throughout the entire SCN. After a phase advancing LP in the late night, Per1 was first induced in the core and then extended into the whole SCN, accompanied by a weak Per2 induction. This compartmentalized expression pattern is very similar to that observed in nocturnal rodents, suggesting that the same molecular and intercellular pathways underlying acute photic responses are present in both diurnal and nocturnal species. However, after an LP in early subjective day, which induces phase advances in diurnal grass rats, but not in nocturnal rodents, we did not observe any Per1 or Per2 induction in the SCN. This result suggests that in spite of remarkable similarities in the SCN of diurnal and nocturnal rodents, unique mechanisms are involved in mediating the phase shifts of diurnal animals during the subjective day.     

Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2

The sequence of Lettuce chlorosis virus (LCV) (genus Crinivirus) was determined and found to contain unique open reading frames (ORFs) and ORFs similar to those of other criniviruses, as well as 3′ non-coding regions that shared a high degree of identity. Northern blot analysis of RNA extracted from LCV-infected plants identified subgenomic RNAs corresponding to six prominent internal ORFs and detected several novel LCV-single stranded RNA species. Virus replication in tobacco protoplasts was investigated and results indicated that LCV replication proceeded with novel crinivirus RNA accumulation kinetics, wherein viral genomic RNAs exhibited a temporally similar expression pattern early in the infection. This was noticeably distinct from the asynchronous RNA accumulation pattern previously observed for Lettuce infectious yellows virus (LIYV), the type member of the genus, suggesting that replication of the two viruses likely operate via dissimilar mechanisms.