Distinct Amino Acid Substitutions Research Articles

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Rh flow cytometry: An updated methodology for D antigen density applied to weak D types 164 and 165.

An original methodology for determining the D antigen density on red cells was published in 2000 and has been applied in many publications since. This flow cytometry-based assay remained largely unrevised utilizing monoclonal anti-Ds that are not readily available anymore. We updated the methodology to quantify erythrocyte D antigen sites using microspheres and monoclonal anti-Ds that are commercially available today. The absolute D antigen density of a frozen standard CcDEe cell, drawn in 2003, a fresh blood donation from the same individual, drawn in 2022, and an internal control CcDEe cell, was quantified by flow cytometry using fluorescence-labeled microspheres. The internal control CcDEe cell was used in conjunction with 9 commercial anti-Ds to determine D antigen densities of 7 normal D, 4 partial D, and 11 weak D type samples, including 2 novel alleles. The reproducibility of the updated assay was evaluated with red cells of published D antigen densities. The current results matched the known ones closely. The new weak D types 164 and 165 carried 4500 and 1505 D antigens/red cell, respectively. The absolute D antigen density decreased from 27,231 to 26,037 in an individual over 19 years. The updated assay gave highly reproducible results for the D antigen densities of Rh phenotypes. Readily available anti-Ds allowed for the determination of the D antigen densities of 7 weak D types. The assay is suitable to evaluate the effects of distinct amino acid substitutions on the RhD phenotype.

Molecular epidemiology, antibiogram profile and risk factor analysis of pathogenic Escherichia coli associated with pre-weaning diarrhoea in piglets from Haryana, India

BackgroundPiglet diarrhoea is a multifactorial disease with serious implications for the swine industry worldwide, including India. The Escherichia coli (E. coli) pathotypes, i.e., enterotoxigenic E. coli (ETEC) and Shiga toxin-producing E. coli (STEC) are among the major bacterial agents attributed as causative agent for piglet diarrhoea, but studies related to genetic diversity, antibiogram profile and their correlation with risk factors of these pathogens are sparse. Material and methodsA total of 104 faecal swab samples were collected from 32 different piggery units of Haryana, India and confirmed as E. coli by standard microbiological methods. The identified E. coli were characterized as ETEC and/or STEC using PCR assays and were studied for their genetic diversity by phylogenetic analysis of the sequences. All the isolates were subjected to antimicrobial susceptibility testing. Further, the correlation of variables with presence or absence of ETEC and/or STEC was also investigated by using Fisher’s exact test. ResultsMicrobiological isolation led to identification of 208 E. coli isolates. A total of 17.3% (31/208) isolates were characterized as ETEC and 4.8% (10/208) isolates as STEC, whereas 2.4% (5/208) isolates exhibited both ETEC and STEC pathotype. Of the total studied piggery units (n = 32), ETEC were isolated from fourteen and both ETEC and STEC from eight farms. The phylogenetic analysis of Stx2 gene revealed 100% homology with Stx2eA variant from Germany, while analysis of STII gene revealed a distinct nucleotide and amino acid substitution when compared with standard strains. The antibiotic susceptibility testing revealed maximum resistance to moxifloxacin (71.9%) followed by tetracycline (58.1%) and amoxicillin with a total of 41.8% (87/208) E. coli isolates designated as multi-drug resistant (MDR). The multiple antibiotic resistance index varied from 0.05 to 0.75. The statistical analysis suggested three factors viz., number of farm worker(s), frequency of using disinfectant for floor cleaning and use of antibiotic in feed as risk factors significantly associated (p < 0.05) with ETEC associated diarrhoea at piggeries under study. ConclusionCurrent study warrants a need for systematic studies on the ETEC/STEC associated diarrhea and antibiotic resistance among these isolates to understand the mechanisms of origin and dissemination of drug resistant pathogens and to design suitable prevention and control measures to curb emergence of antibiotic resistance in the farm settings.

Unraveling the Structural Changes in the DNA-Binding Region of Tumor Protein p53 (TP53) upon Hotspot Mutation p53 Arg248 by Comparative Computational Approach

The vital tissue homeostasis regulator p53 forms a tetramer when it binds to DNA and regulates the genes that mediate essential biological processes such as cell-cycle arrest, senescence, DNA repair, and apoptosis. Missense mutations in the core DNA-binding domain (109-292) simultaneously cause the loss of p53 tumor suppressor function and accumulation of the mutant p53 proteins that are carcinogenic. The most common p53 hotspot mutation at codon 248 in the DNA-binding region, where arginine (R) is substituted by tryptophan (W), glycine (G), leucine (L), proline (P), and glutamine (Q), is reported in various cancers. However, it is unclear how the p53 Arg248 mutation with distinct amino acid substitution affects the structure, function, and DNA binding affinity. Here, we characterized the pathogenicity and protein stability of p53 hotspot mutations at codon 248 using computational tools PredictSNP, Align GVGD, HOPE, ConSurf, and iStable. We found R248W, R248G, and R248P mutations highly deleterious and destabilizing. Further, we subjected all five R248 mutant-p53-DNA and wt-p53-DNA complexes to molecular dynamics simulation to investigate the structural stability and DNA binding affinity. From the MD simulation analysis, we observed increased RMSD, RMSF, and Rg values and decreased protein-DNA intermolecular hydrogen bonds in the R248-p53-DNA than the wt-p53-DNA complexes. Likewise, due to high SASA values, we observed the shrinkage of proteins in R248W, R248G, and R248P mutant-p53-DNA complexes. Compared to other mutant p53-DNA complexes, the R248W, R248G, and R248P mutant-p53-DNA complexes showed more structural alteration. MM-PBSA analysis showed decreased binding energies with DNA in all five R248-p53-DNA mutants than the wt-p53-DNA complexes. Henceforth, we conclude that the amino acid substitution of Arginine with the other five amino acids at codon 248 reduces the p53 protein's affinity for DNA and may disrupt cell division, resulting in a gain of p53 function. The proposed study influences the development of rationally designed molecular-targeted treatments that improve p53-based therapeutic outcomes in cancer.

Multivalent display of engineered HIV-1 envelope trimers on silica nanoparticles for targeting and in vitro activation of germline VRC01 B cells

Selective targeting of germline B cells with specifically designed germline-targeting HIV-1 envelope immunogens (GT-Env) is considered a feasible vaccination strategy to elicit broadly neutralizing antibodies (bnAbs). BnAbs are extremely valuable because they neutralize genetically distant viral strains at the same time. To overcome its inherently low affinity to germline B cells, the aim of the study was to present GT-Env via different immobilization strategies densely arrayed on the surface of nanoparticles. We engineered a prefusion-stabilized GT-Env trimer with affinity to VRC01 germline B cells using a bioinformatics-supported design approach. Distinct glycan modifications and amino acid substitutions yielded a GT-Env trimer which bound to the receptor with a KD of 11.5 µM. Silica nanoparticles with 200 nm diameter (SiNPs) were used for the multivalent display of the novel GT-Env with a 15 nm mean centre-to-centre spacing either by site-specific, covalent conjugation or at random, non-specific adsorption. Oriented, covalent GT-Env conjugation revealed better binding of structure dependent bnAbs as compared to non-specifically adsorbed GT-Env. In addition, GT-Env covalently attached activated a B cell line expressing the germline VRC01 receptor at an EC50 value in the nanomolar range (4 nM), while soluble GT-Env required 1,000-fold higher concentrations to induce signalling. The significantly lower GT-Env concentration was likely required due to avidity effects, which were in the picomolar range. Thus, low affinity antigens may particularly benefit from a particulate and multivalent delivery. In future, SiNPs are ideal to be modified in a modular design with various GT-Env variants that target different stages of germline and bnAb precursor B cells.

Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction

BackgroundSARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants.ResultsHere, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface.ConclusionOur study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

Experimental evolution in morbidostat reveals converging genomic trajectories on the path to triclosan resistance.

Understanding the dynamics and mechanisms of acquired drug resistance across major classes of antibiotics and bacterial pathogens is of critical importance for the optimization of current anti-infective therapies and the development of novel ones. To systematically address this challenge, we developed a workflow combining experimental evolution in a morbidostat continuous culturing device with deep genomic sequencing of population samples collected in time series. This approach was applied to the experimental evolution of six populations of Escherichia coli BW25113 towards acquiring resistance to triclosan (TCS), an antibacterial agent in various consumer products. This study revealed the rapid emergence and expansion (up to 100% in each culture within 4 days) of missense mutations in the fabI gene, encoding enoyl-acyl carrier protein reductase, the known TCS molecular target. A follow-up analysis of isolated clones showed that distinct amino acid substitutions increased the drug IC90 in a 3–16-fold range, reflecting their proximity to the TCS-binding site. In contrast to other antibiotics, efflux-upregulating mutations occurred only rarely and with low abundance. Mutations in several other genes were detected at an earlier stage of evolution. Most notably, three distinct amino acid substitutions were mapped in the C-terminal periplasmic domain of CadC protein, an acid stress-responsive transcriptional regulator. While these mutations do not confer robust TCS resistance, they appear to play a certain, yet unknown, role in adaptation to relatively low drug pressure. Overall, the observed evolutionary trajectories suggest that the FabI enzyme is the sole target of TCS (at least up to the ~50 µm level), and amino acid substitutions in the TCS-binding site represent the main mechanism of robust TCS resistance in E. coli . This model study illustrates the potential utility of the established morbidostat-based approach for uncovering resistance mechanisms and target identification for novel drug candidates with yet unknown mechanisms of action.

Of eyes and embryos: subfunctionalization of the CRX homeobox gene in mammalian evolution.

ETCHbox genes are fast-evolving homeobox genes present only in eutherian (placental) mammals which originated by duplication and divergence from a conserved homeobox gene, Cone-rod homeobox (CRX). While expression and function of CRX are restricted to the retina in eutherian mammals, ETCHbox gene expression is specific to preimplantation embryos. This dramatic difference could reflect the acquisition of new functions by duplicated genes or subfunctionalization of pleiotropic roles between CRX and ETCHbox genes. To resolve between these hypotheses, we compared expression, sequence and inferred function between CRX of metatherian (marsupial) mammals and ETCHbox genes of eutherians. We find the metatherian CRX homeobox gene is expressed in early embryos and in eyes, unlike eutherian CRX, and distinct amino acid substitutions were fixed in the metatherian and eutherian evolutionary lineages consistent with altered transcription factor specificity. We find that metatherian CRX is capable of regulating embryonically expressed genes in cultured cells in a comparable way to eutherian ETCHbox. The data are consistent with CRX having a dual role in eyes and embryos of metatherians, providing an early embryonic function comparable to that of eutherian ETCHbox genes; we propose that subfunctionalization of pleiotropic functions occurred after gene duplication along the placental lineage, followed by functional elaboration.

PmrAB and PhoPQ Variants in Colistin-Resistant Enterobacter spp. Isolates in Korea.

In this study, we investigated the amino acid variations and mRNA expression of PhoPQ and PmrAB two-component regulatory systems in colistin-resistant Enterobacter cloacae isolates from Korea. We determined the nucleotide sequences of phoP, phoQ, pmrA, and pmrB in 51 colistin-resistant, 5 colistin-susceptible, and 8 skip-well isolates and consequently, the corresponding amino acid sequences as well. PhoPQ and PmrAB sequences showed large variations among the isolates (14, 67, 20, and 68 sites, respectively). Although there was some discrepancy between the genes, the colistin-resistant E. cloacae isolates were grouped into four clades and the susceptible isolates were grouped into two clades. We did not find any distinct amino acid substitutions associated with colistin resistance. Furthermore, mRNA expression of phoQ and pmrB was not significantly higher in the colistin-resistant isolates. Our data suggests that the colistin resistance mechanisms might be different in E. cloacae when compared to other gram-negative bacteria.

Molecular Adaptations of Bacterial Mercuric Reductase to the Hypersaline Kebrit Deep in the Red Sea.

The hypersaline Kebrit Deep brine pool in the Red Sea is characterized by high levels of toxic heavy metals. Here, we describe two structurally related mercuric reductases (MerAs) from this site which were expressed in Escherichia coli Sequence similarities suggest that both genes are derived from proteobacteria, most likely the Betaproteobacteria or Gammaproteobacteria We show that one of the enzymes (K35NH) is strongly inhibited by NaCl, while the other (K09H) is activated in a NaCl-dependent manner. We infer from this difference that the two forms might support the detoxification of mercury in bacterial microorganisms that employ the compatible solutes and salt-in strategies, respectively. Three-dimensional structure modeling shows that all amino acid substitutions unique to each type are located outside the domain responsible for formation of the active MerA homodimer, and the vast majority of these are found on the surface of the molecule. Moreover, K09H exhibits the predominance of acidic over hydrophobic side chains that is typical of halophilic salt-dependent proteins. These findings enhance our understanding of how selection pressures imposed by two environmental stressors have endowed MerA enzymes with catalytic properties that can potentially function in microorganisms that utilize distinct mechanisms for osmotic balance in hypersaline environments.IMPORTANCE Analysis of two structurally homologous but catalytically distinct mercuric reductases from the Kebrit Deep brine in the Red Sea sheds light on the adaptations that enable microorganisms to cope simultaneously with extreme salinity and toxic mercury compounds. One is strongly inhibited by high NaCl concentrations, while the other exhibits NaCl-dependent activation. Their different activity profiles imply that they may derive from bacterial microorganisms that utilize compatible solutes and salt-in strategies, respectively, to maintain osmotic balance. Three-dimensional modeling reveals that regions not involved in formation of the active homodimer are conserved between the two. However, in the NaCl-dependent form, distinct amino acid substitutions are found in areas that are critical for stability in high salt. The work provides insights into how two environmental stressors have shaped the structure of orthologous enzymes through selection and adaptation, enabling them to retain their catalytic function in what may be very different cellular contexts.

Molecular modelling of mitofusin 2 for a prediction for Charcot-Marie-Tooth 2A clinical severity

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant neuropathy caused by mutations in the mitofusin 2 gene (MFN2). More than 100 MFN2 gene mutations have been reported so far, with majority located within the GTPase domain encoding region. These domain-specific mutations present wide range of symptoms with differences associated with distinct amino acid substitutions in the same position. Due to the lack of conclusive phenotype-genotype correlation the predictive value of genetic results remains still limited. We have explored whether changes in the protein structure caused by MFN2 mutations can help to explain diseases phenotypes. Using a stable protein model, we evaluated the effect of 26 substitutions on the MFN2 structure and predicted the molecular consequences of such alterations. The observed changes were correlated with clinical features associated with a given mutation. Of all tested mutations positive correlation of molecular modelling with the clinical features reached 73%. Our analysis revealed that molecular modelling of mitofusin 2 mutations is a powerful tool, which predicts associated pathogenic impacts and that these correlate with clinical outcomes. This approach may aid an early diagnosis and prediction of symptoms severity in CMT2A patients.

Clonal spread and azole-resistant mechanisms of non-susceptible Candida albicans isolates from vulvovaginal candidiasis patients in three Shanghai maternity hospitals.

In our multicenter study, 43 fluconazole non-susceptible and 45 fluconazole-susceptible isolates were collected from vulvovaginal candidiasis (VVC) patients from three Shanghai maternity hospitals to analyze their molecular epidemiological features and fluconazole resistant mechanisms. Cross-resistance to fluconazole, itraconazole and voriconazole was observed in 53.5% of the nonsusceptible isolates. Though we acquired 12 clonal complexes (CCs) of diploid sequence types (DSTs) in clinical isolates by a multilocus sequence typing method, fluconazole nonsusceptible isolates all belonged to CC69 with a predominant genotype of DST 79. Increased expressions of efflux pump genes (CDR1, CDR2, and MDR1) were observed only in minor fluconazole non-susceptible isolates by real-time quantitative polymerase chain reaction (PCR). However, ERG11 genes of fluconazole SDD and resistant isolates had significantly higher expression levels than fluconazole-susceptible isolates. Moreover, 13 distinct amino acid substitutions in Erg11p were found in clinical isolates. Three of the substitutions were novel amino acid substitutions (T123I, P98S, and Y286D), which were not in the susceptible isolates. Only two heterozygous amino acid substitutions (A18P/A and R365G/R) in Erg3p were found in two isolates with cross-resistance to fluconazole, itraconazole, and voriconazole. Taken together, we observed the clonal spread of CC69 in fluconazole non-susceptible isolates of Candida albicans from VVC patients with the dominant genotype DST79. ERG11 gene mutations and overexpression predominantly contributed to fluconazole resistance instead of the more common increased expressions of efflux pump genes (CDR1, CDR2, and MDR1).

A novel rhabdovirus, related to Merida virus, in field-collected mosquitoes from Anatolia and Thrace.

Next-generation sequencing technologies have significantly facilitated the discovery of novel viruses, and metagenomic surveillance of arthropods has enabled exploration of the diversity of novel or known viral agents. We have identified a novel rhabdovirus that is genetically related to the recently described Merida virus via next-generation sequencing in a mosquito pool from Thrace. The complete viral genome contains 11,798 nucleotides with 83% genome-wide nucleotide sequence similarity to Merida virus. Five major putative open reading frames that follow the canonical rhabdovirus genome organization were identified. A total of 1380 mosquitoes comprising 13 species, collected from Thrace and the Mediterranean and Aegean regions of Anatolia were screened for the novel virus using primers based on the N and L genes of the prototype genome. Eight positive pools (6.2%) exclusively comprised Culex pipiens sensu lato specimens originating from all study regions. Infections were observed in pools with female as well as male or mixed-sex individuals. The overall and Cx. pipiens-specific minimal infection rates were calculated to be 5.7 and 14.8, respectively. Sequencing of the PCR products revealed marked diversity within a portion of the N gene, with up to 4% divergence and distinct amino acid substitutions that were unrelated to the collection site. Phylogenetic analysis of the complete and partial viral polymerase (L gene) amino acid sequences placed the novel virus and Merida virus in a distinct group, indicating that these strains are closely related. The strain is tentatively named "Merida-like virus Turkey". Studies are underway to isolate and further explore the host range and distribution of this new strain.

Amino acid substitutions in random mutagenesis libraries: lessons from analyzing 3000 mutations.

The quality of amino acid substitution patterns in random mutagenesis libraries is decisive for the success in directed evolution campaigns. In this manuscript, we provide a detailed analysis of the amino acid substitutions by analyzing 3000 mutations of three random mutagenesis libraries (1000 mutations each; epPCR with a low-mutation and a high-mutation frequency and SeSaM-Tv P/P) employing lipase A from Bacillus subtilis (bsla). A comparison of the obtained numbers of beneficial variants in the mentioned three random mutagenesis libraries with a site saturation mutagenesis (SSM) (covering the natural diversity at each amino acid position of BSLA) concludes the diversity analysis. Seventy-six percent of the SeSaM-Tv P/P-generated substitutions yield chemically different amino acid substitutions compared to 64% (epPCR-low) and 69% (epPCR-high). Unique substitutions from one amino acid to others are termed distinct amino acid substitutions. In the SeSaM-Tv P/P library, 35% of all theoretical distinct amino acid substitutions were found in the 1000 mutation library compared to 25% (epPCR-low) and 26% (epPCR-high). Thirty-six percent of distinct amino acid substitutions found in SeSaM-Tv P/P were unobtainable by epPCR-low. Comparison with the SSM library showed that epPCR-low covers 15%, epPCR-high 18%, and SeSaM-Tv P/P 21% of obtainable beneficial amino acid positions. In essence, this study provides first insights on the quality of epPCR and SeSaM-Tv P/P libraries in terms of amino acid substitutions, their chemical differences, and the number of obtainable beneficial amino acid positions.

Resistance Surveillance in Candida albicans: A Five-Year Antifungal Susceptibility Evaluation in a Brazilian University Hospital.

Candida albicans caused 44% of the overall candidemia episodes from 2006 to 2010 in our university tertiary care hospital. As different antifungal agents are used in therapy and also immunocompromised patients receive fluconazole prophylaxis in our institution, this study aimed to perform an antifungal susceptibility surveillance with the C.albicans bloodstream isolates and to characterize the fluconazole resistance in 2 non-blood C.albicans isolates by sequencing ERG11 gene. The study included 147 C. albicans bloodstream samples and 2 fluconazole resistant isolates: one from oral cavity (LIF 12560 fluconazole MIC: 8μg/mL) and one from esophageal cavity (LIF-E10 fluconazole MIC: 64μg/mL) of two different patients previously treated with oral fluconazole. The in vitro antifungal susceptibility to amphotericin B (AMB), 5-flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CASP) was performed by broth microdilution methodology recommended by the Clinical and Laboratory Standards Institute documents (M27-A3 and M27-S4, CLSI). All blood isolates were classified as susceptible according to CLSI guidelines for all evaluated antifungal agents (MIC range: 0,125–1.00 μg/mL for AMB, ≤0.125–1.00 μg/mL for 5FC, ≤0.125–0.5 μg/mL for FLC, ≤0.015–0.125 μg/mL for ITC, ≤0.015–0.06 μg/mL for VRC and ≤0.015–0.125 μg/mL for CASP). In this study, we also amplified and sequenced the ERG11 gene of LIF 12560 and LIF-E10 C.albicans isolates. Six mutations encoding distinct amino acid substitutions were found (E116D, T128K, E266D, A298V, G448V and G464S) and these mutations were previously described as associated with fluconazole resistance. Despite the large consumption of antifungals in our institution, resistant blood isolates were not found over the trial period. Further studies should be conducted, but it may be that the very prolonged direct contact with the oral antifungal agent administered to the patient from which was isolated LIF E-10, may have contributed to the development of resistance.

Diversity of Meq gene from clinical Marek's disease virus infection in Saudi Arabia.

Aim:The aim of this study was to demonstrate the genomic features of Meq gene of Marek’s disease virus (MDV) recently circulating in Saudi Arabia (SA).Materials and Methods:Two poultry flocks suffering from mortalities and visceral tumors were presented to the Veterinary Teaching Hospital, King Faisal University, SA. Subjected to different diagnostic procedures: Case history, clinical signs, and necropsy as well as polymerase chain reaction followed by Meq gene sequence analysis.Results:Case history, clinical signs, and necropsy were suggestive of MDV infection. The Meq gene was successfully detected in liver and spleen of infected chickens. A 1062 bp band including the native Meq ORF in addition to a 939 bp of S-Meq (short isoform of Meq) were amplified from Saudi 01-13 and Saudi 02-13, respectively. The nucleotide and deduced amino acids sequences of the amplified Meq genes of both Saudi isolates showed distinct polymorphism when compared with the standard USA virulent isolates Md5 and GA. The sequence analysis of the S-Meq gene showed a 123 bp deletion representing 41 amino acids between two proline-rich areas without any frameshift. The Meq gene encoded four repeats of proline-rich repeats (PRRs sequences), whereas the S-Meq contains only two PRRs. Interestingly, the phylogenetic analysis revealed that both of SA MDV isolates are closely related to the MDV strains from Poland.Conclusion:The two MDV isolates contain several nucleotide polymorphisms resulting in distinct amino acid substitutions. It is suggested that migratory and wild birds, as well as world trading of poultry and its by-products, have a great contribution in the transmission of MDVs overseas.

Spring viraemia of carp virus: recent advances.

Spring viraemia of carp is an environmentally and economically important disease affecting cyprinids, primarily common carp (Cyprinus carpio). The causative agent of this disease is Spring viraemia of carp virus (SVCV) - a member of the genus Vesiculovirus of the family Rhabdoviridae. The disease is presently endemic in Europe, America and several Asian countries, where it causes significant morbidity and mortality in affected fish. SVCV infection is generally associated with exophthalmia; abdominal distension; petechial haemorrhage of the skin, gills, eyes and internal organs; degeneration of the gill lamellae; a swollen and coarse-textured spleen; hepatic necrosis; enteritis; and pericarditis. The SVCV genome is composed of linear, negative-sense, ssRNA containing five genes in the order 3'-N-P-M-G-L-5', encoding a nucleoprotein, phosphoprotein, matrix protein, glycoprotein and RNA-dependent RNA polymerase, respectively. Fully sequenced SVCV strains exhibit distinct amino acid substitutions at unique positions, which may contribute to as-yet unknown strain-specific characteristics. To advance the study of SVCV and the control of spring viraemia of carp disease in the future, this review summarizes our current understanding of SVCV in terms of its genomic characteristics, genetic diversity and pathogenesis, and provides insights into antiviral immunity against SVCV, diagnosis of SVCV and vaccination strategies to combat SVCV.

Phylogenetic analysis of human influenza A/H3N2 viruses isolated in 2015 in Germany indicates significant genetic divergence from vaccine strains.

Infections by H3N2-type influenza A viruses (IAV) resulted in significant numbers of hospitalization in several countries in 2014-2015, causing disease also in vaccinated individuals and, in some cases, fatal outcomes. In this study, sequence analysis of H3N2 viruses isolated in Germany from 1998 to 2015, including eleven H3N2 isolates collected early in 2015, was performed. Compared to the vaccine strain A/Texas/50/2012 (H3N2), the 2015 strains from Germany showed up to 4.5% sequence diversity in their HA1 protein, indicating substantial genetic drift. The data further suggest that two distinct phylogroups, 3C.2 and 3C.3, with 1.6-2.3% and 0.3-2.4% HA1 nucleotide and amino acid sequence diversity, respectively, co-circulated in Germany in the 2014/2015 season. Distinct glycosylation patterns and amino acid substitutions in the hemagglutinin and neuraminidase proteins were identified, possibly contributing to the unusually high number of H3N2 infections in this season and providing important information for developing vaccines that are effective against both genotypes.

Genetic and phenotypic characterization of Candida albicans strains isolated from infectious disease patients in Shanghai.

Candida albicans, as an opportunistic pathogen, can cause superficial and life-threatening candidiasis in immunocompromised individuals. The formation of surface-associated biofilms and the appearance of drug resistance pose a significant challenge for clinical intervention. In this study, a total of 104 hospital-acquired C. alibcans clinical isolates were collected from sterile sites and mucosal lesions of 92 infectious disease patients in the Shanghai Public Health Clinical Center and analysed. The resistance rates to fluconazole, itraconazole and voriconazole were 12.5 %, 15.4 % and 11.5 % respectively. Multilocus sequence typing (MLST) analysis identified 63 diploid sequence types (DSTs) with a decentralized phylogeny, of which 37 DSTs (58.7 %) had not been reported in the online MLST database. Loss of heterozygosity was observed in ACC1 and ADP1 sequences obtained from six sequential isolates from a patient receiving antifungal treatment, which exemplified the effect of microevolution on C. albicans genetic alterations. Biofilm formation capability, an important virulence trait of C. albicans, was variable among strains isolated from different anatomical sites (P = 0.0302) and affected by genotypes (P = 0.0185). The mRNA levels of the azole antifungal target ERG11 gene and efflux pump genes (CDR1, CDR2 and MDR1) were detected in 9-18.1 % of azole-resistant and susceptible-dose dependent (S-DD) isolates. Twelve mutations encoding distinct amino acid substitutions in ERG11 were found in azole-resistant and S-DD isolates. Among them, A114S, Y132H and Y257H substitution in the ERG11 gene may be primarily related to azole resistance. Taken together, we observed a high level of diversity within C. albicans isolates. Multiple inter-related underlying mechanisms, including genetic and environmental factors, may account for high surface adhesion or azole resistance in clinical C. albicans infections.

Erg11mutations associated with azole resistance in clinical isolates ofCandida albicans

The widespread use of azoles has led to increasing azole resistance among Candida albicans strains. One mechanism of azole resistance involves point mutations in the ERG11 gene, which encodes the target enzyme (cytochrome P450 lanosterol 14α-demethylase). In the present study, we amplified and sequenced the ERG11 gene of 23 C. albicans clinical isolates. Seventeen mutations encoding distinct amino acid substitutions were found, of which seven (K143Q, Y205E, A255V, E260V, N435V, G472R, and D502E) were novel. We further verified the contribution of the amino acid substitutions to azole resistance using site-directed mutagenesis of the ERG11 gene to recreate these mutations for heterologous expression in Saccharomyces cerevisiae. We observed that substitutions A114S, Y132H, Y132F, K143R, Y257H, and a new K143Q substitution contributed to significant increases (≧fourfold) in fluconazole and voriconazole resistance; changes in itraconazole resistance were not significant (≦twofold).