Clade A1 Research Articles

Natural Variation Reveals a Key Role for Rhamnogalacturonan I in Seed Outer Mucilage and Underlying Genes.

On imbibition, Arabidopsis (Arabidopsis thaliana) seeds release polysaccharides from their epidermal cells that form a two-layered hydrogel, termed mucilage. Analysis of a publicly available data set of outer seed mucilage traits of over 300 accessions showed little natural variation in composition. This mucilage is almost exclusively made up of rhamnogalacturonan I (RGI), highlighting the importance of this pectin for outer mucilage function. In a genome-wide association study, observed variations in polymer amount and macromolecular characteristics were linked to several genome polymorphisms, indicating the complexity of their genetic regulation. Natural variants with high molar mass were associated with a gene encoding a putative glycosyltransferase called MUCILAGE-RELATED70 (MUCI70). muci70 insertion mutants produced many short RGI polymers that were highly substituted with xylan, confirming that polymorphism in this gene can affect RGI polymer size. A second gene encoding a putative copper amine oxidase of clade 1a (CuAOα1) was associated with natural variation in the amount of RGI present in the outer mucilage layer; cuaoα1 mutants validated its role in pectin production. As the mutant phenotype is unique, with RGI production only impaired for outer mucilage, this indicates that CuAOα1 contributes to a further mechanism controlling mucilage synthesis.

Severe cases of seasonal influenza in Russia in 2017-2018

Aim. Evaluation of herd immunity prior to the 2017-2018 influenza season, and characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. Materials and methods. Evaluation of herd immunity in hemagglutination inhibition assay. Isolation of influenza viruses. Antigenic and genetic analysis. Results. Prior to epidemic season 33-47% of blood sera samples collected on the territory of Russia showed presence of protective antibody titers against vaccine strains of influenza A, 24-30% of samples — against B/Victoria. During 2017-2018 epidemic season 87 influenza A and B viruses were isolated. A(H1N1)pdm09 strains belonged to clade 6B.1, B/Yamagata strains to clade 3, and B/Victoria strains to clade 1A; they were antigenically similar to corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically. One strain of influenza virus А(H1N1pdm09) was resistant to oseltamivir and had H275Y amino acid substitution in neuraminidase. All other isolates were susceptible to neuraminidase inhibitors. Conclusion. Influenza vaccination with vaccine effective against current circulating strains and treatment with neuraminidase inhibitor drugs at first manifestation of clinical signs of influenza disease are effective means of population protection against influenza.

Severe cases of seasonal influenza in Russia in 2017-2018.

The 2017–2018 influenza epidemic season in Russia was characterized by a relatively low morbidity and mortality. We evaluated herd immunity prior to the 2017–2018 influenza season in hemagglutination inhibition assay, and performed characterization of influenza viruses isolated from severe or fatal influenza cases and from influenza cases in people vaccinated in the fall of 2017. During the 2017–2018 epidemic season, 87 influenza A and B viruses were isolated and viruses of the 75 influenza cases, including selected viral isolates and viruses analyzed directly from the original clinical material, were genetically characterized. The analyzed A(H1N1)pdm09 viruses belonged to clade 6B.1, B/Yamagata-like viruses belonged to clade 3, and B/Victoria-like viruses belonged to clade 1A and they were antigenically similar to the corresponding vaccine strains. A(H3N2) viruses belonged to clade 3C.2a and were difficult to characterize antigenically and the analysis indicated antigenic differences from the corresponding egg-grown vaccine strain. The next generation sequencing revealed the presence of D222/G/N polymorphism in the hemagglutinin gene in 32% of the analyzed A(H1N1)pdm09 lethal cases. This study demonstrated the importance of monitoring D222G/N polymorphism, including detection of minor viral variants with the mutations, in the hemagglutinin gene of A(H1N1)pdm09 for epidemiological surveillance. One strain of influenza virus A(H1N1)pdm09 was resistant to oseltamivir and had the H275Y amino acid substitution in the NA protein. All other isolates were susceptible to NA inhibitors. Prior to the 2017–2018 epidemic season, 67.4 million people were vaccinated, which accounted for 46.6% of the country's population. Just before the epidemic season 33–47% and 24–30% of blood sera samples collected within the territory of Russia showed the presence of protective antibody titers against vaccine strains of influenza A and influenza B/Victoria-like, respectively. Mass vaccination of the population had evidently reduced the severity of the flu epidemic during the 2017–2018 influenza epidemic season in Russia.

Dispersal of linezolid-resistant enterococci carrying poxtA or optrA in retail meat and food-producing animals from Tunisia.

The epidemiology of Enterococcus resistant to priority antibiotics including linezolid has mainly been investigated in developed countries and especially in hospitals. We aimed to evaluate the contribution of different non-human reservoirs for the burden of MDR enterococci in Tunisia, where scarce data are available. Samples (n = 287) were collected from urban wastewater (n = 57), retail meat (n = 29; poultry/bovine/ovine), milk (n = 89; bovine/ovine), farm animal faeces (n = 80; poultry/bovine/ovine) and pets (n = 32; rabbit/dogs/cats/birds) in different Tunisian regions (2014-17). They were plated onto Slanetz-Bartley agar after pre-enrichment without antibiotics. Standard methods were used for bacterial identification and characterization of antibiotic resistance and virulence genes (PCR), antibiotic susceptibility testing (disc diffusion/broth microdilution; EUCAST/CLSI) and clonality (SmaI-PFGE/MLST). All samples carried Enterococcus (n = 377 isolates) resistant to antibiotics considered to be critical or highly important by WHO. Even without antibiotic selection, 38% of Enterococcus faecalis (Efs) and 22% of Enterococcus faecium (Efm) were identified as MDR. Linezolid-resistant isolates (5%; MIC = 8 mg/L) comprised six poxtA-carrying Efm (cow milk), seven optrA-carrying Efs (chicken faeces/meat) and five Efm lacking cfr/optrA/poxtA (poultry/bovine/ovine/wastewater). Clinically relevant Efm clones (clade A1) were identified in animal/meat sources. Ampicillin resistance (1%) was confined to ST18/ST78-like MDR Efm clones from bovine meat/milk samples carrying relevant virulence markers (e.g. ptsD/IS16). This study provides evidence of the contribution of livestock and foodstuffs to the dispersal of acquired linezolid resistance genes including poxtA and optrA. We report the first poxtA-carrying Efm in Tunisia, and for the first time in bovine samples, stressing the urgent need for alternative measures to counteract the spread of linezolid-resistant enterococci globally.

Enterococcus faecium genome dynamics during long-term asymptomatic patient gut colonization.

Enterococcus faecium is a gut commensal of humans and animals. In addition, it has recently emerged as an important nosocomial pathogen through the acquisition of genetic elements that confer resistance to antibiotics and virulence. We performed a whole-genome sequencing-based study on 96 multidrug-resistant E. faecium strains that asymptomatically colonized five patients with the aim of describing the genome dynamics of this species. The patients were hospitalized on multiple occasions and isolates were collected over periods ranging from 15 months to 6.5 years. Ninety-five of the sequenced isolates belonged to E. faecium clade A1, which was previously determined to be responsible for the vast majority of clinical infections. The clade A1 strains clustered into six clonal groups of highly similar isolates, three of which consisted entirely of isolates from a single patient. We also found evidence of concurrent colonization of patients by multiple distinct lineages and transfer of strains between patients during hospitalization. We estimated the evolutionary rate of two clonal groups that each colonized single patients at 12.6 and 25.2 single-nucleotide polymorphisms (SNPs)/genome/year. A detailed analysis of the accessory genome of one of the clonal groups revealed considerable variation due to gene gain and loss events, including the chromosomal acquisition of a 37 kbp prophage and the loss of an element containing carbohydrate metabolism-related genes. We determined the presence and location of 12 different insertion sequence (IS) elements, with ISEfa5 showing a unique pattern of location in 24 of the 25 isolates, suggesting widespread ISEfa5 excision and insertion into the genome during gut colonization. Our findings show that the E. faecium genome is highly dynamic during asymptomatic colonization of the human gut. We observed considerable genomic flexibility due to frequent horizontal gene transfer and recombination, which can contribute to the generation of genetic diversity within the species and, ultimately, can contribute to its success as a nosocomial pathogen.

Manifestations of enterovirus D68 and high seroconversion among children attending a kindergarten.

Enterovirus D68(EV-D68) is an emerging disease that affects mostly children. There have been few relevant investigations to clarify transmission and seroprevalence within daycares and kindergartens. This prospective cohort study investigated respiratory viral transmission among preschool children in a public kindergarten in Taipei City of Taiwan between September 2006 and June 2008. After children were enrolled, daily monitoring of illness and regular biweekly physical examinations were performed. We performed viral isolation to detect acute EV-D68 infection and neutralization tests to detect specific EV-D68 antibodies and to measure the seroprevalence and seroconversion rates. Among 190 kindergarten attendees aged between two to five years old, nine children had acute EV-D68 infection in September 2007. The clinical manifestations included pharyngitis, cough and other unspecified upper respiratory tract infection. None of the infected children had acute flaccid paralysis or severe respiratory illness. The phylogenetic tree of partial viral protein 3 and viral protein 1 was clustered in clade A1. The EV-D68 seropositive rate increased from 19% (25/130) at the beginning to 67% (83/124) at the end of the study. The seroconversion rate of 49 children with initial seronegative and paired sera was 73% (36/49). A high seroconversion rate (73%) for EV-D68 was found among kindergarten attendees, which indicates preschool-aged children are highly susceptible to EV-D68 infection and that the disease burden may be extremely underestimated. Once EV-D 68 circulates, preventive measures may be advocated, especially within kindergartens or daycares, to reduce transmission and subsequent development of severe cases.

Arboviral screening of invasive Aedes species in northeastern Turkey: West Nile virus circulation and detection of insect-only viruses.

BackgroundThe recent reports of Aedes aegypti and Ae. albopictus populations in Turkey, in parallel with the territorial expansion identified in several surrounding countries, have raised concerns about the establishment and re-establishment of these invasive Aedes mosquitoes in Turkey. This cross-sectional study was performed to detect Aedes aegypti and Ae. albopictus in regions of recent incursions, and screen for viral pathogens known to be transmitted elsewhere by these species.MethodologyMosquitoes were collected at several locations in Artvin, Rize and Trabzon provinces of the Black Sea region during 2016–2017, identified morphologically, pooled and analyzed via generic or specific nucleic acid amplification assays. Viruses in positive pools were identified by product sequencing, cell culture inoculation and next generation sequencing (NGS) in selected specimens.Principal findingsThe study group comprised 791 specimens. Aedes albopictus was the most abundant species in all locations (89.6%), followed by Ae. aegypti (7.8%) and Culex pipiens (2.5%). Mosquitoes were screened for viruses in 65 pools where fifteen (23.1%) were reactive. The infecting strains was identified as West Nile virus (WNV) in 5 pools (7.7%) with Ae. albopictus or Cx. pipiens mosquitoes. The obtained WNV sequences phylogenetically grouped with local and global lineage 1 clade 1a viruses. In 4 (6.2%) and 6 (9.2%) pools, respectively, cell fusing agent virus (CFAV) and Aedes flavivirus (AEFV) sequences were characterized. NGS provided a near-complete AEFV genome in a pool of Ae. albopictus. The strain is provisionally called “AEFV-Turkey”, and functional analysis of the genome revealed several conserved motifs and regions associated with virus replication. Merida-like virus Turkey (MERDLVT), a recently-described novel rhabdovirus, was also co-detected in a Cx. pipiens pool also positive for WNV.Conclusions/SignificanceInvasive Aedes mosquitoes are established in certain locations of northeastern Turkey. Herein we conclusively show the role of these species in WNV circulation in the region. Biosurveillance is imperative to monitor the spread of these species further into Asia Minor and to detect possible introduction of pathogens.

Ongoing activity of invasive aedes species in Northern Anatolia: lack of Chikungunya despite West Nile virus circulation

Purpose: In order to screen for invasive mosquito species and associated viruses, we performed a field survey for mosquitoes in the Black Sea region of Anatolia where Aedes aegypti and Aedes albopictus were previously recorded. Methods & Materials: Mosquitoes were collected from 31 sites in Artvin, Trabzon and Rize provinces during 2016-2017. The specimens were identified morphologically and pooled according to collection site and species. Selected specimens were processed for DNA barcoding via cytochrome oxidase I amplification and sequencing, for confirmation of the morphological species identification. Virus screening was carried out using polymerase chain reaction (PCR) assays targeting alpha and flaviviruses, as well as recently-described novel rhabdovirus Merida-like virus Turkey (MERDLVT), followed by sequencing for characterization. Results: A total of 756 mosquitoes that comprise Ae. albopictus (675, 89,2%), Ae. aegypti (61, 8,1%) and Culex pipiens sensu lato (20, 2,6%) were collected and grouped in 65 pools. No amplification was observed in any pool via generic alphavirus PCR. Generic flavivirus PCR was reactive in 7 and 8 pools collected in 2016 and 2017, respectively. Cell fusing agent virus (CFAV) sequences were characterized in 4 pools (6,1%) of Ae. albopictus (n = 2) and Ae. aegypti (n = 2), collected in 2016. Aedes flavivirus (AFEV) sequences were characterized in 6 pools (9,2%) of Ae. albopictus (n = 5) and and Ae. aegypti (n = 1), collected in 2016 (n = 3) and 2017 (n = 3). Sequences of West Nile virus (WNV) was detected in 5 pools (7,6%) of Ae. albopictus (n = 4) and and Cx. pipiens s.l. (n = 1), collected in 2017. In phylogenetic analyses, the WNV sequences clustered with local and global lineage 1 clade 1a strains. Moreover, partial L and N gene sequences of MERDLVT were identified in the Cx. pipiens s.l. pool, coinfected with WNV. Conclusion: This is the initial detection of WNV and MERDLVT in field-collected mosquitoes from the Black Sea region. Although no alphavirus sequence could be demonstrated, presence of Ae. albopictus and Ae. aegypti indicates ongoing risk for potential spread.

The Molecular Basis of pH-Modulated HIV gp120 Binding Revealed

Decades of research has yet to provide a vaccine for HIV, the virus which causes AIDS. Recent theoretical research has turned attention to mucosa pH levels over systemic pH levels. Previous research in this field developed a computational approach for determining pH sensitivity that indicated higher potential for transmission at mucosa pH levels present during intercourse. The process was extended to incorporate a principal component analysis (PCA)-based machine learning technique for classification of gp120 proteins against a known transmitted variant called Biomolecular Electro-Static Indexing (BESI). The original process has since been extended to the residue level by a process we termed Electrostatic Variance Masking (EVM) and used in conjunction with BESI to determine structural differences present among various subspecies across Clades A1 and C. Results indicate that structures outside of the core selected by EVM may be responsible for binding affinity observed in many other studies and that pH modulation of select substructures indicated by EVM may influence specific regions of the viral envelope protein (Env) involved in protein-protein interactions.

Evolution and mutations predisposing to daptomycin resistance in vancomycin-resistant Enterococcus faecium ST736 strains.

We recently identified a novel vancomycin-resistant Enterococcus faecium (VREfm) clone ST736 with reduced daptomycin susceptibility. The objectives of this study were to assess the population dynamics of local VREfm strains and genetic alterations predisposing to daptomycin resistance in VREfm ST736 strains. Multilocus sequence typing and single nucleotide variant data were derived from whole-genome sequencing of 250 E. faecium isolates from 1994–1995 (n = 43), 2009–2012 (n = 115) and 2013 (n = 92). A remarkable change was noticed in the clonality and antimicrobial resistance profiles of E. faecium strains between 1994–1995 and 2013. VREfm sequence type 17 (ST17), the prototype strain of clade A1, was the dominant clone (76.7%) recognized in 1994–1995. By contrast, clone ST736 accounted for 46.7% of VREfm isolates, followed by ST18 (26.1%) and ST412 (20.7%) in 2013. Bayesian evolutionary analysis suggested that clone ST736 emerged between 1996 and 2009. Co-mutations (liaR.W73C and liaS.T120A) of the liaFSR system were identified in all ST736 isolates (n = 111, 100%) examined. Thirty-eight (34.2%) ST736 isolates exhibited daptomycin-resistant phenotype, of which 13 isolates had mutations in both the liaFSR and cardiolipin synthase (cls) genes and showed high level of resistance with a daptomycin MIC50 of 32 μg/mL. The emergence of ST736 strains with mutations predisposing to daptomycin resistance and subsequent clonal spread among inpatients contributed to the observed high occurrence of daptomycin resistance in VREfm at our institution. The expanding geographic distribution of ST736 strains in other states and countries raises concerns about its global dissemination.

Bioinformatic characterisation of the effector repertoire of the strawberry pathogen Phytophthora cactorum.

The oomycete pathogen Phytophthora cactorum causes crown rot, a major disease of cultivated strawberry. We report the draft genome of P. cactorum isolate 10300, isolated from symptomatic Fragaria x ananassa tissue. Our analysis revealed that there are a large number of genes encoding putative secreted effectors in the genome, including nearly 200 RxLR domain containing effectors, 77 Crinklers (CRN) grouped into 38 families, and numerous apoplastic effectors, such as phytotoxins (PcF proteins) and necrosis inducing proteins. As in other Phytophthora species, the genomic environment of many RxLR and CRN genes differed from core eukaryotic genes, a hallmark of the two-speed genome. We found genes homologous to known Phytophthora infestans avirulence genes including Avr1, Avr3b, Avr4, Avrblb1 and AvrSmira2 indicating effector sequence conservation between Phytophthora species of clade 1a and clade 1c. The reported P. cactorum genome sequence and associated annotations represent a comprehensive resource for avirulence gene discovery in other Phytophthora species from clade 1 and, will facilitate effector informed breeding strategies in other crops.

Genetic characterization of influenza B virus in Cameroon and high frequency of reassortant strains.

Influenza B is broadly divided into B/Victoria and B/Yamagata lineages based on its genetic and antigenic properties. We describe in this study the first report on genome characterization of type B influenza virus in the Cameroon National Influenza Center (NIC) between 2014 and 2017. Respiratory samples were collected as part of the influenza surveillance activity in the NIC. RNA products were tested for the presence of influenza using the CDC Influenza A/B typing panel. Thirty-five samples positive for influenza B were selected for sequencing three gene segments (HA, NA, and M) and phylogenetic trees were generated by MEGA version 6.0. Nucleotide phylogenetic analysis of the HA gene revealed the presence of three major clades among Cameroonian strains. All Victoria lineages grouped into B/Victoria clade 1A, while, Yamagata lineages grouped into Yamagata clade 2 (2014 strains) and Yamagata clade 3 (2015-2017). We observed a high frequency of reassortant viruses with Yamagata-like HA gene and Victoria-like NA gene (27.4%; 23/84). The results from this study confirm variations in the genome composition of type B influenza virus and emphasize on the relevance of molecular surveillance for spotting peculiar genetic variants of public health and clinical significance.

Identification and Characterization of Positively Selected Mutations in Nef of Four HIV-1 Major Subtypes from Los Alamos National Laboratory.

Human immunodeficiency virus-1 (HIV-1) mutates rapidly to escape host immune pressure. This results in the generation of positively selected mutations (PSM) throughout the viral genome. Escape mutations in Nef, one of the accessory proteins of HIV-1, which plays an important role in viral pathogenicity have previously been identified in several large cohort studies, but the evolution of PSMs overtime in various HIV-1 subtypes remains unknown. 161 clade A1, 3093 clade B, 647 clade C and 115 clade D HIV-1 nef sequences were obtained from the HIV Database of Los Alamos National Laboratory and aligned using MEGA 6.0. The sequences from each clade were grouped based on the year of collection. Quasi analysis was used to identify PSMs and the number and locations of PSMs were compared among different subtypes. PSMs for all four subtypes were distributed across the sequence of Nef, and conserved residues F90, W113, PxxPxR (a.a 72-77) remain unaltered overtime. The frequency of PSMs was stable among subtype B sequences but increased overtime for other subtypes. Phylogenetic analysis shows that sequences containing PSMs tend to cluster together at both inter and intra- subtype levels. Identification of PSMs and their changes overtime within various subtypes of HIV-1 is important in defining global viral evolutionary patterns that can provide insights for designing therapeutic strategies.

Rapid whole-genome based typing and surveillance of avipoxviruses using nanopore sequencing

Avian pox is an infectious disease caused by avipoxviruses (APV), resulting in cutaneous and/or tracheal lesions. Poxviruses share large genome sizes (from 130 to 360 kb), featuring repetitions, deletions or insertions as a result of a long-term recombination history.The increasing performances of next-generation sequencing (NGS) opened new opportunities for surveillance of poxviruses, based on timely and affordable workflows. We investigated the application of the 3rd generation Oxford Nanopore Minion technology to achieve real-time whole-genome sequencing directly from lesions, without any enrichment or isolation step.Fowlpox lesions were sampled on hens, total DNA was extracted and processed for sequencing on a MinION, Oxford Nanopore. We readily generated whole APV genomes from cutaneous or tracheal lesions, without any isolation or PCR-based enrichment: Fowlpox virus reads loads ranged from 0.75% to 2.62% and reads up to 61 kbp were generated and readily assembled into 3 APV complete genomes. This long read size eases the assembly step and lowers the bioinformatics capacity requirements and processing time compared to huge sets of short reads. The complete genome analysis confirmed that these Fowlpox viruses cluster within clade A1 and host full length reticuloendotheliovirus (REV) inserts. The pathobiological relevance of REV insert, although a classical feature of fowlpoxviruses (FPVs), should be further investigated. Surveillance of emerging poxviruses could greatly benefit from real-time whole genome sequencing.

Molecular characterization of avipoxviruses circulating in Mozambique, 2016-2018.

Samples from 45 chickens, two turkeys, one peacock and one quail with symptoms of fowlpox were collected in Mozambique between November 2016 and January 2018. Phylogenetic analysis revealed that the samples contained avipoxviruses belonging to both clade A1 and clade A2. In addition, all of the Clade A1 viruses were positive by PCR for the integration of reticuloendotheliosis virus, while the clade A2 avipoxvirus samples were negative. This study confirms the circulation of clade A1 avipoxviruses in Mozambique in addition to identifying clade A2 for the first time in the country.

Distribution of putative virulence markers in Enterococcus faecium: towards a safety profile review.

The criteria for identification of Enterococcus faecium (Efm) with the ability to cause human infections are currently being debated by the European Food Safety Authority (EFSA). Strains that have an MIC of ampicillin of ≤ 2 mg/L and lack IS16/esp/hyl genes should be regarded as safe for use as feed additives in animal nutrition, despite the lack of knowledge about putative virulence marker (PVM) distribution in community Efm. We analysed the distribution of major PVM and ampicillin phenotypes in large Efm collections to investigate further the safety of strains from a public health perspective. Thirty-three PVM were assessed by PCR/sequencing among clonally disparate Efm (n = 328; 1986-2015) from different origins. We analysed ampicillin susceptibility (Etest/broth microdilution) according to EUCAST guidelines, clonal relationship (MLST) and genomic location of PVM (S1-PFGE/hybridization). Infection-derived Efm were more enriched in PVM and the increase in ampicillin MIC was positively correlated with an enrichment in different PVM. PVM coding for surface (esp/sgrA/ecbA/complete acm) and pili proteins, or others enhancing colonization (hyl/ptsD/orf1481) or plasticity (IS16), were strongly associated with clinical Efm (mostly clade A1), but also observed in clades A2/B at different rates. ptsD was a good marker of ampicillin-resistant Efm. ptsD, IS16, orf1481, sgrA and hospital variants of complete pili gene clusters are proposed as markers to assess the safety of Efm strains. Our study expands on the distribution of PVM in diverse Efm lineages and demonstrates the enrichment in infection-derived strains of PVM not previously included in EFSA's list of Efm safety criteria. The evidence of relevant Efm infection markers can impact the risk assessment of Efm strains in different public health contexts.

West Nile virus, Anopheles flavivirus, a novel flavivirus as well as Merida-like rhabdovirus Turkey in field-collected mosquitoes from Thrace and Anatolia

Mosquitoes are involved in the transmission and maintenance of several viral diseases with significant health impact. Biosurveillance efforts have also revealed insect-specific viruses, observed to cocirculate with pathogenic strains. This report describes the findings of flavivirus and rhabdovirus screening, performed in eastern Thrace and Aegean region of Anatolia during 2016, including and expanding on locations with previously-documented virus activity. A mosquito cohort of 1545 individuals comprising 14 species were collected and screened in 108 pools via generic and specific amplification and direct metagenomics by next generation sequencing. Seven mosquito pools (6.4%) were positive in the flavivirus screening. West Nile virus lineage 1 clade 1a sequences were characterized in a pool Culex pipiens sensu lato specimens, providing the initial virus detection in Aegean region following 2010 outbreak. In an Anopheles maculipennis sensu lato pool, sequences closely-related to Anopheles flaviviruses were obtained, with similarities to several African and Australian strains of this new insect-specific flavivirus clade. In pools comprising Uranotaenia unguiculata (n=3), Cx. pipiens s.l. (n=1) and Aedes caspius (n=1) mosquitoes, sequences of a novel flavivirus, distantly-related to Flavivirus AV2011, identified previously in Spain and Turkey, were characterized. Moreover, DNA forms of the novel flavivirus were detected in two Ur. unguiculata pools. These sequences were highly-similar to the sequences amplified from viral RNA, with undisrupted reading frames, suggest the occurrence of viral DNA forms in natural conditions within mosquito hosts. Rhabdovirus screening revealed sequences of a recently-described novel virus, named the Merida-like virus Turkey (MERDLVT) in 5 Cx. pipiens s.l. pools (4.6%). Partial L and N gene sequences of MERDLVT were well-conserved among strains, with evidence for geographical clustering in phylogenetic analyses. Metagenomics provided the near-full genomic sequence in a specimen, revealing an identical genome organization and limited divergence from the prototype MERDLVT isolate.

Complete Genome Sequence of West Nile Virus (WNV) from the First Human Case of Neuroinvasive WNV Infection in Cyprus

ABSTRACTWe report here the complete genome sequence of a West Nile virus (WNV) strain from the first laboratory-confirmed human case of neuroinvasive WNV infection in Cyprus. Phylogenetic analysis showed that this WNV strain grouped clearly into genetic lineage 1, clade 1a, cluster 2.

Association of coral algal symbionts with a diverse viral community responsive to heat shock

BackgroundStony corals provide the structural foundation of coral reef ecosystems and are termed holobionts given they engage in symbioses, in particular with photosynthetic dinoflagellates of the genus Symbiodinium. Besides Symbiodinium, corals also engage with bacteria affecting metabolism, immunity, and resilience of the coral holobiont, but the role of associated viruses is largely unknown. In this regard, the increase of studies using RNA sequencing (RNA-Seq) to assess gene expression provides an opportunity to elucidate viral signatures encompassed within the data via careful delineation of sequence reads and their source of origin.ResultsHere, we re-analyzed an RNA-Seq dataset from a cultured coral symbiont (Symbiodinium microadriaticum, Clade A1) across four experimental treatments (control, cold shock, heat shock, dark shock) to characterize associated viral diversity, abundance, and gene expression. Our approach comprised the filtering and removal of host sequence reads, subsequent phylogenetic assignment of sequence reads of putative viral origin, and the assembly and analysis of differentially expressed viral genes. About 15.46% (123 million) of all sequence reads were non-host-related, of which <1% could be classified as archaea, bacteria, or virus. Of these, 18.78% were annotated as virus and comprised a diverse community consistent across experimental treatments. Further, non-host related sequence reads assembled into 56,064 contigs, including 4856 contigs of putative viral origin that featured 43 differentially expressed genes during heat shock. The differentially expressed genes included viral kinases, ubiquitin, and ankyrin repeat proteins (amongst others), which are suggested to help the virus proliferate and inhibit the algal host’s antiviral response.ConclusionOur results suggest that a diverse viral community is associated with coral algal endosymbionts of the genus Symbiodinium, which prompts further research on their ecological role in coral health and resilience.

Global phylogenetic relationships, population structure and gene flow estimation of Trialeurodes vaporariorum (Greenhouse whitefly).

Trialeurodes vaporariorum (Westwood, 1856) (Greenhouse whitefly) is an agricultural pest of global importance. It is associated with damage to plants during feeding and subsequent virus transmission. Yet, global phylogenetic relationships, population structure, and estimation of the rates of gene flow within this whitefly species remain largely unexplored. In this study, we obtained and filtered 227 GenBank records of mitochondrial cytochrome c oxidase I (mtCOI) sequences of T. vaporariorum, across various global locations to obtain a final set of 217 GenBank records. We further amplified and sequenced a ~750 bp fragment of mtCOI from an additional 31 samples collected from Kenya in 2014. Based on a total of 248 mtCOI sequences, we identified 16 haplotypes, with extensive overlap across all countries. Population structure analysis did not suggest population differentiation. Phylogenetic analysis indicated the 2014 Kenyan collection of samples clustered with a single sequence from the Netherlands to form a well-supported clade (denoted clade 1a) nested within the total set of sequences (denoted clade 1). Pairwise distances between sequences show greater sequence divergence between clades than within clades. In addition, analysis using migrate-n gave evidence for recent gene flow between the two groups. Overall, we find that T. vaporariorum forms a single large group, with evidence of further diversification consisting primarily of Kenyan sequences and one sequence from the Netherlands forming a well-supported clade.