Identification Of Clades Research Articles

P-60. Lineage dynamics are not the major drivers of Streptococcus pneumoniae serotype 3 vaccine escape

Abstract Background Streptococcus pneumoniae serotype 3 (Spn3) is included in the pneumococcal 13-valent conjugate vaccine (PCV13) but carriage and invasive disease persist worldwide. Previous work showed that the prevalent clonal complex 180 (CC180) of Spn3 is comprised of 3 genetically distinct lineages: clade Iα, Iβ, and II; a post-PCV13 shift towards increased incidence of clade II strains with a concomitant decrease in clade Iα and 1β incidence has been observed in the United States, England, and Wales. It was hypothesized that PCV13 is controlling clade Iα and 1β but not clade II, resulting in clade II expansion and ultimately rendering the vaccine ineffective against Spn3. It is imperative to test this hypothesis in other regional contexts given the implications on vaccine design and efficacy. Therefore, our group assessed the dynamics of Spn3 population structure and PCV13 in Portugal. Prevalence of Spn3 clonal complexes before and after the introduction of PCV13 in Portugal Methods We whole-genome sequenced 501 Spn3 strains from Portugal and analyzed over 1400 Spn3 whole genome sequences from published datasets for global comparisons. Phylogenetic tree construction, clade identification, clonal complex typing, and annotation of antimicrobial resistance (AMR) and virulence genes was used to evaluate changes in bacterial genetics. Prevalence of Spn3 clades before and after the introduction of PCV13 in Portugal Results Consistent with previous work, the prevalence of clonal complexes significantly changed before and after the introduction of PCV13 in Portugal (p=4.94-12): CC180 increased from 29% of Spn3 strains to 65%. The prevalence of lineages also changed (p=2.33-13): clade Iα increased from 21% to 54% of Spn3 strains, while clade II was not detected pre-PCV13 but increased to only 5% post-PCV13. This contrasts with studies conducted elsewhere. The phylogenetic distribution of AMR alleles is similar across different regions, so altered antibiotic fitness does not explain the continued success of clade Iα over clade II in Portugal. Conclusion Our data suggests clade II is not the global driver of Spn3 PCV13 vaccine escape as clade Iα remains dominant in Portugal. Current work aims to determine if clade Iα allelic variants augment virulence and thus competitive fitness against clade II in Portugal. Informed vaccine design requires an understanding of the region-specific bacterial population structure and genetic features to optimize efficacy. Disclosures Richard Malley, MD, Amplitude Therapeutics: Board Member|Corner Therapeutics: Board Member|GSK: Advisor/Consultant|Merck: Advisor/Consultant Mario Ramirez, PhD, GSK: Advisor/Consultant|Merck Sharp and Dohme: Advisor/Consultant|Pfizer: Honoraria

Candida auris Antifungal Resistance, Virulence and Susceptibility to a Novel Nitric Oxide-Releasing Microparticle and Its Correlations to Clade Identification

Candida auris is a globally emerging pathogenic fungus described in Japan in 2009. This fungus has been identified mainly in nosocomial environments, associated with a high frequency of multidrug-resistant strains, and mortality rates reach 60%. C. auris is divided into 6 biogeographic clades, and there is a correlation between the clades and resistance against antifungals. In the current report, 8 strains of C. auris isolated in the Montefiore Medical Center, Bronx were analyzed to assess their clade (via ClaID) and common molecular determinants of antifungal resistance. We assessed antifungal resistance as well as the efficacy of a novel nitric oxide-donating microparticle as an alternative approach against C. auris in vitro through microplate susceptibility tests. Virulence was also determined in a Galleria mellonella model. Our results indicate that 7 out of 8 strains, belonging to clade 1, were resistant to fluconazole, while clade 2 was susceptible. Additionally, the clade 2 strain was more susceptible to treatment with the microparticle, while also being more virulent in an invertebrate model of infection. Our findings were then correlated to visualize parallels between clade identification and resistance/virulence patterns.

Varicella Vaccination: a Potential Strategy for Preventing Multiple Sclerosis

Multiple sclerosis (MS) is a debilitating neurological condition affecting approximately 2.9 million people worldwide. Its cause remains unclear but environmental factors, such as post-childhood Epstein-Barr virus (EBV) infection, are thought to contribute to MS incidence. This study presents a new hypothesis that MS may be initiated by primary infection with clade 3 varicella-zoster virus (VZV) preceding primary EBV infection. The hypothesis may explain several MS epidemiological features: geographic variation in prevalence, changes in incidence and sex ratios over time, migration patterns, higher EBV and VZV seropositivity rates in patients versus healthy controls, extremely high MS rates in Sardinia, and identification of clade 3 VZV in a single MS patient. If validated, the hypothesis predicts exclusive detection of clade 3 VZV in MS patients, declining MS incidence in countries implementing universal childhood varicella vaccination since the early 2000s, and potential therapeutic benefits for existing MS patients from treatments that prevent herpesvirus reactivation.

Reflective Evaluation of Next-Generation Sequencing Data during Early Phase Detection of the Delta Variant

During the SARS-CoV-2 pandemic, next-generation sequencing (NGS) technologies like the Ion Torrent S5 and Illumina MiSeq, alongside advanced software, improved genomic surveillance in South Africa. This study analysed anonymized samples from the Eastern Cape using Genome Detective and NextClade, showing Ion Torrent S5 and Illumina MiSeq success rates of 96% and 94%, respectively. The study focused on genomic coverage (above 80%) and mutation detection (below 100), with the Ion Torrent S5 achieving 99% coverage compared to Illumina MiSeq's 80%, likely due to different primers used in amplification. The Ion Torrent S5 was more effective in sequencing varied viral loads, whereas Illumina MiSeq had difficulties with lower loads. Both platforms were adept at identifying clades, successfully differentiating between Beta (<45%) and Delta variants (<30%), despite minor discrepancies in assignments due to Illumina MiSeq's lower coverage, leading to a failure rate of up to 6%. Manual library preparation showed similar sample processing and clade identification capabilities for both platforms. However, differences in sequencing duration (3.5 vs. 36 hours), automation level, genomic coverage (80% vs. 99%), and viral load compatibility were noted, highlighting each platform's unique advantages and challenges in SARS-CoV-2 genomic surveillance. In conclusion, the Illumina MiSeq and Ion Torrent S5 platforms are both efficacious in executing whole-genome sequencing (WGS) via amplicons, facilitating precise, accurate, and high-throughput examinations of SARS-CoV-2 viral genomes. However, it is important to note the existence of disparities in the quality of data produced by each platform. Each system offers unique benefits and limitations, rendering them viable choices for the genomic surveillance of SARS-CoV-2.

AutoPhy: Automated phylogenetic identification of novel protein subfamilies.

Phylogenetic analysis of protein sequences provides a powerful means of identifying novel protein functions and subfamilies, and for identifying and resolving annotation errors. However, automation of functional clustering based on phylogenetic trees has been challenging and most of it is done manually. Clustering phylogenetic trees usually requires the delineation of tree-based thresholds (e.g., distances), leading to an ad hoc problem. We propose a new phylogenetic clustering approach that identifies clusters without using ad hoc distances or other pre-defined values. Our workflow combines uniform manifold approximation and projection (UMAP) with Gaussian mixture models as a k-means like procedure to automatically group sequences into clusters. We then apply a "second pass" clade identification algorithm to resolve non-monophyletic groups. We tested our approach with several well-curated protein families (outer membrane porins, acyltransferase, and nuclear receptors) and showed our automated methods recapitulated known subfamilies. We also applied our methods to a broad range of different protein families from multiple databases, including Pfam, PANTHER, and UniProt, and to alignments of RNA viral genomes. Our results showed that AutoPhy rapidly generated monophyletic clusters (subfamilies) within phylogenetic trees evolving at very different rates both within and among phylogenies. The phylogenetic clusters generated by AutoPhy resolved misannotations and identified new protein functional groups and novel viral strains.

Emergence of a new genotype of clade 2.3.4.4b H5N1 highly pathogenic avian influenza A viruses in Bangladesh

ABSTRACT Influenza virological surveillance was conducted in Bangladesh from January to December 2021 in live poultry markets (LPMs) and in Tanguar Haor, a wetland region where domestic ducks have frequent contact with migratory birds. The predominant viruses circulating in LPMs were low pathogenic avian influenza (LPAI) H9N2 and clade 2.3.2.1a highly pathogenic avian influenza (HPAI) H5N1 viruses. Additional LPAIs were found in both LPM (H4N6) and Tanguar Haor wetlands (H7N7). Genetic analyses of these LPAIs strongly suggested long-distance movement of viruses along the Central Asian migratory bird flyway. We also detected a novel clade 2.3.4.4b H5N1 virus from ducks in free-range farms in Tanguar Haor that was similar to viruses first detected in October 2020 in The Netherlands but with a different PB2. Identification of clade 2.3.4.4b HPAI H5N1 viruses in Tanguar Haor provides continued support of the role of migratory birds in transboundary movement of influenza A viruses (IAV), including HPAI viruses. Domestic ducks in free range farm in wetland areas, like Tangua Haor, serve as a conduit for the introduction of LPAI and HPAI viruses into Bangladesh. Clade 2.3.4.4b viruses have dominated in many regions of the world since mid-2021, and it remains to be seen if these viruses will replace the endemic clade 2.3.2.1a H5N1 viruses in Bangladesh.

P414 Diagnostic allele-specific PCR for the identification of Candida auris clades and common resistance mutations

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Candida auris (C. auris) is an opportunistic pathogenic yeast that emerged worldwide during the past decade. This fungal pathogen poses a significant public health threat due to common multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have identified five distinct clades that are linked to five geographic areas of origin and characterized by differences in several phenotypic traits such as virulence and drug resistance. Typing of C. auris strains and the identification of clades can be a powerful tool in molecular epidemiology and might be of clinical importance by estimating outbreak and MDR potential. As C. auris has caused global outbreaks, including in low-income countries, typing C. auris strains quickly and inexpensively is highly valuable. We report five allele-specific multiplex polymerase chain reaction (AS-multiplex PCR) assays for the identification of C. auris and each of the five described clades of C. auris based on conserved mutations in the internal transcribed spacer (ITS) rDNA region and a clade-specific gene cluster. Additionally, we developed AS-PCR assays for the identification of SNPs in FKS1 and ERG11 that are commonly linked to echinocandin and azole resistance respectively. This PCR method provides a fast, cheap, sequencing-free diagnostic tool for the identification of C. auris, C. auris clades, and common resistance mutations.

Phylogenetic and Evolutionary Genomic Analysis of Listeria monocytogenes Clinical Strains in the Framework of Foodborne Listeriosis Risk Assessment.

Listeria monocytogenes is one of the most important foodborne pathogens responsible for listeriosis, a severe disease with symptoms ranging from septicemia, meningoencephalitis, and abortion. Given the strong impact of listeriosis on human health and the difficulty of controlling L. monocytogenes along the food production chain, listeriosis has become a priority subjected to molecular surveillance in European Union/European Economic Area since 2007. From 2018, surveillance is based on whole-genome sequence using the core genome multilocus sequence type. The complete sequences of 132 clinical strains were used to define the evolutionary relatedness among subtypes of L. monocytogenes isolated in Italy from 2010 to 2016, allowing the identification of clades and/or clusters associated with outbreaks or sporadic cases of listeriosis. All the strains analyzed are clustered in lineages I and II, and the majority of the strains were classified as lineage II. A probable epidemic entrance in different years for every clade and cluster from each different region was defined. The persistence of the same specific clonal complexes of L. monocytogenes has been found over long periods; this may be related to the fact that some strains are able to survive better than others in a food production environment. Phylogenic studies, using whole-genome sequence data, are able to identify the emergence of highly persistent pathogenic variants, contributing to improving the hazard characterization of L. monocytogenes.

Phylogenetic organization in the assimilation of chemically distinct substrates by soil bacteria.

Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non-random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition.

Diagnostic Allele-Specific PCR for the Identification of Candida auris Clades.

Candida auris is an opportunistic pathogenic yeast that emerged worldwide during the past decade. This fungal pathogen poses a significant public health threat due to common multidrug resistance (MDR), alarming hospital outbreaks, and frequent misidentification. Genomic analyses have identified five distinct clades that are linked to five geographic areas of origin and characterized by differences in several phenotypic traits such as virulence and drug resistance. Typing of C. auris strains and the identification of clades can be a powerful tool in molecular epidemiology and might be of clinical importance by estimating outbreak and MDR potential. As C. auris has caused global outbreaks, including in low-income countries, typing C. auris strains quickly and inexpensively is highly valuable. We report five allele-specific polymerase chain reaction (AS-PCR) assays for the identification of C. auris and each of the five described clades of C. auris based on conserved mutations in the internal transcribed spacer (ITS) rDNA region and a clade-specific gene cluster. This PCR method provides a fast, cheap, sequencing-free diagnostic tool for the identification of C. auris, C. auris clades, and potentially, the discovery of new clades.

Linking 16S rRNA Gene Classification to amoA Gene Taxonomy Reveals Environmental Distribution of Ammonia-Oxidizing Archaeal Clades in Peatland Soils.

A highly resolved taxonomy for ammonia-oxidizing archaea (AOA) based on the alpha subunit of ammonia monooxygenase (amoA) was recently established, which uncovered novel environmental patterns of AOA, challenging previous generalizations. However, many microbiome studies target the 16S rRNA gene as a marker; thus, the usage of this novel taxonomy is currently limited. Here, we exploited the phylogenetic congruence of archaeal amoA and 16S rRNA genes to link 16S rRNA gene classification to the novel amoA taxonomy. We screened publicly available archaeal genomes and contigs for the co-occurring amoA and 16S rRNA genes and constructed a 16S rRNA gene database with the corresponding amoA clade taxonomy. Phylogenetic trees of both marker genes confirmed congruence, enabling the identification of clades. We validated this approach with 16S rRNA gene amplicon data from peatland soils. We succeeded in linking 16S rRNA gene amplicon sequence variants belonging to the class Nitrososphaeria to seven different AOA (amoA) clades, including two of the most frequently detected clades (Nitrososphaerales γ and δ clades) for which no pure culture is currently available. Water status significantly impacted the distribution of the AOA clades as well as the whole AOA community structure, which was correlated with pH, nitrate, and ammonium, consistent with previous clade predictions. Our study emphasizes the need to distinguish among AOA clades with distinct ecophysiologies and environmental preferences, for a better understanding of the ecology of the globally abundant AOA. IMPORTANCE The recently established phylogeny of amoA provides a finer resolution than previous studies, allowing clustering of AOA beyond the order level and thus revealing novel clades. While the 16S rRNA gene is mostly appreciated in microbiome studies, this novel phylogeny is in limited use. Here, we provide an alternative path to identifying AOA with this novel and highly resolved amoA taxonomy by using 16S rRNA gene sequencing data. We constructed a 16S rRNA gene database with the associated amoA clade taxonomy based on their phylogenetic congruence. With this database, we were able to assign 16S rRNA gene amplicons from peatland soils to different AOA clades, with a level of resolution provided previously only by amoA phylogeny. As 16S rRNA gene amplicon sequencing is still widely employed in microbiome studies, our database may have a broad application for interpreting the ecology of globally abundant AOA.

Clade distribution of Candida auris in South Africa using whole genome sequencing of clinical and environmental isolates

ABSTRACT In South Africa, Candida auris was the third most common cause of candidemia in 2016–2017. We performed single nucleotide polymorphism (SNP) genome-wide analysis of 115 C. auris isolates collected between 2009 and 2018 from national laboratory-based surveillance, an environmental survey at four hospitals and a colonization study during a neonatal unit outbreak. The first known South African C. auris strain from 2009 clustered in clade IV. Overall, 98 strains clustered within clade III (85%), 14 within clade I (12%) and three within clade IV (3%). All environmental and colonizing strains clustered in clade III. We also identified known clade-specific resistance mutations in the ERG11 and FKS1 genes. Identification of clade I strains between 2016 and 2018 suggests introductions from South Asia followed by local transmission. SNP analysis characterized most C. auris strains into clade III, the clade first reported from South Africa, but the presence of clades I and IV strains also suggest early introductions from other regions.

Isolation of Harveyi clade Vibrio spp. collected in aquaculture farms: How can the identification issue be addressed?

Aquaculture is a fast growing industry with its development hampered by bacterial diseases. Vibriosis caused by Harveyi clade strains is known for causing heavy loss especially in shrimp aquaculture farms. For farm treatment and pathogen spread management, veterinarians and researchers need reliable bacterial identification tools. A range of identification methods have been presented for Vibrio spp. in recent literature but little feedback on their performance have been made available to this day.This study aims at comparing Vibrio spp. identification methods and providing guidance on their use.Fifty farms were sampled and bacterial colonies were isolated using specific culture media before microscopic analysis and genomic profiling using ERIC-PCR. A preliminary identification step was carried out using MALDI-ToF mass spectrometry. Four methods were compared for strain identification on 14 newly isolated Harveyi clade Vibrio spp. strains: whole genome sequencing (digital DNA DNA Hybridization (dDDH)), 5 MLSA schemes, ferric uptake regulation (fur) and lecithin-dependent haemolysin (ldh) single gene based identification methods.Apart from dDDH which is a reference method, no technique could identify all the isolates to the species level. The other tested techniques allowed a faster, cheaper but sub genus clade identification which can be interesting when absolute precision is not required. In this regard, MALDI-ToF and fur based identification seemed especially promising.

Introductions and early spread of SARS-CoV-2 in France, 24 January to 23 March 2020.

Following SARS-CoV-2 emergence in China, a specific surveillance was implemented in France. Phylogenetic analysis of sequences retrieved through this surveillance suggests that detected initial introductions, involving non-clade G viruses, did not seed local transmission. Nevertheless, identification of clade G variants subsequently circulating in the country, with the earliest from a patient who neither travelled to risk areas nor had contact with travellers, suggests that SARS-CoV-2 might have been present before the first recorded local cases.

Plastid-encoded gene comparison reveals usefulness of atpB, psaA, and rbcL for identification and phylogeny of plastid-containing cryptophyte clades

ABSTRACTCryptophytes are a ubiquitous algal group and are important primary producers in aquatic ecosystems. Identification of lineages within the group is difficult because of their simple morphology and they are often enumerated at the class level in ecological research. Despite the increasing number of molecular probes used for identification of microorganisms, rRNA genes (i.e. nuclear 18S, ITS, and 28S, and nucleomorph 18S) are the markers most commonly used to identify cryptophytes. To provide a broader choice of markers, we compared and characterised the utility of 13 plastid genes in 23 culture strains, which cover the spectrum cryptophycean lineages (Clades 1–5). The genes include 11 protein-coding (atpA, atpB, chlI, clpC, dnaK, psaA, psbA, rbcL, rpoC1, secA, and tufA) and two ribosomal RNA (plastid 16S and 23S) genes. Most of the genes were successfully amplified and sequenced using newly designed primers, and the sequences aligned with minimal indels. The protein-coding genes showed higher levels of divergence (P-distance, and synonymous and nonsynonymous substitution rates) and higher identification index than those of rRNAs. Considering divergence and phylogenetic usefulness, we selected atpB, psaA, and rbcL as the best candidates for cryptophycean clade identification and further field research.

Genotyping and phylogenetic analysis of Pythium insidiosum causing human corneal ulcer.

Pythium insidiosum belongs to the Oomycetes, which are known to cause serious life-threatening infectious condition in humans and animals. Corneal infections caused by P. insidiosum are rare and difficult to treat. The molecular-based diagnosis of Pythium is employed for the species identification and to study molecular phylogenetic relationship. Based on Cytochrome oxidase II (cox II) gene, P. insidiosum is categorized into three clades or groups: Clade-I or ATH (American strains), Clade-II or BTH (American, Asian, and Australian strains), and Clade-III or CTH (mostly Thailand strains). This study focused on the molecular identification of Pythium insidiosum from patients with corneal ulcer using ITS regions and clade identification by cox II gene sequencing and correlated with the clinical outcome. The isolates were collected from Aravind Eye Hospital, Madurai, India, from April to December 2018. Through the microbiological laboratory reports, 15 isolates of Pythium sp. from keratitis patient were selected, followed by DNA extraction, ITS, and cox II gene sequencing and phylogenetic analysis using the reference sequences from NCBI database. All 15 P. insidiosum isolates were phylogenetically clustered together as a single group and where also placed distantly from other Pythium species (outgroup). Most ocular isolates fell into either clade BTH or clade CTH, and none of our ocular isolates were in clade ATH. Two of the strains were very distinct and did not match any of the clusters indicating different lineages. There was no significant difference between clinical outcome and genotype of P. insidiosum.

Inter- and intracontinental migrations and local differentiation have shaped the contemporary epidemiological landscape of canine parvovirus in South America.

Canine parvovirus (CPV) is a fast-evolving single-stranded DNA virus that causes one of the most significant infectious diseases of dogs. Although the virus dispersed over long distances in the past, current populations are considered to be spatially confined and with only a few instances of migration between specific localities. It is unclear whether these dynamics occur in South America where global studies have not been performed. The aim of this study is to analyze the patterns of genetic variability in South American CPV populations and explore their evolutionary relationships with global strains. Genomic sequences of sixty-three strains from South America and Europe were generated and analyzed using a phylodynamic approach. All the obtained strains belong to the CPV-2a lineage and associate with global strains in four monophyletic groups or clades. European and South American strains from all the countries here analyzed are representative of a widely distributed clade (Eur-I) that emerged in Southern Europe during 1990–98 to later spread to South America in the early 2000s. The emergence and spread of the Eur-I clade were correlated with a significant rise in the CPV effective population size in Europe and South America. The Asia-I clade includes strains from Asia and Uruguay. This clade originated in Asia during the late 1980s and evolved locally before spreading to South America during 2009–10. The third clade (Eur-II) comprises strains from Italy, Brazil, and Ecuador. This clade appears in South America as a consequence of an early introduction from Italy to Ecuador in the middle 1980s and has experienced extensive local genetic differentiation. Some strains from Argentina, Uruguay, and Brazil constitute an exclusive South American clade (SA-I) that emerged in Argentina in the 1990s. These results indicate that the current epidemiological scenario is a consequence of inter- and intracontinental migrations of strains with different geographic and temporal origins that set the conditions for competition and local differentiation of CPV populations. The coexistence and interaction of highly divergent strains are the main responsible for the drastic epidemiological changes observed in South America in the last two decades. This highlights the threat of invasion from external sources and the importance of whole-genome resolution to robustly infer the origin and spread of new CPV variants. From a taxonomic standpoint, the findings herein show that the classification system that uses a single amino acid to identify variants (2a, 2b, and 2c) within the CPV-2a lineage does not reflect phylogenetic relationships and is not suitable to analyze CPV evolution. In this regard, the identification of clades or sublineages within circulating CPV strains is the first step towards a genetic and evolutionary classification of the virus.

Clade-specific responses regulate phenological patterns in Neotropical Myrtaceae

Understanding plant reproduction from an eco-phylogenetic perspective is essential because it allows us to predict how phenology could change in response to climatic changes. Unfortunately, reproductive patterns have been poorly explored in contemporary phylogenetic context until now. Closely related species are expected to reproduce in a similar ecological niche if phylogeny constrains the time of flowering and fruiting. In contrast, if climate determines phenology regardless of species affinities, species would randomly reproduce along favourable temporal axes. In this study, we used a clade of Neotropical Myrtaceae to test the influence of phylogenetic signal (PS) in phenology. Myrtaceae is the eighth largest angiosperm plant family in the world and the most speciose in the Atlantic rainforest. We reconstructed phylogenetic relationships among 57 Myrteae species plus an outgroup and quantified PS across three Atlantic rainforest communities: Saibadela, Cardoso and Picinguaba. Ecological factors were the main drivers of plant phenology. However, PS in plant reproduction was detected for flowering in Cardoso, suggesting a clade-specific response because flowering was conservative in the Gomidesia group. Fruiting in Saibadela also exhibited PS with phenological divergence between taxa with similar embryo shape and conservative fruiting time inside these groups, indicating that seed germination requirement can affect fruiting phenology. We suggest that investigation of PS in phenology should consider phylogenetic non-stationarity because evolutionary rates and ecological patterns are variable across taxa and often variable in short time scales. The identification of clades with conservative phenology, indicates the taxa most sensitive to climate change that can guide future conservation initiatives.

87. Towards Renal-Targeted Gene Delivery: Identification of Clades A, B, E and F Adeno-Associated Virus Sequences from Human Urine Samples

Adeno-associated virus (AAV) vectors have been successful in gene therapy for a range of inherited and acquired diseases. However, the development of efficient AAV-based gene delivery systems to treat renal diseases is lagging behind, mainly due to the unavailability of highly kidney-tropic AAV serotypes. To identify novel AAV capsid mutants with a natural renal tropism, we tested human urine samples for the presence of AAV sequences. We treated a combination of 293T, HeLa and Vero cells with 93 individual clinical urine samples, followed by one day of infection with high titer adenovirus 5 for AAV expansion. The majority of samples, 69%, were positive for AAV DNA by capsid specific, N-terminus nested PCR. Out of these samples 63% were AAV2-like (Clade B), 27% AAV8-like (Clade E), 1.6% AAV1-like (Clade A) and 8.7% had chimeric cap sequences (Clades B-E, B-F, F-B-E, F-E and E-B hybrids). In characterizing the AAV rep-cap junction region from DNA samples that were not uniquely AAV2 cap positive, the sequences were most closely related to the following: AAV1 rep-AAV1 cap, AAV2 rep-AAV2cap, AAV9 rep-AAV9 cap and AAV2 rep-AAV8 cap. Analysis of full length capsid sequences from overlapping PCRs revealed AAV8-like sequences, AAV2-like sequences and chimeric sequences of AAV8, AAV9 and rhesus isolates. We have cloned these AAV variants into the pXR plasmid and are currently testing their tissue tropisms in vivo. This study is the first to report frequent detection of AAV sequences in human urine. AAV vectors with novel AAV capsid variants may accelerate renal-targeted AAV gene therapy applications.

Detection of highly pathogenic zoonotic influenza virus H5N6 by reverse-transcriptase quantitative polymerase chain reaction.

BackgroundVariant high pathogenicity avian influenza (HPAI) H5 viruses have recently emerged as a result of reassortment of the H5 haemagglutinin (HA) gene with different neuraminidase (NA) genes, including NA1, NA2, NA5, NA6 and NA8. These viruses form a newly proposed HA clade 2.3.4.4 (previously provisionally referred to as clade 2.3.4.6), and have been implicated in disease outbreaks in poultry in China, South Korea, Laos, Japan and Vietnam and a human fatality in China. There is real concern that this new clade may be wide spread and not readily identified using existing diagnostic algorithms.FindingsFluorescent probe based reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were developed to facilitate the identification of novel clade 2.3.4.4 viruses of H5N6 subtype emerging in Asia. Assays were aimed at the haemagglutinin (HA) gene for clade identification and at the NA gene to identify N6. The HA assay employing a minor groove binder (MGB) probe was able to detect and differentiate A/duck/Laos/XBY004/2014(H5N6) and related influenza A(H5N6) virus isolates belonging to the proposed clade 2.3.4.4 from other H5 HPAI viruses. In addition, an Eurasian N6 assay was able to differentiate N6 from other NA subtypes.ConclusionsLaos influenza A(H5N6) virus representative of proposed clade 2.3.4.4, was detected and differentiated from viruses in other H5N1 clades using a clade-specific HA RT-qPCR assay whereas the N6-NA subtype was determined by an Eurasian N6 RT-qPCR assay. Such a clade-specific assay would be of particular value for surveillance and in diagnostic laboratories where sequencing is not readily available.