Salivary Microbial Dysbiosis Is Associated With Peri-Implantitis: A Case-Control Study in a Brazilian Population

Background and ObjectivesThe aim of this study was to examine the salivary microbiome in healthy peri-implant sites and those with peri-implantitis.MethodsSaliva samples were collected from 21 participants with healthy peri-implant sites and 21 participants with peri-implantitis. The V4 hypervariable region of the 16S rRNA gene was sequenced using the Ion Torrent PGM System (Ion 318™ Chip v2 400). The NGS analysis and composition of the salivary microbiome were determined by taxonomy assignment. Downstream bioinformatic analyses were performed in QIIME (v 1.9.1).ResultsClinical differences according to peri-implant condition status were found. Alpha diversity metrics revealed that the bacterial communities of participants with healthy peri-implant sites tended to have a richer microbial composition than individuals with peri-implantitis. In terms of beta diversity, bleeding on probing (BoP) may influence the microbial diversity. However, no clear partitioning was noted between the salivary microbiome of volunteers with healthy peri-implant sites or volunteers with peri-implantitis. The highest relative abundance of Stenotrophomonas, Enterococcus and Leuconostoc genus, and Faecalibacterium prausnitzii, Haemophilus parainfluenzae, Prevotella copri, Bacteroides vulgatus, and Bacteroides stercoris bacterial species was found in participants with peri-implantitis when compared with those with healthy peri-implant sites.ConclusionDifferences in salivary microbiome composition were observed between patients with healthy peri-implant sites and those with peri-implantitis. BoP could affect the diversity (beta diversity) of the salivary microbiome.

Photocontrol of GTPase Cycle and Multimerization of the Small G-Protein H-Ras using Photochromic Azobenzene Derivatives

Ras is a small G protein known as a central regulator of cellular signal transduction that induces processes, such as cell division, transcription. The hypervariable region (HVR) is one of the functional parts of this G protein, which induces multimerization and interaction between Ras and the plasma membrane. We introduced two highly different in polarity photochromic SH group-reactive azobenzene derivatives, N-4-phenyl-azophenyl maleimide (PAM) and 4-chloroacetoamido-4-sulfo-azobenzene (CASAB), into three cysteine residues in HVR to control Ras GTPase using light. PAM stoichiometrically reacted with the SH group of cysteine residues and induced multimerization. The mutants modified with PAM exhibited reversible changes in GTPase activity accelerated by the guanine nucleotide exchange factor and GTPase activating protein and multimerization accompanied by cis- and trans-photoisomerization upon ultraviolet and visible light irradiation. CASAB was incorporated into two of the three cysteine residues in HVR but did not induce multimerization. The H-Ras GTPase modified with CASAB was photo controlled more effectively than PAM-H-Ras. In this study, we revealed that the incorporation of azobenzene derivatives into the functional site of HVR enables photo reversible control of Ras function. Our findings may contribute to the development of a method to control functional biomolecules with physiologically important roles.

Genetic Diversity and Molecular Epidemiology of Circulating Respiratory Syncytial Virus in Central Taiwan, 2008–2017

In this study, we investigated the molecular evolution and phylodynamics of respiratory syncytial virus (RSV) over 10 consecutive seasons (2008–2017) and the genetic variability of the RSV genotypes ON1 and BA in central Taiwan. The ectodomain region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the second hypervariable region of the G protein in RSV ON1 and BA were analyzed. A total of 132 RSV-A and 81 RSV-B isolates were obtained. Phylogenetic analysis revealed that the NA1, ON1, and BA9 genotypes were responsible for the RSV epidemics in central Taiwan in the study period. For RSV-A, the NA1 genotype predominated during the 2008–2011 seasons. The ON1 genotype was first detected in 2011 and replaced NA1 after 2012. For RSV-B, the BA9 and BA10 genotypes cocirculated from 2008 to 2010, but the BA9 genotype has predominated since 2012. Amino acid sequence alignments revealed the continuous evolution of the G gene in the ectodomain region. The predicted N-glycosylation sites were relatively conserved in the ON1 (site 237 and 318) and BA9 (site 296 and 310) genotype strains. Our results contribute to the understanding and prediction of the temporal evolution of RSV at the local level.

Size-Fractionated Filtration Combined with Molecular Methods Reveals the Size and Diversity of Picophytoplankton

In this study, flow cytometry (FCM) and size-fractionated filtration, together with high-throughput molecular sequencing methods (SM), were used to investigate picophytoplankton. A particle separation filter and a higher-throughput sequencing method were used to evaluate the composition of a euphotic zone of picophytoplankton—especially picoeukaryotic phytoplankton—in the Western Pacific, and the results of flow cytometry, which is a classic way to detect picophytoplankton, were used as a standard to evaluate the reliability of the results of the SMs. Within a water column of 200 m, six water depths (5, 25, 50, 113 (DCM), 150, and 200 m) were established. In order to further study the particle size spectra of the picophytoplankton, size-fractionated filtration was used to separate water samples from each water depth into three particle size ranges: 0.2–0.6, 0.6–1.2, and 1.2–2 μm. A total of 36 (6 × 3 × 2) samples were obtained through PCR amplification of the 18S rRNA V4 hypervariable region and 16S rRNA, which were biased toward phytoplankton plastids, and then high-throughput sequencing was performed. The estimation of the picophytoplankton diameter relied on forward scattering (FSC) through FCM. The estimation of the vertical distribution and diameter of the picophytoplankton using the SM was consistent with the results with FCM; thus, we believe that the estimation of picophytoplankton composition with the SM has value as a reference, although the size-fractionated filtration seemed to cause some deviations. In addition to Prochlorococcus and Synechococcus, the SM was used to evaluate the composition of picoeukaryotic phytoplankton, which mainly included Prymnesiophycea (Haptophyta) (38.15%), Cryptophyceae (Cryptophyta) (22.36%), Dictyochophyceae (Chrysophyta) (12.22%), and Mamiellophyceae (Chlorophyta) (3.31%). In addition, the SM also detected Dinophyceae (Dinoflagellata) (11.69%) sequences and a small number of Bacillariophyceae (Diatom) (1.64%) sequences, which are generally considered to have large particle sizes. The results of the SM also showed that the picoeukaryotic phytoplankton were not evenly distributed in the euphotic layer, and the vertical distributions of the different picoeukaryotic phytoplankton were different. An analysis of correlations with environmental factors showed that temperature was the main environmental factor controlling the vertical distribution of picophytoplankton.

No part gets left behind: Tiled nanopore sequencing of whole ASFV genomes stitched together using Lilo

African Swine Fever virus (ASFV) is the causative agent of a deadly, panzootic disease, infecting wild and domesticated suid populations. Contained for a long time to the African continent, an outbreak of a particularly infectious variant in Georgia in 2007 initiated the spread of the virus around the globe, severely impacting pork production and local economies. The virus is highly contagious and has a mortality of up to 100% in domestic pigs. It is critical to track the spread of the virus, detect variants associated with pathology, and implement biosecurity measures in the most effective way to limit its spread. Due to its size and other limitations, the 170-190kbp large DNA virus has not been well sequenced with fewer than 200 genome sequences available in public repositories. Here we present an efficient, low-cost method of sequencing ASFV at scale. The method uses tiled PCR amplification of the virus to achieve greater coverage, multiplexability and accuracy on a portable sequencer than achievable using shotgun sequencing. We also present Lilo, a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly other methods fail on. The resulting ASFV genomes are near complete, lacking only parts of the highly repetitive 3’- and 5’telomeric regions, and have a high level of accuracy. Our results will allow sequencing of ASFV at optimal efficiency and high throughput to monitor and act on the spread of the virus.

Characterization of the hepatitis E virus replicase

Hepatitis E virus (HEV) is the major cause of acute hepatitis worldwide. HEV is a positive-sense RNA virus expressing 3 open reading frames (ORFs). ORF1 encodes the ORF1 non– structural polyprotein, the viral replicase which transcribes the full-length genome and a subgenomic RNA that encodes the structural ORF2 and ORF3 proteins. The present study is focused on the replication step with the aim to determine whether the ORF1 polyprotein is processed during the HEV lifecycle and to identify where the replication takes place inside the host cell. As no commercial antibody recognizes ORF1 in HEV-replicating cells, we aimed at inserting epitope tags within the ORF1 protein without impacting the virus replication efficacy. Two insertion sites located in the hypervariable region were thus selected to tolerate the V5 epitope while preserving HEV replication efficacy. Once integrated into the infectious full-length Kernow C-1 p6 strain, the V5 epitopes did neither impact the replication of genomic nor the production of subgenomic RNA. Also, the V5-tagged viral particles remained as infectious as the wildtype particles to Huh-7.5 cells. Next, the expression pattern of the V5-tagged ORF1 was compared in heterologous expression and replicative HEV systems. A high molecular weight protein (180 kDa) that was expressed in all 3 systems and that likely corresponds to the unprocessed form of ORF1 was detected up to 25 days after electroporation in the p6 cell culture system. Additionally, less abundant products of lower molecular weights were detected in both in cytoplasmic and nuclear compartments. Concurrently, the V5-tagged ORF1 was localized by confocal microscopy inside the cell nucleus but also as compact perinuclear substructures in which ORF2 and ORF3 proteins were detected. Importantly, using in situ hybridization (RNAScope®), positive and negative-strand HEV RNAs were localized in the perinuclear substructures of HEV-producing cells. Finally, by simultaneous detection of HEV genomic RNAs and viral proteins in these substructures, we identified candidate HEV factories.

Genotyping and Molecular Characterization of Infectious Bursal Disease Virus Identified in Important Poultry-Raising Areas of China During 2019 and 2020

Infectious bursal disease (IBD) is an acute and highly contagious immunosuppressive disease caused by the infectious bursal disease virus (IBDV), which seriously threatens the healthy development of the poultry industry. Since its spread to China in the early 1990s, the very virulent IBDV (vvIBDV) characterized by high lethality, has been the focus of prevention and control. However, the novel variant IBDV (nVarIBDV), which has been widely prevalent in China since 2017, has brought a new threat to the poultry industry. In this study, the prevalence of IBDV in the important poultry-raising areas of China from 2019 to 2020 was detected. Of these, 45.1% (101/224) of the samples and 61.9% (26/42) of the chicken flocks were shown to be positive for IBDV. For 50 IBDVs, the sequences of the hypervariable region of the VP2 gene in segment A and of the B-marker of the VP1 gene in segment B were analyzed. The results revealed the coexistence of a number of different IBDV genotypes, including A2dB1 (nVar, 26/50, 52.0%), A3B3 (HLJ0504-like, 15/50, 30.0%), A1B1 (classical, 1/50, 2.0%), and A8B1 (attenuated, 1/50, 2.0%). This indicated that the newly emerging nVarIBDV of A2dB1 and the persistently circulating HLJ0504-like vvIBDV of A3B3 are the two important epidemic strains. Furthermore, we established that segment reassortment has occurred among these circulating strains. This study is the first to reveal the novel epidemic characteristics of IBDV since the report of the emerging nVarIBDV of A2dB1 in China.

Association between Parkinson’s disease and the faecal eukaryotic microbiota

Parkinson’s disease (PD) is one of the most common neurodegenerative disease, and is so far not considered curable. PD patients suffer from several motor and non-motor symptoms, including gastrointestinal dysfunctions and alterations of the enteric nervous system. Constipation and additional intestinal affections can precede the classical motor symptoms by several years. Recently, we reported effects of PD and related medications on the faecal bacterial community of 34 German PD patients and 25 age-matched controls. Here, we used the same collective and analysed the V6 and V7 hypervariable region of PCR-amplified, eukaryotic 18S rRNA genes using an Illumina MiSeq platform. In all, 53% (18) of the PD samples and 72% (18) of the control samples yielded sufficient amplicons for downstream community analyses. The PD samples showed a significantly lower alpha and a different beta eukaryotic diversity than the controls. Most strikingly, we observed a significantly higher relative abundance of sequence affiliated with the Geotrichum genus in the PD samples (39.7%), when compared to the control samples (0.05%). In addition, we observed lower relative abundances of sequences affiliated with Aspergillus/Penicillium, Charophyta/Linum, unidentified Opisthokonta and three genera of minor abundant zooflagellates in the PD samples. Our data add knowledge to the small body of data about the eukaryotic microbiota of PD patients and suggest a potential association of certain gut eukaryotes and PD.

Geographic patterns of koala retrovirus genetic diversity, endogenization and subtype distributions

Koala retrovirus subtype A is the youngest endogenized retrovirus, providing a unique system to elucidate retroviral-host co-evolution. We characterised KoRV geography using faecal DNA from 192 samples across 20 populations throughout the koala’s range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/NSW state border. In northern koalas, pol gene copies were ubiquitously present at greater than 5 per-cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers less than one, while the env gene was detected in all animals and in a majority at copy numbers of greater than one per-cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The previously described exogenous KoRV subtypes (B-K), two novel subtypes (L and M), and intermediate or hybrid subtypes were detected in all northern koala populations but strikingly absent from all southern animals tested. Apart from KoRV-D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes has occurred within northern Australia, but this has not extended into animals within southern Australia.