Molecular determinants of Ras-mTORC2 signaling.

Recent research has identified the mechanistic Target of Rapamycin Complex 2 (mTORC2) as a conserved direct effector of Ras proteins. While previous studies suggested the involvement of the Switch I (SWI) effector domain of Ras in binding mTORC2 components, the regulation of the Ras-mTORC2 pathway is not entirely understood. In Dictyostelium, mTORC2 is selectively activated by the Ras protein RasC, and the RasC-mTORC2 pathway then mediates chemotaxis to cAMP and cellular aggregation by regulating the actin cytoskeleton and promoting cAMP signal relay. Here, we investigated the role of specific residues in RasC's SWI, C-terminal allosteric domain, and hypervariable region (HVR) related to mTORC2 activation. Interestingly, our results suggest that RasC SWI residue A31, which was previously implicated in RasC-mediated aggregation, regulates RasC's specific activation by the Aimless RasGEF. On the other hand, our investigation identified a crucial role for RasC SWI residue T36, with secondary contributions from E38 and allosteric domain residues. Finally, we found that conserved basic residues and the adjacent prenylation site in the HVR, which are crucial for RasC's membrane localization, are essential for RasC-mTORC2 pathway activation by allowing for both RasC's own cAMP-induced activation and its subsequent activation of mTORC2. Therefore, our findings revealed new determinants of RasC-mTORC2 pathway specificity in Dictyostelium, contributing to a deeper understanding of Ras signaling regulation in eukaryotic cells.

Development of Recombinant Infectious Hepatitis E Virus Harboring the nanoKAZ Gene and Its Application in Drug Screening.

Hepatitis E virus (HEV) is a quasi-enveloped virus with a single-stranded positive-sense RNA genome belonging to the family Hepeviridae. Studies of the molecular aspects of HEV and drug screening have benefited from the discovery of bioluminescent reporter genes. However, the stability of large foreign genes is difficult to maintain after insertion into the viral genome. Currently, ribavirin is used to treat HEV-infected patients who require antiviral therapy. This has several major drawbacks. Thus, the development of novel anti-HEV drugs is of great importance. We developed a system consisting of recombinant infectious HEV harboring a small luciferase gene (nanoKAZ) in the hypervariable region (HVR) of the open reading frame 1 (ORF1) (HEV-nanoKAZ). It replicated efficiently in cultured cells, was genetically stable, and had morphological characteristics similar to those of the parental virus. Both membrane-associated (eHEV-nanoKAZ) and membrane-unassociated (neHEV-nanoKAZ) particles were infectious. HEV particles circulating in the bloodstream and attaching to hepatocytes in HEV-infected patients are membrane-associated; thus, eHEV-nanoKAZ was applied in drug screening. The eHEV-nanoKAZ system covers at least the inhibitor of HEV entry and inhibitor of HEV RNA replication. Four drugs with anti-HEV activity were identified. Their effectiveness in cultured cells was confirmed in naive and HEV-producing PLC/PRF/5 cells. Two hit drugs (azithromycin and ritonavir) strongly inhibited HEV production in culture supernatants, as well as intracellular expression of ORF2 protein, and may therefore be candidate novel anti-HEV drugs. The HEV-nanoKAZ system was developed and applied in drug screening and is expected to be useful for investigating the HEV life cycle. IMPORTANCE Bioluminescent reporter viruses are essential tools in molecular virological research. They have been widely used to investigate viral life cycles and in the development of antiviral drugs. For drug screening, the use of a bioluminescent reporter virus helps shorten the time required to perform the assay. A system, consisting of recombinant infectious HEV harboring the nanoKAZ gene in the HVR of ORF1 (HEV-nanoKAZ), was developed in this study and was successfully applied to drug screening in which four hit drugs with anti-HEV activity were identified. The results of this study provide evidence supporting the use of this system in more variable HEV studies. In addition, both forms of viral particles (eHEV-nanoKAZ and neHEV-nanoKAZ) are infectious, which will enable their application in HEV studies requiring both forms of viral particles, such as in the investigation of unknown HEV receptors and the elucidation of host factors important for HEV entry.

Insertions and deletions in the hypervariable region of the hepatitis E virus genome in individuals with acute and chronic infection.

BACKGROUND AND AIMS: Hepatitis E virus is a major cause of acute hepatitis worldwide and can progress to chronicity in immunocompromised individuals. Various virus-host recombination events have been reported in the hypervariable region of the hepatitis E virus genome, but the patterns of assembly and selection remain unclear. METHODS: To gain further insight into viral evolution, we assessed the presence of low abundance variants in 16 samples from individuals with acute or chronic infection using a targeted next-generation sequencing approach. RESULTS: In seven samples, different variants with insertions and/or deletions were identified. Among them, eight insertions originating either from human genes or from the hepatitis E virus genome. Five different deletions could be identified. The amino acid composition of sequences with insertions showed a higher frequency of lysine and a lower abundance of proline, and additionally acetylation and ubiquitination sites were more frequent than in hepatitis E virus wild-type sequences. CONCLUSIONS: These findings suggest that the nucleotide composition of insertions and sites for post-translational modification may contribute to recombination events. Although the impact of low-level hepatitis E virus variants is uncertain, our results highlight the importance of a highly sensitive next-generation sequencing approach to capture the full diversity of hypervariable region.

A Customized Human Mitochondrial DNA Database (hMITO DB v1.0) for Rapid Sequence Analysis, Haplotyping and Geo-Mapping.

The field of mitochondrial genomics has advanced rapidly and has revolutionized disciplines such as molecular anthropology, population genetics, and medical genetics/oncogenetics. However, mtDNA next-generation sequencing (NGS) analysis for matrilineal haplotyping and phylogeographic inference remains hindered by the lack of a consolidated mitogenome database and an efficient bioinformatics pipeline. To address this, we developed a customized human mitogenome database (hMITO DB) embedded in a CLC Genomics workflow for read mapping, variant analysis, haplotyping, and geo-mapping. The database was constructed from 4286 mitogenomes. The macro-haplogroup (A to Z) distribution and representative phylogenetic tree were found to be consistent with published literature. The hMITO DB automated workflow was tested using mtDNA-NGS sequences derived from Pap smears and cervical cancer cell lines. The auto-generated read mapping, variants track, and table of haplotypes and geo-origins were completed in 15 min for 47 samples. The mtDNA workflow proved to be a rapid, efficient, and accurate means of sequence analysis for translational mitogenomics.

Plastome structure, phylogenomics, and divergence times of tribe Cinnamomeae (Lauraceae).

BACKGROUND: Tribe Cinnamomeae is a species-rich and ecologically important group in tropical and subtropical forests. Previous studies explored its phylogenetic relationships and historical biogeography using limited loci, which might result in biased molecular dating due to insufficient parsimony-informative sites. Thus, 15 plastomes were newly sequenced and combined with published plastomes to study plastome structural variations, gene evolution, phylogenetic relationships, and divergence times of this tribe. RESULTS: Among the 15 newly generated plastomes, 14 ranged from 152,551 bp to 152,847 bp, and the remaining one (Cinnamomum chartophyllum XTBGLQM0164) was 158,657 bp. The inverted repeat (IR) regions of XTBGLQM0164 contained complete ycf2, trnICAU, rpl32, and rpl2. Four hypervariable plastid loci (ycf1, ycf2, ndhF-rpl32-trnLUAG, and petA-psbJ) were identified as candidate DNA barcodes. Divergence times based on a few loci were primarily determined by prior age constraints rather than by DNA data. In contrast, molecular dating using complete plastid protein-coding genes (PCGs) was determined by DNA data rather than by prior age constraints. Dating analyses using PCGs showed that Cinnamomum sect. Camphora diverged from C. sect. Cinnamomum in the late Oligocene (27.47 Ma). CONCLUSIONS: This study reports the first case of drastic IR expansion in tribe Cinnamomeae, and indicates that plastomes have sufficient parsimony-informative sites for molecular dating. Besides, the dating analyses provide preliminary insights into the divergence time within tribe Cinnamomeae and can facilitate future studies on its historical biogeography.

Inactivated whole hepatitis C virus vaccine employing a licensed adjuvant elicits cross-genotype neutralizing antibodies in mice.

BACKGROUND & AIMS: A prophylactic vaccine is required to eliminate HCV as a global public health threat. We developed whole virus inactivated HCV vaccine candidates employing a licensed adjuvant. Further, we investigated the effects of HCV envelope protein modifications (to increase neutralization epitope exposure) on immunogenicity. METHODS: Whole virus vaccine antigen was produced in Huh7.5 hepatoma cells, processed using a multistep protocol and formulated with adjuvant (MF-59 analogue AddaVax or aluminium hydroxide). We investigated the capacity of IgG purified from the serum of immunized BALB/c mice to neutralize genotype 1-6 HCV (by virus neutralization assays) and to bind homologous envelope proteins (by ELISA). Viruses used for immunizations were (i) HCV5aHi with strain SA13 envelope proteins and modification of an O-linked glycosylation site in E2 (T385P), (ii) HCV5aHi(T385) with reversion of T385P to T385, featuring the original E2 sequence determined in vivo and (iii) HCV5aHi(ΔHVR1) with deletion of HVR1. For these viruses, epitope exposure was investigated using human monoclonal (AR3A and AR4A) and polyclonal (C211 and H06) antibodies in neutralization assays. RESULTS: Processed HCV5aHi formulated with AddaVax induced antibodies that efficiently bound homologous envelope proteins and broadly neutralized cultured genotype 1-6 HCV, with half maximal inhibitory concentrations of between 14 and 192 µg/ml (mean of 36 µg/ml against the homologous virus). Vaccination with aluminium hydroxide was less immunogenic. Compared to HCV5aHi(T385) with the original E2 sequence, HCV5aHi with a modified glycosylation site and HCV5aHi(ΔHVR1) without HVR1 showed increased neutralization epitope exposure but similar immunogenicity. CONCLUSION: Using an adjuvant suitable for human use, we developed inactivated whole HCV vaccine candidates that induced broadly neutralizing antibodies, which warrant investigation in further pre-clinical studies. LAY SUMMARY: A vaccine against hepatitis C virus (HCV) is needed to prevent the estimated 2 million new infections and 400,000 deaths caused by this virus each year. We developed inactivated whole HCV vaccine candidates using adjuvants licensed for human use, which, following immunization of mice, induced antibodies that efficiently neutralized all HCV genotypes with recognized epidemiological importance. HCV variants with modified envelope proteins exhibited similar immunogenicity as the virus with the original envelope proteins.

The complete chloroplast genome of Onobrychis gaubae (Fabaceae-Papilionoideae): comparative analysis with related IR-lacking clade species.

BACKGROUND: Plastome (Plastid genome) sequences provide valuable markers for surveying evolutionary relationships and population genetics of plant species. Papilionoideae (papilionoids) has different nucleotide and structural variations in plastomes, which makes it an ideal model for genome evolution studies. Therefore, by sequencing the complete chloroplast genome of Onobrychis gaubae in this study, the characteristics and evolutionary patterns of plastome variations in IR-loss clade were compared. RESULTS: In the present study, the complete plastid genome of O. gaubae, endemic to Iran, was sequenced using Illumina paired-end sequencing and was compared with previously known genomes of the IRLC species of legumes. The O. gaubae plastid genome was 122,688 bp in length and included a large single-copy (LSC) region of 81,486 bp, a small single-copy (SSC) region of 13,805 bp and one copy of the inverted repeat (IRb) of 29,100 bp. The genome encoded 110 genes, including 76 protein-coding genes, 30 transfer RNA (tRNA) genes and four ribosome RNA (rRNA) genes and possessed 83 simple sequence repeats (SSRs) and 50 repeated structures with the highest proportion in the LSC. Comparative analysis of the chloroplast genomes across IRLC revealed three hotspot genes (ycf1, ycf2, clpP) which could be used as DNA barcode regions. Moreover, seven hypervariable regions [trnL(UAA)-trnT(UGU), trnT(GGU)-trnE(UUC), ycf1, ycf2, ycf4, accD and clpP] were identified within Onobrychis, which could be used to distinguish the Onobrychis species. Phylogenetic analyses revealed that O. gaubae is closely related to Hedysarum. The complete O. gaubae genome is a valuable resource for investigating evolution of Onobrychis species and can be used to identify related species. CONCLUSIONS: Our results reveal that the plastomes of the IRLC are dynamic molecules and show multiple gene losses and inversions. The identified hypervariable regions could be used as molecular markers for resolving phylogenetic relationships and species identification and also provide new insights into plastome evolution across IRLC.

Complete chloroplast genomes of two medicinal Swertia species: the comparative evolutionary analysis of Swertia genus in the Gentianaceae family.

MAIN CONCLUSION: The complete chloroplast genome of Swertia kouitchensis has been sequenced and assembled, compared with that of S. bimaculata to determine the evolutionary relationships among species of the Swertia in the Gentianaceae family. Swertia kouitchensis and S. bimaculata are from the Gentianaceae family. The complete chloroplast genome of S. kouitchensis was newly assembled, annotated, and analyzed by Illumina Hiseq 2500 platform. The chloroplast genomes of the two species encoded a total of 133, 134 genes, which included 88-89 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA genes. One intron was contained in each of the eight protein-coding genes and eight tRNA-coding genes, whereas two introns were found in two genes (ycf3 and clpP). The most abundant codon of the two species was for isoleucine, and the least abundant codon was for cysteine. The number of microsatellite repeat sequences was twenty-eight and thirty-two identified in the chloroplast genomes of S. kouitchensis and S. bimaculata, respectively. A total of 1127 repeat sequences were identified in all the 23 Swertia chloroplast genomes, and they fell into four categories. Furthermore, five divergence hotspot regions can be applied to discriminate these 23 Swertia species through genomes comparison. One pair of genus-specific DNA barcodes primer has been accurately identified. Therefore, the diverse regions cloned by a specific primer may become an effective and powerful molecular marker for the identification of Swertia genus. Moreover, four genes (ccsA, ndhK, rpoC1, and rps12) were positive selective pressure. The phylogenetic tree showed that the 23 Swertia species were clustered into a large clade including four evident subbranches, whereas the two species of S. kouitchensis and S. bimaculata were separately clustered into the diverse but correlated species group.

Mitochondrial DNA (CA)n dinucleotide repeat variations in Sinhalese and Vedda populations in Sri Lanka.

Sinhalese and Vedda people are respectively the major ethnic group and the descendants of the probably earliest inhabitants of Sri Lanka, both believed to have a long history of settlement on the island. However, very little information is available on the origin and possible migration patterns of the two populations. Some studies have focused on (CA) dinucleotide repeat variations located in the mitochondrial hypervariable region 3 (HVS3) (base pairs 514-524) as a useful biomarker to understand migration patterns of different populations. Hence, here we analyze these repeat variations in these two ethnic groups to understand their historical roots and possible patterns of gene flow. Blood samples were collected from healthy, maternally unrelated individuals (N = 109) and mitochondrial D-loop was amplified and sequenced. The (CA)4 dinucleotide repeat in hypervariable region 3 was detected in the majority of Vedda samples while the remaining samples were defined by a (CA)5 cluster. In contrast, the (CA)5 repeat was the most frequent among Sinhalese followed by (CA)4 and (CA)7 repeats. Haplogroup diversity of (CA)4 variation indicated that the majority of Sinhalese individuals grouped into the M30 haplogroup while Vedda clustered into the R5a2b and U7a2 haplogroups. No significant differences in diversity measures were observed among the two populations. However, Multidimensional Scaling indicated a separate clustering for aboriginal Vedda and contemporary Sinhalese populations. Results from this study can be used together with mitochondrial DNA information from hypervariable regions 1 and 2 to perform anthropological and forensic investigations in the two populations studied.

The complete chloroplast genome sequence of Clerodendranthus spicatus, a medicinal plant for preventing and treating kidney diseases from Lamiaceae family.

BACKGROUND: Clerodendranthus spicatus (Thunb.) C. Y. Wu ex H. W. Li is one of the most important medicines for the treatment of nephrology in the southeast regions of China. To understand the taxonomic classification of Clerodendranthus species and identify species discrimination markers, we sequenced and characterized its chloroplast genome in the current study. METHODS AND RESULTS: Total genomic DNA were isolated from dried leaves of C. spicatus and sequenced using an Illumina sequencing platform. The data were assembled and annotated by the NOVOPlasty software and CpGAVAS2 web service. The complete chloroplast genome of C. spicatus was 152,155 bp, including a large single-copy region of 83,098 bp, a small single-copy region of 17,665 bp, and a pair of inverted repeat regions of 25,696 bp. The Isoleucine codons are the most abundant, accounting for 4.17% of all codons. The codons of AUG, UUA, and AGA demonstrated a high degree of usage bias. Twenty-eight simple sequence repeats, thirty-six tandem repeats, and forty interspersed repeats were identified. The distribution of the specific rps19, ycf1, rpl2, trnH, psbA genes were analyzed. Analysis of the genetic distance of the intergenic spacer regions shows that ndhG-ndhI, accD-psaI, rps15-ycf1, rpl20-clpP, ccsA-ndhD regions have high K2p values. Phylogenetic analysis showed that C. spicatu is closely related to two Lamiaceae species, Tectona grandis, and Glechoma longituba. CONCLUSIONS: In this study, we sequenced and characterized the chloroplast genome of C. spicatus. Phylogenomic analysis has identified species closely related to C. spicatus, which represent potential candidates for the development of drugs improving renal functions.

Developing an efficient DNA barcoding system to differentiate between Lilium species.

BACKGROUND: Lilium is an important ornamental bulb, possesses medicinal properties, and is also edible. Species within the Lilium genus share very similar morphology and macroscopic characteristics, thus they cannot be easily and clearly distinguished from one another. To date, no efficient species-specific markers have been developed for classifying wild lily species, which poses an issue with further characterizing its medicinal properties. RESULTS: To develop a simple and reliable identification system for Lilium, 45 representative species from 6 sections were used to develop a DNA barcoding system, which was based on DNA sequence polymorphisms. In this study, we assessed five commonly used DNA barcode candidates (ITS, rbcL, ycf1b, matK and psbA-trnH) and five novel barcode candidates obtained from highly variable chloroplast genomic regions (trnL-trnF, trnS-trnG, trnF-ndhJ, trnP-psaJ-rpI33 and psbB-psbH). We showed that a set of three novel DNA barcodes (ITS + trnP-psaJ-rpI33 + psbB-psbH) could be efficiently used as a genetic marker to distinguish between lily species, as assessed by methods including DNAsp, BI and ML tree, and Pair Wise Group (PWG). CONCLUSIONS: A rapid and reliable DNA barcoding method was developed for all 45 wild Lilium species by using ITS, trnP-psaJ-rpI33, and psbB-psbH as DNA barcoding markers. The method can be used in the classification of wild Lilium species, especially endangered species, and also provides an effective method for selective lily breeding.

Comparative plastid genomics of four Pilea (Urticaceae) species: insight into interspecific plastid genome diversity in Pilea.

BACKGROUND: Pilea is a genus of perennial herbs from the family Urticaceae, and some species are used as courtyard ornamentals or for medicinal purposes. At present, there is no information about the plastid genome of Pilea, which limits our understanding of this genus. Here, we report 4 plastid genomes of Pilea taxa (Pilea mollis, Pilea glauca 'Greizy', Pilea peperomioides and Pilea serpyllacea 'Globosa') and performed comprehensive comparative analysis. RESULTS: The four plastid genomes all have a typical quartile structure. The lengths of the plastid genomes ranged from 150,398 bp to 152,327 bp, and each genome contained 113 unique genes, including 79 protein-coding genes, 4 rRNA genes, and 30 tRNA genes. Comparative analysis showed a rather high level of sequence divergence in the four genomes. Moreover, eight hypervariable regions were identified (petN-psbM, psbZ-trnG-GCC, trnT-UGU-trnL-UAA, accD-psbI, ndhF-rpl32, rpl32-trnL-UAG, ndhA-intron and ycf1), which are proposed for use as DNA barcode regions. Phylogenetic relationships based on the plastid genomes of 23 species of 14 genera of Urticaceae resulted in the placement of Pilea in the middle and lower part of the phylogenetic tree, with 100% bootstrap support within Urticaceae. CONCLUSION: Our results enrich the resources concerning plastid genomes. Comparative plastome analysis provides insight into the interspecific diversity of the plastid genome of Pilea. The identified hypervariable regions could be used for developing molecular markers applicable in various research areas.

Structural changes in the gut microbiota community of the black-necked crane (Grus nigricollis) in the wintering period.

During overwintering of black-necked cranes (Grus nigricollis), the composition and function of the gut microbiota changes are of considerable interest for understanding its environmental adaption mechanism. In this study, we characterized the structure of the gut microbiota from the black-necked crane in the Dashanbao wintering area, and compared the early-winter (November) microbiota to the late-winter (March of the next year) microbiota. The results showed that the gut microbiota diversity of black-necked crane in the early-overwintering stage was higher than that in the late-overwintering stage, but it did not reach a significant level. Gut microbiota taxonomic composition analysis showed that relative abundance of Bacteroidota increased significantly, and showed decreased Firmicutes to Bacteroidota ratio at the phylum level, meanwhile, the abundance of Lactobacillus decreased significantly at the genus level. Explain gut microbiota between the early- and late-wintering showed some differences in microbiota richness but maintained a relatively conservative microbiota structure. PICRUSt2 method was used to predict and analyze the KEGG functional abundance of 16S rDNA sequences of bacteria, it was found that the changes in gut microbiota composition increased the abundance of bacteria associated with amino acid biosynthesis and acid metabolism in the late stage of overwintering. This work provides basic data for black-necked crane gut microbiota study, which might further contribute to their protection.

Electrostatic Forces Mediate the Specificity of RHO GTPase-GDI Interactions.

Three decades of research have documented the spatiotemporal dynamics of RHO family GTPase membrane extraction regulated by guanine nucleotide dissociation inhibitors (GDIs), but the interplay of the kinetic mechanism and structural specificity of these interactions is as yet unresolved. To address this, we reconstituted the GDI-controlled spatial segregation of geranylgeranylated RHO protein RAC1 in vitro. Various biochemical and biophysical measurements provided unprecedented mechanistic details for GDI function with respect to RHO protein dynamics. We determined that membrane extraction of RHO GTPases by GDI occurs via a 3-step mechanism: (1) GDI non-specifically associates with the switch regions of the RHO GTPases; (2) an electrostatic switch determines the interaction specificity between the C-terminal polybasic region of RHO GTPases and two distinct negatively-charged clusters of GDI1; (3) a non-specific displacement of geranylgeranyl moiety from the membrane sequesters it into a hydrophobic cleft, effectively shielding it from the aqueous milieu. This study substantially extends the model for the mechanism of GDI-regulated RHO GTPase extraction from the membrane, and could have implications for clinical studies and drug development.

The nsp2 Hypervariable Region of Porcine Reproductive and Respiratory Syndrome Virus Strain JXwn06 Is Associated with Viral Cellular Tropism to Primary Porcine Alveolar Macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a major threat to global pork production and has been notorious for its rapid genetic evolution in the field. The nonstructural protein 2 (nsp2) replicase protein represents the fastest evolving region of PRRSV, but the underlying biological significance has remained poorly understood. By deletion mutagenesis, we discovered that the nsp2 hypervariable region plays an important role in controlling the balance of genomic mRNA and a subset of subgenomic mRNAs. More significantly, we revealed an unexpected link of the nsp2 hypervariable region to viral tropism. Specifically, a mutant of the Chinese highly pathogenic PRRSV strain JXwn06 carrying a deletion spanning nsp2 amino acids 323 to 521 (nsp2Δ323-521) in its hypervariable region was found to lose infectivity in primary porcine alveolar macrophages (PAMs), although it could replicate relatively efficiently in the supporting cell line MARC-145. Consequently, this mutant failed to establish an infection in piglets. Further dissection of the viral life cycle revealed that the mutant had a defect (or defects) lying in the steps between virus penetration and negative-stranded RNA synthesis. Taken together, our results reveal novel functions of nsp2 in the PRRSV life cycle and provide important insights into the mechanisms of PRRSV RNA synthesis and cellular tropism.IMPORTANCE The PRRSV nsp2 replicase protein undergoes rapid and broad genetic variations in its middle region in the field, but the underlying significance has remained enigmatic. Here, we demonstrate that the nsp2 hypervariable region not only plays an important regulatory role in maintaining the balance of different viral mRNA species but also regulates PRRSV tropism to primary PAMs. Our results reveal novel functions for PRRSV nsp2 and have important implications for understanding the mechanisms of PRRSV RNA synthesis and cellular tropism.

A Recombinant Hepatitis C Virus Genotype 1a E1/E2 Envelope Glycoprotein Vaccine Elicits Antibodies That Differentially Neutralize Closely Related 2a Strains through Interactions of the N-Terminal Hypervariable Region 1 of E2 with Scavenger Receptor B1.

The global health burden for hepatitis C virus (HCV) remains high, despite available effective treatments. To eliminate HCV, a prophylactic vaccine is needed. One major challenge in the development of a vaccine is the genetic diversity of the virus, with 7 major genotypes and many subtypes. A global vaccine must be effective against all HCV genotypes. Our previous data showed that the 1a E1/E2 glycoprotein vaccine component elicits broad cross-neutralizing antibodies in humans and animals. However, some variation is seen in the effectiveness of these antibodies to neutralize different HCV genotypes and isolates. Of interest was the differences in neutralizing activity against two closely related isolates of HCV genotype 2a, the J6 and JFH-1 strains. Using site-directed mutagenesis to generate chimeric viruses between the J6 and JFH-1 strains, we found that variant amino acids within the core E2 glycoprotein domain of these two HCV genotype 2a viruses do not influence isolate-specific neutralization. Further analysis revealed that the N-terminal hypervariable region 1 (HVR1) of the E2 protein determines the sensitivity of isolate-specific neutralization, and the HVR1 of the resistant J6 strain binds scavenger receptor class-B type-1 (SR-B1), while the sensitive JFH-1 strain does not. Our data provide new information on mechanisms of isolate-specific neutralization to facilitate the optimization of a much-needed HCV vaccine.IMPORTANCE A vaccine is still urgently needed to overcome the hepatitis C virus (HCV) epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis, and eventually, end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies; however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicate that the HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.

Molecular epidemiology of Aleutian mink disease virus from fecal swab of mink in northeast China.

BACKGROUND: Aleutian mink disease parvovirus (AMDV) causes Aleutian mink disease (AMD), which is a serious infectious disease of mink. The aim of this study was to get a better understanding of the molecular epidemiology of AMDV in northeast China to control and prevent AMD from further spreading. This study for the first time isolated AMDV from fecal swab samples of mink in China. RESULTS: A total of 157/291 (54.0%) of the fecal swab samples were positive for AMDV. Of these, 23 AMDV positive samples were randomly selected for sequence alignment and phylogenetic analysis based on the acquired partial fragments of VP2 gene with the hypervariable region. Comparative DNA sequence analysis of 23 AMDV isolates with a reference nonpathogenic (AMDV-G) strain revealed 8.3% difference in partial VP2 nucleotide sequences. Amino acid alignment indicated the presence of several genetic variants, as well as one single amino acid residue deletion. The most concentrated area of variation was located in the hypervariable region of VP2 protein. According to phylogenetic analysis, the Chinese AMDV strains and the other reference AMDV strains from different countries clustered into three groups (clades A, B and C). Most of the newly sequenced strains were found to form a Chinese-specific group, which solely consisted of Chinese AMDV strains. CONCLUSION: These findings indicated that a high genetic diversity was found in Chinese AMDV strains and the virus distribution were not dependent on geographical origin. Both local and imported AMDV positive species were prevalent in the Chinese mink farming population. The genetic evidence of AMDV variety and epidemic isolates have importance in mink farming practice.

Variation of Mitochondrial DNA HV1 AND HV2 of the Vietnamese Population.

BACKGROUND: The sequence polymorphism of mitochondrial DNA (mtDNA) hypervariable segment 1 (HV1) and hypervariable segment 2 (HV2) is studied and applied to genetic diversity and human evolution assessment, forensic genetics, consanguinity determination, and mitochondrial disease diagnosis. METHODS: The study identified the variations of HV1 and HV2 of 517 unrelated Vietnamese individuals in Kinh, Muong, Cham, and Khmer ethnic. We performed sequencing of two hypervariable segments of mitochondrial DNA: HV1 and HV2. RESULTS: Fifty haplogroups were identified in which F1a haplogroup frequency was highest at 15.7%, followed by B5a (10.8%), M (8.9%), and M7b1 (7.7%). The most frequently encountered SNPs in this study were A263G (100%), A73G (99.6%), 315insC (96%), 309insC (56%), C16223T (41%), and T16189C (39%). The genetic diversity was calculated at 99.83%, and the probability of random match of two individuals sharing the same mtDNA haplotype was 0.37%. CONCLUSION: We have assessed the genetic polymorphism of mtDNA HV1 and HV2 of 517 Kinh, Muong, Cham, and Khmer ethnic samples. The result will help in better understanding of Vietnamese's mitochondrial genome diversity and aid in population as well as forensic science.

Genetic structure of Kho population from north-western Pakistan based on mtDNA control region sequences.

The Kho population speaking Khowar language reside since long ago in Chitral District of North-western Pakistan. So far, no report is available about their genetic structure and relationship with surrounding population groups. We partially sequenced the mitochondrial DNA control region from 16 unrelated Kho male and female individuals of different ages. The D-Loop region sequences of Kho were aligned and compared with the revised Cambridge Reference Sequence (rCRS). The genetic data of Kho was compared with surrounding north-western Pakistani population groups including Pathan, Kashmiri, and Hazara. Comparison with rCRS identified overall 49 different haplotypes for Kho samples. Among these 21 haplotypes were shared by more than one Kho individuals. The genetic diversity and power of discrimination observed for Kho group were 0.215 and 0.202 respectively indicating the Kho tribe as a least differentiated group among north-western Pakistani populations. The haplogroup mapping, phylogenetic and haplotype network analysis revealed the nearby maternal ancestral relationship between Kho and Kashmiri populations. The haplogroups analysis demonstrates the western Eurasian ancestral origin of Kho samples. However, the appearance of a few South Asian haplogroups with low frequency speculate the Kho tribe as an admixed population of western Eurasian and South Asian genetic components.

The Hypervariable Region of K-Ras4B Governs Molecular Recognition and Function.

The flexible C-terminal hypervariable region distinguishes K-Ras4B, an important proto-oncogenic GTPase, from other Ras GTPases. This unique lysine-rich portion of the protein harbors sites for post-translational modification, including cysteine prenylation, carboxymethylation, phosphorylation, and likely many others. The functions of the hypervariable region are diverse, ranging from anchoring K-Ras4B at the plasma membrane to sampling potentially auto-inhibitory binding sites in its GTPase domain and participating in isoform-specific protein-protein interactions and signaling. Despite much research, there are still many questions about the hypervariable region of K-Ras4B. For example, mechanistic details of its interaction with plasma membrane lipids and with the GTPase domain require further clarification. The roles of the hypervariable region in K-Ras4B-specific protein-protein interactions and signaling are incompletely defined. It is also unclear why post-translational modifications frequently found in protein polylysine domains, such as acetylation, glycation, and carbamoylation, have not been observed in K-Ras4B. Expanding knowledge of the hypervariable region will likely drive the development of novel highly-efficient and selective inhibitors of K-Ras4B that are urgently needed by cancer patients.