Molecular characterization of the complete genome of a novel ormycovirus infecting the ectomycorrhizal fungus Hortiboletus rubellus.

Advancements in high-throughput sequencing and the development of new bioinformatics tools for large-scale data analysis play a crucial role in uncovering virus diversity and enhancing our understanding of virus evolution. The discovery of the ormycovirus clades, a group of RNA viruses that are phylogenetically distinct from all known Riboviria members and are found in fungi, highlights the value of these tools for the discovery of novel viruses. The aim of this study was to examine viral populations in fungal hosts to gain insights into the diversity, evolution, and classification of these viruses. Here, we report the molecular characterization of a newly discovered ormycovirus, which we have named "Hortiboletus rubellus ormycovirus 1" (HrOMV1), that was found in the ectomycorrhizal fungus Hortiboletus rubellus. The bipartite genome of HrOMV1, whose nucleotide sequence was determined by HTS and RLM-RACE, consists of two RNA segments (RNA1 and RNA2) that exhibit similarity to those of previously studied ormycoviruses in their organization and the proteins they encode. The presence of upstream, in-frame AUG triplets in the 5' termini of both RNA segments suggests that HrOMV1, like certain other ormycoviruses, employs a non-canonical translation initiation strategy. Phylogenetic analysis showed that HrOMV1 is positioned within the gammaormycovirus clade. Its putative RNA-dependent RNA polymerase (RdRp) exhibits sequence similarity to those of other gammaormycovirus members, the most similarity to that of Termitomyces ormycovirus 1, with 33.05% sequence identity. This protein was found to contain conserved motifs that are crucial for RNA replication, including the distinctive GDQ catalytic triad observed in gammaormycovirus RdRps. The results of this study underscore the significance of investigating the ecological role of mycoviruses in mycorrhizal fungi. This is the first report of an ormycovirus infecting a member of the ectomycorrhizal genus Hortiboletus.

Full-length genome characterization of a novel mitovirus isolated from the root rot fungus Armillaria mellea.

Members of the genus Armillaria belong to the group of pathogenic and facultative saprotrophic fungi that are generally known as one of the causative agents of white root rot in infected plants including deciduous and evergreen trees and shrubs. Although several single-stranded RNA mycoviruses were previously described in different Armillaria species, there is no report on mitoviruses (one of the simplest RNA viruses of fungal hosts) known to infect Armillaria taxa. In this study, a new mitovirus denominated "Armillaria mellea mitovirus 1" (AmMV1) was identified in the sporophore samples of Armillaria mellea, commonly known as honey mushroom. AmMV1 has a genome length of 4440 nucleotides and a G + C content of 48%. It encompasses a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp). Comparison through BLASTp analysis revealed that the RdRp domain of AmMV1 shares a sequence identity ranging from 33.43% to 43.27% with RdRp domains of Duamitovirus genus members, having the highest similarity (43.27%) to Rhizoctonia solani mitovirus 94. According to phylogenetic analysis, AmMV1 is classified as a member of the genus Duamitovirus belonging to the Mitoviridae family. This marks the initial instance of a mitovirus identified in Armillaria spp..

Diverse partitiviruses hosted by the ectomycorrhizal agaric Hebeloma mesophaeum and the natural transmission of a partitivirus between phylogenetically distant, sympatric fungi.

Mycorrhizal fungi host diverse mycoviruses that contribute to our understanding of their diversity and evolution. Here we report on the identification and complete genome characterization of three novel partitiviruses naturally infecting the ectomycorrhizal fungus Hebeloma mesophaeum. During NGS derived viral sequence analyses, we identified a partitivirus that is conspecific with the previously reported partitivirus (LcPV1) described from a saprotrophic fungus Leucocybe candicans. The two distinct fungal specimens inhabited the same vicinity of a campus garden. RdRp sequences encoded by the LcPV1 isolates from both host fungi was found to be identical. Bio-tracking studies revealed that viral loads of LcPV1 drop significantly in L. candicans but not in H. mesophaeum within four years period. The physical proximity of the mycelial networks of both fungal specimens implied the occurrence of a virus transmission event with unknown mechanism. Nature of this virus transmission was discussed in relation to transient interspecific mycelial contact hypothesis.

Identification and complete genome sequencing of a novel betapartitivirus naturally infecting the mycorrhizal desert truffle Terfezia claveryi.

Viruses that naturally infect fungal species and capable of establishing mycorrhizae are largely unknown. In this study, we identified and characterized a new partitivirus inhabiting the ascomycete, mycorrhizal desert truffle species Terfezia claveryi, and named it "Terfezia claveryi partitivirus 1" (TcPV1). The entire genome of TcPV1, sequenced by both high throughput sequencing of the total dsRNA extracts and by Sanger sequencing of the RLM-RACE PCR products comprised two dsRNA segments of 2404 bp and 2374 bp, respectively. Both dsRNA genome segments harbored a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. The BLASTp search of the RdRp and CP sequences revealed the highest sequence identities (41.92% and 24.13% identity, respectively) to those of Bipolaris maydis partitivirus 2 and Plasmopara viticola lesion associated partitivirus 5. Molecular phylogenetic analyses of the RdRp sequence showed that TcPV1 fall within a clade composed entirely of members of the genus Betapartitivirus, belonging to the family Partitiviridae. In light of this molecular evidence, TcPV1 is a new member of the genus Betapartitivirus. This is the first report of a new partitivirus hosted by the ascomycete, mycorrhizal fungus T. claveryi.

Molecular characterization of a new mitovirus hosted by the ectomycorrhizal fungus Albatrellopsis flettii.

The complete genome of a novel mycovirus, Albatrellopsis flettii mitovirus 1 (AfMV1), hosted by the basidiomycetous ectomycorrhizal fungus Albatrellopsis flettii (Morse ex Pouzar) Audet, was sequenced and analyzed. The full-length cDNA sequence, obtained from a dsRNA replication intermediate of the AfMV1 genome, is 3037 bp in length with a predicted G+C content of 40.66%. Sequence analysis revealed that a single large open reading frame (ORF) is present on the positive strand when the mold mitochondrial genetic code is applied. The single ORF encodes a putative RNA-dependent RNA polymerase of 859 amino acids with a predicted molecular weight of 97.05 kDa that shares the closest similarity with the corresponding protein of Entomophthora muscae mitovirus 7, with 43.38% sequence identity. Phylogenetic analysis showed that AfMV1 could be classified as a new member of the genus Mitovirus within the family Mitoviridae. This is the first report of the complete genome sequence of a new mitovirus, AfMV1, isolated from the basidiomycetous ectomycorrhizal fungus A. flettii.

Full-length genome characterization of a new fusagravirus hosted by the spring orange peel fungus Caloscypha fulgens.

Mycoviruses widely exist in diverse lineages of fungi, yet there are only a few studies on mycovirus infection in uncultivated fungi. We here report the presence of a dsRNA mycovirus in saprotrophic spring orange peel fungus Caloscypha fulgens. A novel dsRNA virus, named "Caloscypha fulgens fusagravirus 1" (CfFV1), was isolated from a single ascocarp of C. fulgens, and its molecular features were revealed. The full-length cDNA of CfFV1 comprises 9,548 nucleotides with a calculated GC content of 47.9% and two discontinuous open reading frames (ORF 1 and 2). A-1 ribosomal frameshift region with two distinctive elements, including a canonical slippery heptanucleotide (AAAAAAC) and a pseudoknot structure, predicted as a Recoding Stimulatory Element, was detected in the junction region of ORF1 and ORF2. The deduced amino acid sequence of ORF1 and ORF2 showed the highest similarity to the putative structural protein and RNA-dependent RNA polymerase (RdRp) of Rosellinia necatrix fusagravirus 4 (RnFGV4). Genome organization, sequence similarity, and phylogenetic analysis indicate that this virus belongs to a new member of the proposed family Fusagraviridae. This is the first report of the presence of a mycovirus in the spring orange peel fungus C. fulgens. Keywords: mycovirus; dsRNA; proposed Fusagraviridae; uncultivated fungi; Caloscypha fulgens.

Molecular characterization of the complete genome of a novel partitivirus hosted by the saprobic mushroom Leucocybe candicans.

Virus communities of uncultivated fungi stay largely unknown. In the current study, we characterized a new partitivirus species detected in the basidiomycetous, saprobic mushroom Leucocybe candicans, named "Leucocybe candicans partitivirus 1" (LcPV1). The full-length genome of LcPV1, determined using deep sequencing and RLM-RACE approaches, consists of two dsRNA segments with each having the same size of 1984 bp. Both dsRNA genome segments comprise a single open reading frame (ORF), encoding an RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP show the highest identity (50.09% and 35.71% similarity, respectively) to those of partitiviruses reported from an oomycetous, plant pathogenic, stramenopile algae Plasmopara viticola and basidiomycetous, plant pathogenic fungus Ceratobasidium sp., respectively. Phylogenetic analyses performed based on the RdRp and CP sequences revealed that LcPV1 falls within a cluster that includes different alphapartitivirus species from the family Partitiviridae. In this study, we propose that LcPV1 is a new member of a species belonging to the genus Alphapartitivirus. To our knowledge, this is the first study reporting on a new fungal virus (mycovirus) identified in the basidiomycetous, saprobic mushroom Leucocybe candicans.

The unique genome organization of two novel fusariviruses hosted by the true morel mushroom Morchella esculenta.

Two putative mycoviruses belonging to the proposed family "Fusariviridae" were identified in Morchella esculenta by sequencing of double-stranded RNAs extracted from the morel mushroom. These viruses were tentatively named "Morchella esculenta fusarivirus 1″ (MeFV1) and "Morchella esculenta fusarivirus 2″ (MeFV2). Including the poly(A) tail the complete genomes of MeFV1 and MeFV2 are composed of 9096 and 9011 nucleotides (nt) respectively. Both genomes contain four non-overlapping open reading frames (ORFs) in which the largest and the smallest ORFs are ORF2 and ORF3 for both genomes respectively. The ORF1 of MeFV1 and MeFV2 are preceded by the 5' untranslated regions (UTRs) of 27 and 37 nt respectively and encode 341 and 339 aa long proteins that do not exhibit significant similarity to any of the protein sequences present in GenBank database. The 1502 and 1511 aa long proteins encoded by ORF2 of MeFV1 and MeFV2 share 84.42% sequence identity to each other and are 58.54% and 58.57% identical to the RNA-dependent RNA polymerase (RdRp) of Morchella importuna fusarivirus 1 (MiFV1) respectively. Interestingly, a Promethin/LDAF1 protein domain that is associated with the endoplasmic reticulum (ER) and lipid droplet (LD) membranes was identified at the N terminal regions of MeFV1 and MeFV2 RdRps, implying that the replication of these viruses is linked to the lipid membranes. The ORF3 and ORF4 of MeFV1 and MeFV2 encode proteins (268 and 333 aa long, and 645 and 647 aa long respectively) that only share significant sequence similarities with the proteins encoded by the ORF2 and ORF3 of MiFV1 respectively. The 3' UTRs of MeFV1 and MeFV2 are 162 and 159 nt long respectively and both of them have 51 nt long terminal poly(A) traits. To our knowledge, MeFV1 and MeFV2 are the first fusariviruses identified in M. esculenta and this is the first study reporting on the presence of Promethin/LDAF1 domain in viral RdRps.

Molecular characterization of a new endornavirus inhabiting the ectomycorrhizal fungus Hygrophorus penarioides.

Viruses hosted by uncultivated fungi have been poorly studied. We carried out studies to characterize a large dsRNA segment (~20 kbp) detected in the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides. The dsRNA was gel-purified and its randomly amplified cDNA fragments were used for high throughput sequencing (HTS). Reads were de novo assembled and BLASTx analysis revealed sequence similarity to viruses of the family Endornaviridae. The 5' and 3' terminal sequences of the dsRNA segment were determined by performing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The full-length cDNA sequence of the putative endornavirus comprises 16,785 nt and contains a single, long open reading frame which encodes for a polyprotein of 5522 aa with conserved domains for cysteine-rich region, helicase, glycosyltransferase, and RNA-dependent RNA polymerase. The virus was named Hygrophorus penarioides endornavirus 1 (HpEnV1). A BLASTp search performed using the polyprotein sequence revealed that the most closely related, fully sequenced endornavirus to HpEnV1 is Ceratobasidium endornavirus B.

Molecular characterization of a novel partitivirus hosted by the false morel mushroom Gyromitra esculenta.

Virus populations of uncultivated fungi remain scarcely studied. In the present study, we characterized a new partitivirus isolated from the false morel mushroom Gyromitra esculenta, named "Gyromitra esculenta partitivirus 1" (GePV1). The complete genome of GePV1, whose sequence was determined by combining high-throughput sequencing and RLM-RACE approaches, comprises two dsRNA segments of 1971 bp and 1799 bp, respectively. Each dsRNA genome segment contains a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP), respectively. The sequences of the RdRp and CP exhibited the highest similarity (69.77% and 47.00% identity, respectively) to those of Rosellinia necatrix partitivirus 2 (RnPV2). Phylogenetic analysis based on the CP and RdRp sequences demonstrated that GePV1 clusters within a clade that includes members of the genus Alphapartitivirus, family Partitiviridae. We propose that GePV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified in the false morel mushroom Gyromitra esculenta.

Full-length genome characterization of a novel alphapartitivirus detected in the ectomycorrhizal fungus Hygrophorus penarioides.

Virus populations of ectomycorrhizal fungi remain poorly studied. In the present study, we characterized a new partitivirus isolated from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides, named "Hygrophorus penarioides partitivirus 1" (HpPV1). The whole genome of HpPV1, determined by merging deep sequencing and RLM-RACE approaches, comprised two dsRNA segments of 2053 bp and 2072 bp, respectively. Both dsRNA genome segments included a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp), and a capsid protein (CP), respectively. Based on BLASTp search, the sequences of the RdRp and CP exhibits the highest similarity (67.49% and 75.61% identity, respectively) to those of partitiviruses identified from an ascomycetous ectomycorrhizal fungus Sarcosphaera coronaria. Phylogenetic analyses performed based on the CP and RdRp sequences demonstrated that HpPV1 clusters within a clade that includes members of the genus Alphapartitivirus, belonging to the family Partitiviridae. Here, we propose that HpPV1 is a new member of the genus Alphapartitivirus. This is the first study reporting on a new partitivirus identified from the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides.

Novel and diverse mycoviruses co-inhabiting the hypogeous ectomycorrhizal fungus Picoa juniperi.

Viruses hosted by ectomycorrhizal fungi remain poorly studied. In this study, we detected eight new fungal viruses co-infecting a single isolate of the hypogeous ectomycorrhizal fungus Picoa juniperi using high-throughput sequencing. Phylogenetic analysis of one identified virus abbreviated as PjMTV1 revealed its closest relatives as members of the newly proposed family "Megatotiviridae". Phylogenetic analyses of two identified viruses abbreviated as PjV1 and PjV2 showed that these viruses are associated with members of the proposed family "Fusagraviridae". Phylogenetic analysis of the identified one another virus abbreviated as PjYV1 demonstrated that this virus is related to the members of the proposed family Yadokariviridae. The remaining four identified virus-like contigs were determined as segments of the bipartite dsRNA mycoviruses from the family Partitiviridae. The mycoviruses reported in this study are the first viruses described in Picoa juniperi, and PjMTV1 characterized herein is the secondly reported member of the newly proposed family "Megatotiviridae".

Novel and divergent bipartite mycoviruses associated with the ectomycorrhizal fungus Sarcosphaera coronaria.

Members of the family Partitiviridae are reported from a variety of fungal and plant taxa. After dsRNA-preparation, deep sequencing, and bioinformatics, we here reveal the existence of various divergent partitiviruses co-infecting the ectomycorrhizal fungus Sarcosphaera coronaria, symbiotically associated with the pine species Pinus brutia in Turkey. A total of 75 complete or nearly complete sequences related to the members of Alphapartitivirus and Betapartitivirus, were detected from the ascocarp sample of the fungal isolate. Two of the identified partitivirus genome segments encoding for partitiviral capsid protein represent evolutionarily distinct members of Alphapartitivirus, indicating that they may have diverged in the presence of long spatial isolation. In an attempt to match the two genome segments of the identified partitiviruses and distinguish individual species co-inhabiting a single host, nine possible genome segment pairs were identified.

Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3.

Prostate cancer is a common health problem among men worldwide and most of these prostate cancer cases are related to a dysfunctional mutant Tumor Protein p53 (TP53) gene. However, the CRISPR/Cas9 system can be used for repairing of a dysfunctional mutant TP53 gene in combination with donor single-stranded oligodeoxynucleotide (ssODN) via cells' own homology-directed repair (HDR) mechanism. In this study, we aimed to evaluate the CRISPR/Cas9 repairing efficiency on TP53 414delC (p.K139fs*31) null mutation, located in the TP53 gene, of human prostate cancer cell line PC-3 in combination with ssODNs. According to the next-generation sequencing results, TP53 414delC mutation was repaired with an efficiency of 19.95% and 26.0% at the TP53 414delC position with ssODN1 and ssODN2 accompanied by sgRNA2 guided CRISPR/Cas9, respectively. Besides, qPCR and immunofluorescence analysis showed that PC-3 cells, the TP53 414delC mutation of which were repaired, expressed wild type p53 again. Also, significantly increased number of apoptotic cells, driven by the repaired TP53 gene were detected compared to the control cells by flow cytometry analysis. As a result, sgRNA2 guided CRISPR/Cas9 system accompanied by ssODN was shown to effectively repair the TP53 414delC gene region and inhibit the cell proliferation of PC-3 cells. Therefore, the effects of the TP53 414delC mutation repairment in PC-3 cells will be investigated in the in vivo models for tumor clearance analysis in the near future.

Complete genome sequence of a novel mitovirus from the ectomycorrhizal fungus Geopora sumneriana.

A double-stranded RNA (dsRNA) segment was extracted from the ectomycorrhizal fungus Geopora sumneriana (Cooke) M. Torre, and its full-length cDNA sequence, comprising 3146 nucleotides, was determined. Sequence analysis revealed the presence of a large open reading frame (ORF) on the positive strand of this dsRNA segment when the mold mitochondrial genetic code was applied. The ORF encodes a putative RNA-dependent RNA polymerase (RdRp), which shares the highest degree of similarity with Tuber excavatum mitovirus, with 37.52% identity. This dsRNA segment represents the genome replication intermediate of a novel mitovirus that was tentatively designated as "Geopora sumneriana mitovirus 1" (GsMV1). Phylogenetic analysis further suggested that GsMV1 is a member of the family Narnaviridae. This is the first study reporting on a mitovirus genome sequence in the ectomycorrhizal fungus G. sumneriana.

Introduction of novel thermostable α-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related α-amylases under five individual GH13 subfamilies.

Among the thermophilic Bacillaceae family members, α-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus α-amylase genes displayed ≥ 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (≤ 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (≤ 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 α-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli α-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 α-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around α-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around α-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3.

Viruses infecting macrofungi.

Ever since their discovery just about 56 years ago in the cultivated mushroom Agaricus bisporus, many more viruses infecting fungi have been identified in a wide range of fungal taxa. With mostly being asymptomatic, especially the ones that are detrimental to their phytopathogenic hosts are intensively studied due to their considerable importance in developing novel plant protection measures. Contrary to the rapid accumulation of notable data on viruses of plant pathogenic microfungi, much less information have hitherto been obtained in regards to the viruses whose hosts are macrofungi. According to the current literature, only more than 80 distinct viruses bearing either linear dsRNA or linear positive sense ssRNA genome and infecting a total number of 34 macrofungal species represented with four Ascomycota and 30 Basidiomycota have been identified so far. Among these 34 macrofungal species, 14 are cultivated edible and wild edible mushroom species. According to the 10th ICTV (International Committee on Taxonomy of Viruses) Report, macrofungal viruses with linear dsRNA genome are classified into five families (Partitiviridae, Totiviridae, Chrysoviridae, Endornaviridae and Hypoviridae) and macrofungal viruses with linear positive sense ssRNA genome are classified into seven families (Betaflexiviridae, Gammaflexiviridae, Barnaviridae, Narnaviridae, Virgaviridae, Benyviridae and Tymoviridae). In this review, following a brief overview of some general characteristics of fungal viruses, an up to date knowledge on viruses infecting macrofungal hosts were presented by summarizing the previous, recent and prospective studies of the field.

Oleuropein and rutin protect against 6-OHDA-induced neurotoxicity in PC12 cells through modulation of mitochondrial function and unfolded protein response.

Parkinson's disease (PD) is a highly prevalent neurodegenerative disorder, often associated with oxidative stress-induced transcriptional changes in dopaminergic neurons. Phenolic antioxidants, oleuropein (OLE) and rutin (RUT) have attracted a great interest due to their potential to counteract oxidative protein aggregation and toxicity. This study aimed at examining the effects of OLE and RUT against 6-OHDA-induced stress response in rat pheochromocytoma cells. When differentiated PC12 cells were exposed to oxidative stress composer 6-OHDA (100 µM, 8 h), a decreased mitochondrial membrane potential (ΔΨm) was observed along with a significant loss of cell viability and apoptotic nuclear changes. Exposure to 6-OHDA resulted in unfolded protein response (UPR) in differentiated PC12 cells as evidenced by an increased level of endoplasmic reticulum (ER)-localized transmembrane signal transducer IRE1α, adaptive response proteins ATF-4 and proapoptotic transcription factor CHOP. OLE or RUT pretreatment (24 h) at low doses (1-50 µM) protected the differentiated PC12 cells from 6-OHDA-induced cytotoxicity as assessed by increased viability, improved ΔΨm and inhibited apoptosis, whereas relatively high doses of OLE or RUT (>50 µM) inhibited cell growth and proliferation, indicating a typical hormetic effect. In hormetic doses, OLE and RUT up-regulated 6-OHDA-induced increase in IRE1α, ATF-4 and inhibited CHOP, PERK, BIP and PDI. 6-OHDA-activated XBP1 splicing was also inhibited by OLE or RUT. The presented results suggest that neuroprotection against 6-OHDA-induced oxidative toxicity may be attributable to neurohormetic effects of OLE or RUT at low doses through regulating mitochondrial functions, controlling persistent protein misfolding, activating and/or amplificating the adaptive response-related signaling pathways, leading to UPR prosurvival output.

Evaluation of antiviral resistant hepatitis B virus subpopulations in patients with chronic hepatitis B by using terminal restriction fragment length polymorphism.

Antiviral therapies with nucleotide analogues (NA) is crucial in the treatment of chronic hepatitis B as it substantially protects patients from the complications of the disease . However in most of the available NA therapies, resistance emerges in the patients' HBV populations. Therefore, detection of antiviral resistance as early as possible by means of genotypically monitoring the patients' HBV pool during NA therapy is critical to manage treatment regime. In this research study we have investigated the sensitivity and specificity of the terminal restriction fragment length polymorphism (T-RFLP) method in detecting HBV subpopulations carrying antiviral resistance mutations. For this aim, differentiation of mutant strains from wild type strains was demonstrated by PCR-RFLP method. With using recombinant plasmids containing mutant and wild type HBV genomes, we constructed artificial HBV genome populations in order to determine the sensitivity of PCR-T-RFLP method in detecting antiviral resistant minor HBV populations. Finally by comparing with the DNA sequencing method, we demonstrated the specificity of T-RFLP method in genotyping HBV populations. As a result we showed that T-RFLP is able to detect HBV subpopulations representing as low as 1 % of the whole viral population. Additionally T-RFLP showed 100 % concordance with the DNA sequencing method in genotyping HBV populations. As a conclusion, considering the other genotyping methods used in evaluating HBV populations, T-RFLP showed high sensitivity and specificity profiles in detecting antiviral resistant HBV subpopulations. Therefore T-RFLP method can be easily employed in genotypic evaluation of patients' HBV populations during the course of antiviral treatment.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.