Locally Directed Recombinant Adeno- Associated Virus-Mediated IGF-1 Gene Therapy Enhances Osteochondral Repair and Counteracts Early Osteoarthritis In Vivo.

BACKGROUND: Restoration of osteochondral defects is critical, because osteoarthritis (OA) can arise. HYPOTHESIS: Overexpression of insulin-like growth factor 1 (IGF-1) via recombinant adeno-associated viral (rAAV) vectors (rAAV-IGF-1) would improve osteochondral repair and reduce parameters of early perifocal OA in sheep after 6 months in vivo. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral defects were created in the femoral trochlea of adult sheep and treated with rAAV-IGF-1 or rAAV-lacZ (control) (24 defects in 6 knees per group). After 6 months in vivo, osteochondral repair and perifocal OA were assessed by well-established macroscopic, histological, and immunohistochemical scoring systems as well as biochemical and micro-computed tomography evaluations. RESULTS: Application of rAAV-IGF-1 led to prolonged (6 months) IGF-1 overexpression without adverse effects, maintaining a significantly superior overall cartilage repair, together with significantly improved defect filling, extracellular matrix staining, cellular morphology, and surface architecture compared with rAAV-lacZ. Expression of type II collagen significantly increased and that of type I collagen significantly decreased. Subchondral bone repair and tidemark formation were significantly improved, and subchondral bone plate thickness and subarticular spongiosa mineral density returned to normal. The OA parameters of perifocal structure, cell cloning, and matrix staining were significantly better preserved upon rAAV-IGF-1 compared with rAAV-lacZ. Novel mechanistic associations between parameters of osteochondral repair and OA were identified. CONCLUSION: Local rAAV-mediated IGF-1 overexpression enhanced osteochondral repair and ameliorated parameters of perifocal early OA. CLINICAL RELEVANCE: IGF-1 gene therapy may be beneficial in repair of focal osteochondral defects and prevention of perifocal OA.

Phyllocoptes parviflori is a distinct species and a vector of the pervasive blackberry leaf mottle associated virus.

Several viruses are transmitted by eriophyid mites (Acariformes: Eriophyoidea) including blackberry leaf mottle-associated emaravirus (BLMaV) (Emaravirus rubi). BLMaV is transmitted by an unidentified eriophyid species and is involved in blackberry yellow vein, a devastating disease in the southeastern United States. In this study, we assessed the eriophyid mite Phylocoptes parviflori as a vector of BLMaV and clarified its taxonomic status as it was previously synonymized with Phyllocoptes gracilis. P. parviflori can efficiently transmit BLMaV. The virus was found to cause yellow vein disease symptoms on 'Ouachita' blackberry marking a paradigm shift as disease symptoms have always been associated with multiple virus infections. Therefore, we propose renaming the virus to blackberry leaf mottle virus. The occurrence of P. parviflori on wild and cultivated blackberries, as well as its ability to colonize other Rubus species, enhances its importance as a major contributor to the spread of yellow vein disease.

Presence of CRISPR CAS-Like Sequences as a Proposed Mechanism for Horizontal Genetic Exchanges between Trichomonas vaginalis and Its Associated Virus: A Comparative Genomic Analysis with the First Report of a Putative CRISPR CAS Structures in Eukaryotic Cells.

Introduction: Trichomonas vaginalis genome is among the largest genome size and coding capacities. Combinations of gene duplications, transposon, repeated sequences, and lateral gene transfers (LGTs) have contributed to the unexpected large genomic size and diversity. This study is aimed at investigating genomic exchange and seeking for presence of the CRISPR CAS system as one of the possible mechanisms for some level of genetic exchange. Material and Methods. In this comparative analysis, 398 publicly available Trichomonas vaginalis complete genomes were investigated for the presence of CRISPR CAS. Spacer sequences were also analyzed for their origin using BLAST. Results: We identified a CRISPR CAS (Cas3). CRISPR spacers are highly similar to transposable genetic elements such as viruses of protozoan parasites, especially megavirals, some transposons, and, interestingly, papillomavirus and HIV-1 in a few cases. Discussion. There is a striking similarity between the prokaryotes/Archaean CRISPR and what we find as eukaryotic CRISPR. About 5-10% of the 398 T. vaginalis possess a CRISPR structure. Conclusion: According to sequences and their organization, we assume that these repeated sequences and spacer, along with their mentioned features, could be the eukaryotic homolog of prokaryotes and Archaean CRISPR systems and may involve in a process similar to the CRISPR function.

Detection and characterization of a putative emaravirus infecting Clematis brevicaudata DC. in China.

A novel emaravirus, tentatively named "clematis yellow mottle associated virus" (CYMaV), was identified through transcriptome sequencing and RT-PCR analysis of yellow-mottled leaf samples from Clematis brevicaudata DC. The genome of CYMaV consists of five viral RNAs: RNA1 (6591 nucleotides, nt), RNA2 (1982 nt), RNA3a (1301 nt), RNA3b (1397 nt), and RNA4 (1192 nt). The 13-nt sequences at the 5'- and 3'-termini of the CYMaV RNAs are conserved and have reverse complementary, as typically seen in emaraviruses. The proteins encoded by CYMaV shared the highest amino acid sequence similarity with those of the unclassified Karaka Okahu purepure emaravirus (KOPV), with 60.2% identity in the RNA-dependent RNA polymerase (RdRp), 44.4% in the glycoprotein precursor, and 46.9% in the nucleocapsid protein. A phylogenetic tree based on amino acid sequences of the RdRp revealed that CYMaV is most closely related to KOPV and clusters with ChMaV (chrysanthemum mosaic-associated virus, LC576445) and PCLSaV (pear chlorotic leaf spot-associated virus, MK602177) in one distinct clade. Transmission electron microscopy observation of negatively stained samples from C. brevicaudata revealed spherical virus-like particles (VLPs) approximately 100 nm in diameter. Five primers, specific for each viral RNA, were used to detect CYMaV in 11 symptomatic and two asymptomatic C. brevicaudata samples, but the results failed to show a consistent association of viral infection with symptoms. CYMaV can be considered a putative new member in the genus Emaravirus, and this marks the first report of an emaravirus found infecting C. brevicaudata plants.

Simultaneous entry as an adaptation to virulence in a novel satellite-helper system infecting Streptomyces species.

Satellites are mobile genetic elements that are dependent upon the replication machinery of their helper viruses. Bacteriophages have provided many examples of satellite nucleic acids that utilize their helper morphogenic genes for propagation. Here we describe two novel satellite-helper phage systems, Mulch and Flayer, that infect Streptomyces species. The satellites in these systems encode for encapsidation machinery but have an absence of key replication genes, thus providing the first example of bacteriophage satellite viruses. We also show that codon usage of the satellites matches the tRNA gene content of the helpers. The satellite in one of these systems, Flayer, does not appear to integrate into the host genome, which represents the first example of a virulent satellite phage. The Flayer satellite has a unique tail adaptation that allows it to attach to its helper for simultaneous co-infection. These findings demonstrate an ever-increasing array of satellite strategies for genetic dependence on their helpers in the evolutionary arms race between satellite and helper phages.

Role of human papillomavirus and associated viruses in bladder cancer: An updated review.

Bladder cancer (BC) is a complex disease affecting the urinary system and is regulated by several carcinogenic factors. Viral infection is one such factor that has attracted extensive attention in BC. Human papillomavirus (HPV) is the most common sexually transmitted infection, and although multiple researchers have explored the role of HPV in BC, a consensus has not yet been reached. In addition, HPV-associated viruses (e.g., human immunodeficiency virus, herpes simplex virus, BK virus, and JC virus) appear to be responsible for the occurrence and progression of BC. This study systematically reviews the relationship between HPV-associated viruses and BC to elucidate the role of these viruses in the onset and progression of BC. In addition, the study aims to provide a greater insight into the biology of HPV-associated viruses, and assess potential strategies for treating virus-induced BC. The study additionally focuses on the rapid development of oncolytic viruses that provide a potentially novel option for the treatment of BC.

Molecular Population Genetics of Aspen Mosaic-Associated Virus in Finland and Sweden.

Aspen mosaic-associated virus (AsMaV) is a newly identified Emaravirus, in the family Fimoviridae, Bunyavirales, associated with mosaic symptoms in aspen trees (Populus tremula). Aspen trees are widely distributed in Europe and understanding the population structure of AsMaV may aid in the development of better management strategies. The virus genome consists of five negative-sense single-stranded RNA (-ssRNA) molecules. To investigate the genetic diversity and population parameters of AsMaV, different regions of the genome were amplified and analyzed and full-length sequence of the divergent isolates were cloned and sequenced. The results show that RNA3 or nucleoprotein is a good representative for studying genetic diversity in AsMaV. Developed RT-PCR-RFLP was able to identify areas with a higher number of haplotypes and could be applied for screening the large number of samples. In general, AsMaV has a conserved genome and based on the phylogenetic studies, geographical structuring was observed in AsMaV isolates from Sweden and Finland, which could be attributed to founder effects. The genome of AsMaV is under purifying selection but not distributed uniformly on genomic RNAs. Distant AsMaV isolates displayed amino acid sequence variations compared to other isolates, and bioinformatic analysis predicted potential post-translational modification sites in some viral proteins.

Extracellular vesicles enhance pulmonary transduction of stably associated adeno-associated virus following intratracheal administration.

Adeno-associated virus (AAV) vector has shown multiple clinical breakthroughs, but its clinical implementation in inhaled gene therapy remains elusive due to difficulty in transducing lung airway cells. We demonstrate here AAV serotype 6 (AAV6) associated with extracellular vesicles (EVs) and secreted from vector-producing HEK-293 cells during vector preparation (EVAAV6) as a safe and highly efficacious gene delivery platform for inhaled gene therapy applications. Specifically, we discovered that EVAAV6 provided markedly enhanced reporter transgene expression in mucus-covered air-liquid interface (ALI) cultures of primary human bronchial and nasal epithelial cells as well as in mouse lung airways compared to standard preparations of AAV6 alone. Of note, AAV6 has been previously shown to outperform other clinically tested AAV serotypes, including those approved by the FDA for treating non-lung diseases, in transducing ALI cultures of primary human airway cells. We provide compelling experimental evidence that the superior performance of EVAAV6 is attributed to the ability of EV to facilitate mucus penetration and cellular entry/transduction of AAV6. The tight and stable linkage between AAV6 and EVs appears essential to exploit the benefits of EVs given that a physical mixture of individually prepared EVs and AAV6 failed to mediate EV-AAV6 interactions or to enhance gene transfer efficacy.

Metagenomic Detection of Divergent Insect- and Bat-Associated Viruses in Plasma from Two African Individuals Enrolled in Blood-Borne Surveillance.

Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.

The pollen virome: A review of pollen-associated viruses and consequences for plants and their interactions with pollinators.

The movement of pollen grains from anthers to stigmas, often by insect pollinator vectors, is essential for plant reproduction. However, pollen is also a unique vehicle for viral spread. Pollen-associated plant viruses reside on the outside or inside of pollen grains, infect susceptible individuals through vertical or horizontal infection pathways, and can decrease plant fitness. These viruses are transferred with pollen between plants by pollinator vectors as they forage for floral resources; thus, pollen-associated viral spread is mediated by floral and pollen grain phenotypes and pollinator traits, much like pollination. Most of what is currently known about pollen-associated viruses was discovered through infection and transmission experiments in controlled settings, usually involving one virus and one plant species of agricultural or horticultural interest. In this review, we first provide an updated, comprehensive list of the recognized pollen-associated viruses. Then, we summarize virus, plant, pollinator vector, and landscape traits that can affect pollen-associated virus transmission, infection, and distribution. Next, we highlight the consequences of plant-pollinator-virus interactions that emerge in complex communities of co-flowering plants and pollinator vectors, such as pollen-associated virus spread between plant species and viral jumps from plant to pollinator hosts. We conclude by emphasizing the need for collaborative research that bridges pollen biology, virology, and pollination biology.

The RNA-RNA interactome between a phage and its satellite virus reveals a small RNA that differentially regulates gene expression across both genomes.

Satellite viruses are present across all domains of life, defined as subviral parasites that require infection by another virus for satellite progeny production. Phage satellites exhibit various regulatory mechanisms to manipulate phage gene expression to the benefit of the satellite, redirecting resources from the phage to the satellite, and often inhibiting phage progeny production. While small RNAs (sRNAs) are well documented as regulators of prokaryotic gene expression, they have not been shown to play a regulatory role in satellite-phage conflicts. Vibrio cholerae encodes the phage inducible chromosomal island-like element (PLE), a phage satellite, to defend itself against the lytic phage ICP1. Here, we use Hi-GRIL-seq to identify a complex RNA-RNA interactome between PLE and ICP1. Both inter- and intragenome RNA interactions were detected, headlined by the PLE sRNA, SviR. SviR is involved in regulating both PLE and ICP1 gene expression uniquely, decreasing ICP1 target translation and affecting PLE transcripts. The striking conservation of SviR across all known PLEs suggests the sRNA is deeply rooted in the PLE-ICP1 conflict and implicates sRNAs as unidentified regulators of gene expression in phage-satellite interactions.

Characterization of a putative novel higrevirus infecting Phellodendron amurense Rupr. in China.

Phellodendron-associated higre-like virus (PaHLV) was identified in Phellodendron amurense Rupr. in China. Three near-full-length sequences of the viral genomic RNAs (RNA1-RNA3) were first obtained by RNA-seq, and their complete sequences were then determined by RT-PCR, 5'-RACE, and 3'-RACE. RNA1-3 of PaHLV were determined to be 8,183, 3,062, and 3,998 nucleotides (nt) in length, respectively, excluding the poly(A) tail. All of the viral proteins encoded by PaHLV shared the highest amino acid sequence identity (44.8-78.1%) with the unclassified kitavirid pistachio virus X (PiVX, MT334618-MT334620) from Iranian pistachio. Sequence comparisons and phylogenetic analysis also showed PiVX to be the closest relative of PaHLV and supported their inclusion in the genus Higrevirus, family Kitaviridae. Thus, PaHLV is proposed to be a member of a new species in this genus, for which we suggest the binomial name "Higrevirus amur".

Cohombrillo-associated virus: a novel virus infecting Ecballium elaterium plants.

We determined the complete genome sequence of a new virus infecting Ecballium elaterium ('cohombrillo amargo') plants, a weed species common on the borders of cultivated fields in the Mediterranean region. The genome of this virus is composed of two molecules of monocistronic positive-sense RNA, 6,934 and 3,501 nucleotides in length, excluding their poly(A) tails. The highest amino acid sequence similarity (50 % identity) in the Pro-Pol core region encoded by RNA 1 was observed in the corresponding protein of strawberry latent ringspot virus. Based on pairwise comparisons and phylogenetic analysis, this virus, tentatively named "cohombrillo-associated virus" (CoAV), appears to be a member of a new species in the genus Stralarivirus (family Secoviridae), for which the name "Stralarivirus elaterii" is proposed. This new virus has different putative cleavage patterns from members of other species belonging to this genus.

Transmission of Grapevine Leafroll-Associated Viruses and Grapevine Virus A by Vineyard-Sampled Soft Scales (Parthenolecanium corni, Hemiptera: Coccidae).

Grapevine-infecting ampelo- and vitiviruses are transmitted by scale insects belonging to several species, among which is the European fruit lecanium, Parthenolecanium corni (Bouché) (Hemiptera Coccidae). Our objective was to characterize the transmission biology of grapevine leafroll-associated viruses (GLRaV) and grapevine virus A (GVA) by this soft scale species in order to evaluate its ability to spread these viruses. In transmission experiments with nymphs sampled from different vineyards infected with GLRaV 1, 2, 3 and GVA, P. corni transmitted only GLRaV 1 and GVA to healthy vines. GVA was predominantly transmitted along with GLRaV 1, whereas the latter could be transmitted alone from single or co-infected vines. Vineyard-sampled second instar nymphs were more efficient than first instars at transmitting GLRaV 1, whereas both instars displayed similar transmission rates for GVA. Short virus inoculation access periods and the absence of virus in eggs of females living on infected grapevines fulfilled the criteria of non-circulative semi-persistent transmission mode.

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.

Identification and complete genome sequencing of a divergent olive virus T isolate and an olive leaf yellowing-associated virus isolate naturally infecting olive trees in Greece.

Several new full genome sequences of olive viruses came to light recently via high-throughput sequencing (HTS) analysis. In this study, total RNA HTS analysis of two Greek olive trees revealed the presence of an olive virus T (OlVT) isolate and an olive leaf yellowing-associated virus (OLYaV) isolate. The full viral genome of OlVT isolate (50Ch) is composed of 6862 nucleotides encoding for three proteins (replicase, movement protein, and capsid protein) with typical betaflexiviruses' genomic features. However, both sequence and phylogenetic data analysis exhibited high levels of variability between 50Ch and the previously characterized OlVT isolates. In addition, the almost full genome of the Greek OLYaV isolate (OL2) was obtained, which is composed of 16,693 nucleotides encoding for 11 open reading frames (ORFs) and shares common genomic features with the recently characterized OLYaV isolates from Spain and Brazil. Sequence and phylogenetic analysis revealed high similarity between these three isolates. Due to problems encountered with the detection of both viruses, new nested RT-PCR assays were developed and applied. In addition, recombination events were observed in OlVT isolates (50Ch GR-168), thus highlighting the potential role of this mechanism in the evolution of the virus. This study is adding further knowledge to the limited information available about these recently characterized olive infecting viral pathogens and highlights their widespread distribution in Greece, one of the most important olive producing countries of the world.

[Distribution of mosquito species and associated viruses in Hami City of Xinjiang Uygur Autonomous Region from 2019 to 2020].

OBJECTIVE: To investigate the distribution of mosquito species and their associated viruses, and identify Culex pipiens subspecies in Hami City, Xinjiang Uygur Autonomous Region. METHODS: Mosquitoes were captured using mosquito trapping lamps method in Yizhou District, Yiwu County, and Balikun County of Hami City in mi-July, 2019 and 2020. The species and subspecies of all captured mosquitoes were characterized. In addition, the flavivirus, alphavirus, bunyavirus, Japanese encephalitis virus, Liaoning virus, Tahyna virus, tick-borne encephalitis virus and West Nile virus were detected using reverse-transcription PCR assay in captured mosquitoes. RESULTS: A total of 1 496 mosquitoes were captured from Yizhou District, Yiwu County, and Balikun County of Hami City, belonging to 3 genus and 3 species. Cx. pipiens was the dominant mosquito species (986 mosquitoes, 65.91%), followed by Aedes caspius (457 mosquitoes, 30.55%), while Culiseta alaskaensis had the lowest number (53 mosquitoes, 3.54%). All captured Cx. pipiens mosquitoes were identified as Cx. pipiens pipiens based on the terminalia of male mosquitoes. RT-PCR assay tested negative for flavivirus, alphavirus, bunyavirus, Japanese encephalitis virus, Liaoning virus, Tahyna virus, tick-borne encephalitis or West Nile virus in captured Cx. pipiens mosquitoes. CONCLUSIONS: There were 3 species of mosquitoes in Hami City from 2019 to 2020, including Cx. pipiens, Ae. Caspius and C. alaskaensis, with Cx. pipiens as the dominant mosquito species, and all captured Cx. pipiens mosquitoes were Cx. pipiens pipiens; however, no arboviruses were detected.

Mosquito-Associated Virus Isolation from Field-Collected Mosquitoes.

With the broad application of sequencing technologies, many novel virus-like sequences have been discovered in arthropods, including mosquitoes. The two main categories of these new mosquito-associated viruses are "mosquito-borne viruses (MBVs)" and "mosquito-specific viruses (MSVs)". These novel viruses might be pathogenic to both vertebrates and mosquitoes, or they could just be symbiotic with mosquitoes. Entity viruses are essential to confirm the biological characters of these viruses. Thus, a detailed protocol has been described here for virus isolation and amplification from field-collected mosquitoes. First, the mosquito samples were prepared as supernatants of mosquito homogenates. After centrifugation twice, the supernatants were then inoculated into either mosquito cell line C6/36 or vertebrate cell line BHK-21 for virus amplification. After 7 days, the supernatants were collected as the P1 supernatants and stored at -80 °C. Next, P1 supernatants were passaged twice more in C6/36 or BHK-21 cells while the cell status was being checked daily. When cytopathogenic effect (CPE) on the cells was discovered, these supernatants were collected and used to identify viruses. This protocol serves as the foundation for future research on mosquito-associated viruses, including MBVs and MSVs.

Complete genome sequence of Edgeworthia chrysantha mosaic-associated virus, a tentative new member of the genus Coguvirus (family Phenuiviridae).

A new negative-strand RNA (nsRNA) virus genome was discovered in Edgeworthia chrysantha Lindl. This virus, tentatively named "Edgeworthia chrysantha mosaic-associated virus" (ECMaV), has a bipartite genome that comprises (i) a nsRNA1, encoding the viral RNA-dependent RNA polymerase (RdRp), and (ii) an ambisense RNA2, coding for the putative movement protein (MP) and nucleocapsid protein (NP), with the open reading frames separated by a long AU-rich intergenic region (IR). Sequence comparisons and phylogenetic analysis showed that the RdRp is closely related to those of other recently discovered plant-infecting nsRNA viruses in the new genus Coguvirus and that ECMaV can be classified as a member of a novel species.

Development of a sensitive real-time quantitative RT-PCR assay for the detection of pear chlorotic leaf spot-associated virus.

Pear chlorotic leaf spot associated virus (PCLSaV) belongs to the genus Emaravirus and possesses a genome composed of five negative-sense single-stranded RNA (-ssRNA) segments. This study developed a SYBR green-based reverse transcription quantitative PCR (RT-qPCR) assay for the detection of PCLSaV infecting pear trees. A set of two primers q5-F2/q5-R2 designed based on the viral RNA5 sequences showed high specificity and feasibility for PCLSaV detection. The standard curve was established. RT-qPCR assays showed that PCLSaV content was greatly higher in diseased branch and symptomatic leaf samples than that in un-diseased branch and asymptomatic leaf samples. The RT-qPCR was reliability in the detection of the virus in field and in-vitro cultured pear samples. This technique would be useful for the supervision of the viral disease and the certification of pear planting materials.