The Viral Fraction Metatranscriptomes of Lake Baikal.

This article characterises viral fraction metatranscriptomes (smaller than 0.2 µm) from the pelagic zone of oligotrophic Lake Baikal (Russia). The study revealed the dominance of transcripts of DNA viruses: bacteriophages and algal viruses. We identified transcripts similar to Pithovirus sibericum, a nucleocytoplasmic large DNA virus (NCLDV) isolated from the permafrost region of Eastern Siberia. Among the families detected were RNA viruses assigned to Retroviridae, Metaviridae, Potyviridae, Astroviridae, and Closteroviridae. Using the PHROG, SEED subsystems databases, and the VOGDB, we indicated that the bulk of transcripts belong to the functional replication of viruses. In a comparative unweighted pair group method with arithmetic mean (UPGMA) analysis, the transcripts from Lake Baikal formed a separate cluster included in the clade with transcripts from other freshwater lakes, as well as marine and oceanic waters, while there was no separation based on the trophic state of the water bodies, the size of the plankton fraction, or salinity.

Development and evaluation of a ligation-free sequence-independent, single-primer amplification (LF-SISPA) assay for whole genome characterization of viruses.

Molecular identification and characterization of novel or re-emerging infectious pathogens is critical for disease surveillance and outbreak investigations. Next generation sequencing (NGS) using Sequence-Independent, Single-Primer Amplification (SISPA) is being used extensively in sequencing of viral genomes but it requires an expensive library preparation step. We developed a simple, low-cost method that enriches nucleic acids followed by a ligation-free (LF) 2-step Polymerase Chain Reaction (PCR) procedure for library preparation. A pan-chimeric universal primer (JS15N14) containing 15 nucleotides with a random tetradecamer (14N) attached to the 3'-end was designed. The complimentary primer (JS15) was used for nucleic acid enrichment in a first round PCR. A second PCR was designed to create Illumina sequencer-compatible sequencing-ready libraries for NGS. The new LF-SISPA protocol was tested using six RNA and DNA viral genomes (10.8-229.4 kilobases, kb) from an ATCC virome nucleic acid mix (ATCC® MSA-1008™) followed by analysis using One Codex, an online identification tool. In addition, a human stool sample known to be positive for norovirus GII was sequenced, and de novo assembly was performed using the Genome Detective Virus Tool allowing for near complete genome identification in less than 24 h. The LF-SISPA method does not require prior knowledge of target sequences and does not require an expensive enzymatic library preparation kit, thereby providing a simple, fast, low-cost alternative for the identification of unknown viral pathogens.

Inhibitors of Yellow Fever Virus replication based on 1,3,5-triphenyl-4,5-dihydropyrazole scaffold: Design, synthesis and antiviral evaluation.

By the antiviral screening of an in house library of pyrazoline compounds, 4-(3-(4-phenoxyphenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (5a) was identified as a promising hit compound for the development of anti- Yellow Fever Virus (YFV) agents. Structural optimization studies were focused on the development of 5a analogues which retain the potency as YFV inhibitors and show a reduced cytotoxicity. The synthesized 1-3,5-triphenyl-pyrazolines (4a-j, 5a-j, 6a-j) were evaluated in cell based assays for cytotoxicity and antiviral activity against representative viruses of two of the three genera of the Flaviviridae family, i.e.: Pestivirus (BVDV) and Flavivirus (YFV). These compounds were also tested against a large panel of different pathogenic RNA and DNA viruses. Most of the new 1-3,5-triphenyl-pyrazolines (4a-j, 5a-j, 6a-j) exhibited a specific activity against YFV, showing EC50 values in the low micromolar range with almost a 10-fold improvement in potency compared to the reference inhibitor 6-azauridine. However, the selectivity indexes of the unsubstituted (4a-j) and the phenoxy (5a-j) analogues were generally modest due to the pronounced cytotoxicity against BHK-21 cells. Otherwise, the benzyloxy derivatives (6a-j) generally coupled high potency and selectivity. On the basis of both anti-YFV activity and selectivity index, pyrazolines 6a and 6b were chosen for time of addition experiments. The selected pyrazolines and the reference inhibitor 6-azauridine displayed maximal inhibition when added in the pretreatment or during the infection.

N-((1,3-Diphenyl-1H-pyrazol-4-yl)methyl)anilines: A novel class of anti-RSV agents.

A series of N-((1,3-diphenyl-1H-pyrazol-4-yl)methyl)anilines were synthesized and evaluated in vitro for cytotoxicity and antiviral activity against a large panel of viruses. Most of the tested compounds interfered with RSV replication in the micromolar concentrations (EC50s ranging from 5 µM to 28 µM). SAR studies suggested that the presence of a trifluoromethyl group in R(1) abolished the anti-RSV activity and enhanced the cytotoxicity while the best results in term of both anti-RSV activity and selectivity were obtained by the introduction in R(1) of a chlorine or a bromine atom.

Antiviral activity of benzimidazole derivatives. III. Novel anti-CVB-5, anti-RSV and anti-Sb-1 agents.

A library of eighty-six assorted benzimidazole derivatives was screened for antiviral activity against a panel of ten RNA and DNA viruses. Fifty-two of them displayed different levels of activity against one or more viruses, among which CVB-5, RSV, BVDV and Sb-1 were the most frequently affected. In particular, fourteen compounds exhibited an EC50 in the range 9-17µM (SI from 6 to >11) versus CVB-5, and seven compounds showed an EC50 in the range 5-15µM (SI from 6.7 to ⩾20) against RSV, thus resulting comparable to or more potent than the respective reference drugs (NM108 and ribavirin). Most of these compounds derive from 2-benzylbenzimidazole, but also other molecular scaffolds [as 1-phenylbenzimidazole (2), 2-trifluoromethylbenzimidazole (69), dihydropyrido[3',2':4,5]imidazo[1,2-a][1,4]benzodiazepin-5-one (3), dibenzo[c,e]benzimidazo[1,2-a]azepine (22), and 2-(tetrahydropyran-2-yl)benzimidazole (81, 82 and 86)] are related to interesting levels of activity against these or other viruses (BVDV, Sb-1). Thus, these scaffolds (some of which, so far unexplored), represent valid starting points to develop more efficient agents against pathologies caused by CVB-5, RSV, BVDV and Sb-1 viruses.

Easy-to-use rapid gene amplification method for direct detection of RNA and DNA viruses in sera and feces from various animals.

The development of rapid and simple gene amplification tests is required for detection of pathogens to prevent transmission of infectious diseases between animals or from animals to humans. An easy-to-use rapid gene amplification method that can directly detect RNA and DNA viruses in clinical samples was developed. This method is based on combining loop-mediated isothermal amplification (LAMP) or reverse transcription-LAMP (RT-LAMP) and RNA GEM Tissue, a thermophilic enzyme that extracts nucleic acid by quickly digesting proteins and ribonucleases. The authors named these methods GEM LAMP and GEM RT-LAMP. These methods were able to detect viral DNA and RNA within 70 min in a single tube using only a water bath. The detection capacities were 10-100-fold more sensitive than those of previously established LAMP and RT-LAMP methods. The GEM LAMP and GEM RT-LAMP methods were used to detect macroscopically the presence of DNA and RNA viruses in sera or fecal samples from cattle, pigs, horses, dolphins, penguins, and sea lions using SYBR green I. The GEM LAMP and GEM RT-LAMP methods thus have considerable versatility as tools for detecting pathogens and are applicable to basic human and veterinary medicine, environmental hygiene, and point-of-care-testing.