Biological Evaluation of Uridine Derivatives of 2-Deoxy Sugars as Potential Antiviral Compounds against Influenza A Virus

Influenza virus infection is a major cause of morbidity and mortality worldwide. Due to the limited ability of currently available treatments, there is an urgent need for new anti-influenza drugs with broad spectrum protection. We have previously shown that two 2-deoxy sugar derivatives of uridine (designated IW3 and IW7) targeting the glycan processing steps during maturation of viral glycoproteins show good anti-influenza virus activity and may be a promising alternative approach for the development of new anti-influenza therapy. In this study, a number of IW3 and IW7 analogues with different structural modifications in 2-deoxy sugar or uridine parts were synthesized and evaluated for their ability to inhibit influenza A virus infection in vitro. Using the cytopathic effect (CPE) inhibition assay and viral plaque reduction assay in vitro, we showed that compounds 2, 3, and 4 exerted the most inhibitory effect on influenza virus A/ostrich/Denmark/725/96 (H5N2) infection in Madin-Darby canine kidney (MDCK) cells, with 50% inhibitory concentrations (IC50) for virus growth ranging from 82 to 100 (μM) without significant toxicity for the cells. The most active compound (2) showed activity of 82 μM with a selectivity index value of 5.27 against type A (H5N2) virus. Additionally, compound 2 reduced the formation of HA glycoprotein in a dose-dependent manner. Moreover, an analysis of physicochemical properties of studied compounds demonstrated a significant linear correlation between lipophilicity and antiviral activity. Therefore, inhibition of influenza A virus infection by conjugates of uridine and 2-deoxy sugars is a new promising approach for the development of new derivatives with anti-influenza activities

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Multitemperature Single Stranded Conformational Polymorphism

Baculoviruses have been used as biopesticides for decades. Recently, due to the excessive use of chemical pesticides there is a need for finding new agents that may be useful in biological protection. Sometimes few isolates or species are discovered in one host. In the past few years, many new baculovirus species have been isolated from environmental samples, thoroughly characterized and thanks to next generation sequencing methods their genomes are being deposited in the GenBank database. Next generation sequencing (NGS) methodology is the most certain way of detection, but it has many disadvantages. During our studies, we have developed a method based on Polymerase chain reaction (PCR) followed by Multitemperature Single Stranded Conformational Polymorphism (MSSCP) which allows for distinguishing new granulovirus isolates in only a few hours and at low-cost. On the basis of phylogenetic analysis of betabaculoviruses, representative species have been chosen. The alignment of highly conserved genes—granulin and late expression factor-9, was performed and the degenerate primers were designed to amplify the most variable, short DNA fragments flanked with the most conserved sequences. Afterwards, products of PCR reaction were analysed by MSSCP technique. In our opinion, the proposed method may be used for screening of new isolates derived from environmental samples

Additional file 1: Table S1. of The genome of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) reveals novel genetic connection between baculoviruses infecting moths of the Lymantriidae family

Predicted ORFs and repeat regions in the genome of DapuNPV. This file lists the ORFs predicted (with its direction and promoter sequences) in the genome of DapuNPV and their homologues in 15 other completely sequenced baculoviruses. (XLSX 31 kb

Additional file 2: Table S2. of The genome of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) reveals novel genetic connection between baculoviruses infecting moths of the Lymantriidae family

List of sequenced to date baculoviruses. This file lists all baculoviruses sequenced to date, with their accession number, genome length and source of the sequence. (DOCX 31 kb

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real-Time Polymerase Chain Reaction (Real-Time PCR)

Baculoviridae is a highly diverse family of rod-shaped viruses with double-stranded DNA. To date, almost 100 species have had their complete genomic sequences deposited in the GenBank database, a quarter of which comprises granuloviruses (GVs). Many of the genomes are sequenced using next-generation sequencing, which is currently considered the best method for characterizing new species, but it is time-consuming and expensive. Baculoviruses form a safe alternative to overused chemical pesticides and therefore there is a constant need for identifying new species that can be active components of novel biological insecticides. In this study, we have described a fast and reliable method for the detection of new and differentiation of previously analyzed granulovirus species based on a real-time polymerase chain reaction (PCR) technique with melting point curve analysis. The sequences of highly conserved baculovirus genes, such as granulin and late expression factors 8 and 9 (lef-8 and lef-9), derived from GVs available to date have been analyzed and used for degenerate primer design. The developed method was tested on a representative group of eight betabaculoviruses with comparisons of melting temperatures to allow for quick and preliminary granulovirus detection. The proposed real-time PCR procedure may be a very useful tool as an easily accessible screening method in a majority of laboratories

Additional file 3: Figure S1. of The genome of Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) reveals novel genetic connection between baculoviruses infecting moths of the Lymantriidae family

Alignment of OpMNPV p8.9 ORF and its homologue in DapuNPV genome. Large insertion after nucleotide 201 in DapuNPV gene does not change the reading frame, although it decreases total basicity of translated protein which is a specific feature of p8.9 protein from OpMNPV (MAFT multiple alignment with default settings, visualization in Geneious R7). (PNG 80 kb

Genome Analysis and Genetic Stability of the Cryptophlebia leucotreta Granulovirus (CrleGV-SA) after 15 Years of Commercial Use as a Biopesticide

Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is an indigenous pest in southern Africa which attacks citrus fruits and other crops. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme incorporating the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) as a biopesticide has been implemented. This study investigated the genetic stability of a commercially produced CrleGV-SA product that has been applied in the field since 2000. Seven representative full-genome sequences of the CrleGV-SA isolate spanning a 15-year period were generated and compared with one another. Several open reading frames (ORFs) were identified to have acquired single nucleotide polymorphisms (SNPs) during the 15-year period, with three patterns observed and referred to as “stable”, “reversion”, and “unstable switching”. Three insertion events were also identified, two of which occurred within ORFs. Pairwise multiple alignments of these sequences showed an identity ranging from 99.98% to 99.99%. Concentration-response bioassays comparing samples of CrleGV-SA from 2000 and 2015 showed an increase in virulence toward neonate T. leucotreta larvae. The CrleGV-SA genome sequence generated from the 2015 sample was compared to the Cape Verde reference genome, CrleGV-CV3. Several fusion events were identified between ORFs within these genomes. These sequences shared 96.7% pairwise identity, confirming that CrleGV-SA is a genetically distinct isolate. The results of this study indicate that the genome of CrleGV-SA has remained stable over many years, with implications for its continued use as a biopesticide in the field. Furthermore, the study describes the first complete baculovirus genome to be sequenced with the MinION (Oxford Nanopore, Oxford, UK) platform and the first complete genome sequence of the South African CrleGV isolate