Morphologic Differentiation of Viruses beyond the Family Level

Electron microscopy has been instrumental in the identification of viruses by being able to characterize a virus to the family level. There are a few cases where morphologic or morphogenesis factors can be used to differentiate further, to the genus level. These include viruses in the families Poxviridae, Reoviridae, Retroviridae, Herpesviridae, Filoviridae, and Bunyaviridae.     

Dual selection mechanisms drive efficient single-gene reverse genetics for rotavirus

Current methods for engineering the segmented double-stranded RNA genome of rotavirus (RV) are limited by inefficient recovery of the recombinant virus. In an effort to expand the utility of RV reverse genetics, we developed a method to recover recombinant viruses in which independent selection strategies are used to engineer single-gene replacements. We coupled a mutant SA11 RV encoding a temperature-sensitive (ts) defect in the NSP2 protein with RNAi-mediated degradation of NSP2 mRNAs to isolate a virus containing a single recombinant gene that evades both selection mechanisms. Recovery is rapid and simple; after two rounds of selective passage the recombinant virus reaches titers of ≥10(4) pfu/mL. We used this reverse genetics method to generate a panel of viruses with chimeric NSP2 genes. For one of the chimeric viruses, the introduced NSP2 sequence was obtained from a pathogenic, noncultivated human RV isolate, demonstrating that this reverse genetics system can be used to study the molecular biology of circulating RVs. Combining characterized RV ts mutants and validated siRNA targets should permit the extension of this "two-hit" reverse genetics methodology to other RV genes. Furthermore, application of a dual selection strategy to previously reported reverse genetics methods for RV may enhance the efficiency of recombinant virus recovery.     

Zoonotic infections in communities of the James Bay Cree territory: An overview of seroprevalence

The Cree communities of James Bay are at risk for contracting infectious diseases transmitted by wildlife. Data from serological testing for a range of zoonotic infections performed in the general population (six communities), or trappers and their spouses (one community), were abstracted from four population-based studies conducted in Cree territory (Quebec) between 2005 and 2009. Evidence of exposure to Trichinella species, Toxoplasma gondii, Toxocara canis, Echinococcus granulosus, Leptospira species, Coxiella burnetii and Francisella tularensis was verified in all communities, whereas antibodies against Sin Nombre virus and California serogroup viruses (Jamestown Canyon and snowshoe hare viruses) were evaluated in three and six communities, respectively. Seroprevalence varied widely among communities: snowshoe hare virus (1% to 42%), F tularensis (14% to 37%), Leptospira species (10% to 27%), Jamestown Canyon virus (9% to 24%), C burnetii (0% to 18%), T gondii (4% to 12%), T canis (0% to 10%), E granulosus (0% to 4%) and Trichinella species (0% to 1%). No subject had serological evidence of Sin Nombre virus exposure. These data suggest that large proportions of the Cree population have been exposed to at least one of the targeted zoonotic agents. The Cree population, particularly those most heavily exposed to fauna, as well as the medical staff living in these regions, should be aware of these diseases. Greater awareness would not only help to decrease exposures but would also increase the chance of appropriate diagnostic testing.     

Characterization of MADS-box genes in charophycean green algae and its implication for the evolution of MADS-box genes

The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.     

Isolation and Genome Characterization of the Virulent Staphylococcus aureus Bacteriophage SA97

A novel bacteriophage that infects S. aureus, SA97, was isolated and characterized. The phage SA97 belongs to the Siphoviridae family, and the cell wall teichoic acid (WTA) was found to be a host receptor of the phage SA97. Genome analysis revealed that SA97 contains 40,592 bp of DNA encoding 54 predicted open reading frames (ORFs), and none of these genes were related to virulence or drug resistance. Although a few genes associated with lysogen formation were detected in the phage SA97 genome, the phage SA97 produced neither lysogen nor transductant in S. aureus. These results suggest that the phage SA97 may be a promising candidate for controlling S. aureus.     

Molecular Characterization of a Novel Positive-Sense,Single-Stranded RNA Mycovirus Infecting the Plant Pathogenic Fungus Sclerotinia sclerotiorum

Recent studies have demonstrated that a diverse array of mycoviruses infect the plant pathogenic fungus Sclerotinia sclerotiorum. Here, we report the molecular characterization of a newly identified mycovirus, Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), which was isolated from a sclerotia-defective strain JMTJ14 of S. sclerotiorum. Excluding a poly (A) tail, the genome of SsFV1 comprises 7754 nucleotides (nts) in length with 83 and 418 nts for 5''- and 3''-untranslated regions, respectively. SsFV1 has four non-overlapping open reading frames (ORFs): ORF1 encodes a 191 kDa polyprotein (1664 amino acid residues in length) containing conserved RNA-dependent RNA polymerase (RdRp) and helicase domains; the other three ORFs encode three putative hypothetical proteins of unknown function. Phylogenetic analysis, based on RdRp and Helicase domains, indicated that SsFV1 is phylogenetically related to Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), a cluster of an independent group belonging to a newly proposed family Fusarividae. However, SsFV1 is markedly different from FgV1 and RnFV1 in genome organization and nucleotide sequence. SsFV1 was transmitted successfully to two vegetatively incompatible virus-free strains. SsFV1 is not responsible for the abnormal phenotype of strain JMTJ14.     

Role of hepatitis B virus DNA integration in human hepatocarcinogenesis

Liver cancer ranks sixth in cancer incidence, and is the third leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which arises from hepatocytes and accounts for approximately 70%-85% of cases. Hepatitis B virus (HBV) frequently causes liver inflammation, hepatic damage and subsequent cirrhosis. Integrated viral DNA is found in 85%-90% of HBV-related HCCs. Its presence in tumors from non-cirrhotic livers of children or young adults further supports the role of viral DNA integration in hepatocarcinogenesis. Integration of subgenomic HBV DNA fragments into different locations within the host DNA is a significant feature of chronic HBV infection. Integration has two potential consequences: (1) the host genome becomes altered (“cis” effect); and (2) the HBV genome becomes altered (“trans” effect). The cis effect includes insertional mutagenesis, which can potentially disrupt host gene function or alter host gene regulation. Tumor progression is frequently associated with rearrangement and partial gain or loss of both viral and host sequences. However, the role of integrated HBV DNA in hepatocarcinogenesis remains controversial. Modern technology has provided a new paradigm to further our understanding of disease mechanisms. This review summarizes the role of HBV DNA integration in human carcinogenesis.     

Nomenclature- and Database-Compatible Names for the Two Ebola Virus Variants that Emerged in Guinea and the Democratic Republic of the Congo in 2014

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: “Makona”, Middle Africa: “Lomela”) and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.     

Genetic and epigenetic biomarkers for diagnosis,prognosis and treatment of colorectal cancer

Colorectal cancer(CRC)is one of the most common cancer worldwide and results from the accumulation of mutations and epimutations in colonic mucosa cells ultimately leading to cell proliferation and metastasis.Unfortunately,CRC prognosis is still poor and the search of novel diagnostic and prognostic biomarkers is highly desired to prevent CRC-related deaths.The present article aims to summarize the most recent findings concerning the use of either genetic or epigenetic(mainly related to DNA methylation)biomarkers for CRC diagnosis,prognosis,and response to treatment.Recent large-scale DNA methylation studies suggest that CRC can be divided into several subtypes according to the frequency of DNA methylation and those of mutations in key CRC genes,and that this is reflected by different prognostic outcomes.Increasing evidence suggests that the analysis of DNA methylation in blood or fecal specimens could represent a valuable non-invasive diagnostic tool for CRC.Moreover,a broad spectrum of studies indicates that the inter-individual response to chemotherapeutic treatments depends on both epigenetic modifications and genetic mutations occurring in colorectal cancer cells,thereby opening the way for a personalized medicine.Overall,combining genetic and epigenetic data might represent the most promising tool for a proper diagnostic,prognostic and therapeutic approach.     

The Genetic Characterization of a Novel Natural Recombinant Pseudorabies Virus in China

We sequenced the complete genome of the pseudorabies virus (PRV) FJ epidemic strain, and we studied the characteristics and the differences compared with the classical Chinese strain and that of other countries. Third-generation sequencing and second-generation sequencing technology were used to construct, sequence, and annotate an efficient, accurate PRV library. The complete FJ genome was 143,703 bp, the G+C content was 73.67%, and it encoded a total of 70 genes. The genetic evolution of the complete genome and some key gene sequences of the FJ strain and PRV reference strains were analyzed by the maximum likelihood (ML) method of MEGA 7.0 software. According to the ML tree based on the full-length genome sequences, PRV FJ strain was assigned to the branch of genotype II, and it showed a close evolutionary relationship with PRV epidemic variants isolated in China after 2011. The gB, gC, gD, gH, gL, gM, gN, TK, gI, and PK genes of the FJ strain were assigned to the same branch with other Chinese epidemic mutants; its gG gene was assigned to the same branch with the classic Chinese Fa and Ea strains; and its gE gene was assigned to a relatively independent branch. Potential recombination events were predicted by the RDP4 software, which showed that the predicted recombination sites were between 1694 and 1936 bp, 101,113 and 102,660 bp, and 107,964 and 111,481 bp in the non-coding region. This result broke the previously reported general rule that pseudorabies virus recombination events occur in the gene coding region. The major backbone strain of the recombination event was HLJ8 and the minor backbone strain was Ea. Our results allowed us to track and to grasp the recent molecular epidemiological changes of PRV. They also provide background materials for the development of new PRV vaccines, and they lay a foundation for further study of PRV.     

Recessive Resistance Derived from Tomato cv. Tyking-Limits Drastically the Spread of Tomato Yellow Leaf Curl Virus

The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F₁. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread.     

Genetic and dietary regulation of lipid droplet expansion in Caenorhabditis elegans

Dietary fat accumulates in lipid droplets or endolysosomal compartments that undergo selective expansion under normal or pathophysiological conditions. We find that genetic defects in a peroxisomal β-oxidation pathway cause size expansion in lipid droplets that are distinct from the lysosome-related organelles in Caenorhabditis elegans. Expansion of lipid droplets is accompanied by an increase in triglycerides (TAG) that are resistant to fasting- or TAG lipase-triggered lipolysis. Nevertheless, in mutant animals, a diet poor in vaccenic acid reduced the TAG level and lipid droplet size. Our results implicate peroxisomal dysfunction in pathologic lipid droplet expansion in animals and illustrate how dietary factors modulate the phenotype of such genetic defects.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Haplotype structure strongly affects recombination in a maize genetic interval polymorphic for Helitron and retrotransposon insertions

We have asked here how the remarkable variation in maize haplotype structure affects recombination. We compared recombination across a genetic interval of 9S in 2 highly dissimilar heterozygotes that shared 1 parent. The genetic interval in the common haplotype is ≈100 kb long and contains 6 genes interspersed with gene-fragment-bearing Helitrons and retrotransposons that, together, comprise 70% of its length. In one heterozygote, most intergenic insertions are homozygous, although polymorphic, enabling us to determine whether any recombination junctions fall within them. In the other, most intergenic insertions are hemizygous and, thus, incapable of homologous recombination. Our analysis of the frequency and distribution of recombination in the interval revealed that: (i) Most junctions were circumscribed to the gene space, where they showed a highly nonuniform distribution. In both heterozygotes, more than half of the junctions fell in the stc1 gene, making it a clear recombination hotspot in the region. However, the genetic size of stc1 was 2-fold lower when flanked by a hemizygous 25-kb retrotransposon cluster. (ii) No junctions fell in the hypro1 gene in either heterozygote, making it a genic recombination coldspot. (iii) No recombination occurred within the gene fragments borne on Helitrons nor within retrotransposons, so neither insertion class contributes to the interval''s genetic length. (iv) Unexpectedly, several junctions fell in an intergenic region not shared by all 3 haplotypes. (v) In general, the ability of a sequence to recombine correlated inversely with its methylation status. Our results show that haplotypic structural variability strongly affects the frequency and distribution of recombination events in maize.     

First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis

Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection.     

Genetic dissection of the biosynthetic route to gentamicin A2 by heterologous expression of its minimal gene set

Since the first use of streptomycin as an effective antibiotic drug in the treatment of tuberculosis, aminoglycoside antibiotics have been widely used against a variety of bacterial infections for over six decades. However, the pathways for aminoglycoside biosynthesis still remain unclear, mainly because of difficulty in genetic manipulation of actinomycetes producing this class of antibiotics. Gentamicin belongs to the group of 4,6-disubstituted aminoglycosides containing a characteristic core aminocyclitol moiety, 2-deoxystreptamine (2-DOS), and the recent discovery of its biosynthetic gene cluster in Micromonospora echinospora has enabled us to decipher its biosynthetic pathway. To determine the minimal set of genes and their functions for the generation of gentamicin A(2), the first pseudotrisaccharide intermediate in the biosynthetic pathway for the gentamicin complex, various sets of candidate genes from M. echinospora and other related aminoglycoside-producing strains were introduced into a nonaminoglycoside producing strain of Streptomyces venezuelae. Heterologous expression of different combinations of putative 2-DOS biosynthetic genes revealed that a subset, gtmB-gtmA-gacH, is responsible for the biosynthesis of this core aminocyclitol moiety of gentamicin. Expression of gtmG together with gtmB-gtmA-gacH led to production of 2'-N-acetylparomamine, demonstrating that GtmG acts as a glycosyltransferase that adds N-acetyl-d-glucosamine (GLcNA) to 2-DOS. Expression of gtmM in a 2'-N-acetylparomamine-producing recombinant S. venezuelae strain generated paromamine. Expression of gtmE in an engineered paromamine-producing strain of S. venezuelae successfully generated gentamicin A(2), indicating that GtmE is another glycosyltransferase that attaches d-xylose to paromamine. These results represent in vivo evidence elucidating the complete biosynthetic pathway of the pseudotrisaccharide aminoglycoside.