The lion's share of bacteria in various environments cannot be cloned in the laboratory and thus cannot be sequenced using existing technologies. A major goal of single-cell genomics is to complement gene-centric metagenomic data with whole-genome assemblies of uncultivated organisms. Assembly of single-cell data is challenging because of highly non-uniform read coverage as well as elevated levels of sequencing errors and chimeric reads. We describe SPAdes, a new assembler for both single-cell and standard (multicell) assembly, and demonstrate that it improves on the recently released E+V-SC assembler (specialized for single-cell data) and on popular assemblers Velvet and SoapDeNovo (for multicell data). SPAdes generates single-cell assemblies, providing information about genomes of uncultivatable bacteria that vastly exceeds what may be obtained via traditional metagenomics studies. SPAdes is available online ( http://bioinf.spbau.ru/spades ). It is distributed as open source software.

SPAdes, a new genome assembly algorithm and its applications to single-cell sequencing ( 7th Annual SFAF Meeting, 2012)

http://irceb.asu.edu/amphibians/text/actatrop.pdf

Anthropogenic environmental change and the emergence of infectious diseases in wildlife.

By using the criteria that define emerging infectious diseases (EIDs) of humans, we can identify a similar group of EIDs in wildlife. In the current review we highlight an important series of wildlife EIDs: amphibian chytridiomycosis; diseases of marine invertebrates and vertebrates and two recently-emerged viral zoonoses, Nipah virus disease and West Nile virus disease. These exemplify the varied etiology, pathogenesis, zoonotic potential and ecological impact of wildlife EIDs. Strikingly similar underlying factors drive disease emergence in both human and wildlife populations. These are predominantly ecological and almost entirely the product of human environmental change. The implications of wildlife EIDs are twofold: emerging wildlife diseases cause direct and indirect loss of biodiversity and add to the threat of zoonotic disease emergence. Since human environmental changes are largely responsible for their emergence, the threats wildlife EIDs pose to biodiversity and human health represent yet another consequence of anthropogenic influence on ecosystems. We identify key areas where existing expertise in ecology, conservation biology, wildlife biology, veterinary medicine and the impact of environmental change would augment programs to investigate emerging diseases of humans, and we comment on the need for greater medical and microbiological input into the study of wildlife diseases. © 2001 Elsevier Science B.V. All rights reserved.

Review article Anthropogenic environmental change and the emergence of infectious diseases in wildlife

Large DNA viruses defend against hostile assault executed by the host immune system by producing an array of gene products that systematically sabotage key components of the inflammatory response. Poxviruses target many of the primary mediators of innate immunity including interferons, tumor necrosis factors, interleukins, complement, and chemokines. Poxviruses also manipulate a variety of intracellular signal transduction pathways such as the apoptotic response. Many of the poxvirus genes that disrupt these pathways have been hijacked directly from the host immune system, while others have demonstrated no clear resemblance to any known host genes. Nonetheless, the immunological targets and the diversity of strategies used by poxviruses to disrupt these host pathways have provided important insights into diverse aspects of immunology, virology, and inflammation. Furthermore, because of their anti-inflammatory nature, many of these poxvirus proteins hold promise as potential therapeutic agents for acute or chronic inflammatory conditions.

Poxviruses and Immune Evasion

Viral mechanisms of immune evasion

The parapoxvirus orf virus encodes a novel soluble protein inhibitor of ovine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2). The GM-CSF- and IL-2-inhibitory factor (GIF) gene was expressed as an intermediate-late viral gene in orf virus-infected cells. GIF formed homodimers and tetramers in solution, and it bound ovine GM-CSF with a Kd of 369 pM and ovine IL-2 with a Kd of 1.04 nM. GIF did not bind human GM-CSF or IL-2 in spite of the fact that orf virus is a human pathogen. GIF was detected in afferent lymph plasma draining the skin site of orf virus reinfection and was associated with reduced levels of lymph GM-CSF. GIF expression by orf virus indicates that GM-CSF and IL-2 are important in host antiviral immunity.

Orf Virus Encodes a Novel Secreted Protein Inhibitor of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2

The population of red squirrels (Sciurus vulgaris) in the British Isles is in decline and is being supplanted by the grey squirrel (Sciurus carolinensis). It has been suggested that parapoxvirus-associated disease has caused significant mortality in red squirrels and that grey squirrels are the source of the virus. A direct enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of antibody to squirrel parapoxvirus. We tested 140 sera from red squirrels and 223 from grey squirrels from different populations in the UK. A high percentage (61%) of apparently healthy grey squirrels, were found to have been exposed to the parapoxvirus. Only 2.86% (4/140) of red squirrels had antibody and three of these animals had parapoxvirus-associated disease. We postulate that the grey squirrel may act as a reservoir host for the virus.

Grey squirrels have high seroprevalence to a parapoxvirus associated with deaths in red squirrels

During investigations into recent population decreases in Pyrenean chamois (Rupicapra pyrenaica pyrenaica) 21 animals found dead or dying were necropsied. Immunohistochemistry revealed the presence of a pestivirus in organs from two of the 21 chamois. From one of these animals a pestivirus was isolated from the spleen, skin and serum. The virus had better growth in ovine than in bovine cells and was neutralized most effectively by an anti-border disease virus (BDV) reference antiserum. Using panpestivirus and genotype-specific primers selected from 5′-untranslated region (UTR) of the pestivirus genome, BDV RNA was demonstrated by RT-PCR. Comparison of the chamois sequences from 5′-UTR, entire Npro and E2 gene coding regions with those of other pestivirus genotypes revealed that this virus did not fall into any of the pestivirus genotypes identified so far. Results of phylogenetic analysis suggested that the chamois pestivirus was closely related to BDV and it was typed as BDV-4 genotype.

A novel pestivirus associated with deaths in Pyrenean chamois (Rupicapra pyrenaica pyrenaica)

Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD. A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT). The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.

/pdf/one-step-multiplex-real-time-rt-pcr-for-the-detection-of-3rqg7by64c.pdf

One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully characterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus.

/pdf/the-reactivity-of-monoclonal-antibodies-against-orf-virus-lr67sqa8r2.pdf

Janice Gilray

Papers

Orf Virus Encodes a Novel Secreted Protein Inhibitor of Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin-2

Grey squirrels have high seroprevalence to a parapoxvirus associated with deaths in red squirrels

A novel pestivirus associated with deaths in Pyrenean chamois (Rupicapra pyrenaica pyrenaica)

One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein.