Analysis of a Novel Bacteriophage vB_AchrS_AchV4 Highlights the Diversity of Achromobacter Viruses.

Achromobacter spp. are ubiquitous in nature and are increasingly being recognized as emerging nosocomial pathogens. Nevertheless, to date, only 30 complete genome sequences of Achromobacter phages are available in GenBank, and nearly all of those phages were isolated on Achromobacter xylosoxidans. Here, we report the isolation and characterization of bacteriophage vB_AchrS_AchV4. To the best of our knowledge, vB_AchrS_AchV4 is the first virus isolated from Achromobacter spanius. Both vB_AchrS_AchV4 and its host, Achromobacter spanius RL_4, were isolated in Lithuania. VB_AchrS_AchV4 is a siphovirus, since it has an isometric head (64 ± 3.2 nm in diameter) and a non-contractile flexible tail (232 ± 5.4). The genome of vB_AchrS_AchV4 is a linear dsDNA molecule of 59,489 bp with a G+C content of 62.8%. It contains no tRNA genes, yet it includes 82 protein-coding genes, of which 27 have no homologues in phages. Using bioinformatics approaches, 36 vB_AchrS_AchV4 genes were given a putative function. A further four were annotated based on the results of LC-MS/MS. Comparative analyses revealed that vB_AchrS_AchV4 is a singleton siphovirus with no close relatives among known tailed phages. In summary, this work not only describes a novel and unique phage, but also advances our knowledge of genetic diversity and evolution of Achromobacter bacteriophages.

Antigenicity study of the yeast-generated human parvovirus 4 (PARV4) virus-like particles.

Human parvovirus 4 (PARV4) is a novel tetraparvovirus that was isolated from intravenous drug users in 2005. Recombinant PARV4 capsid protein VP2 can form stable virus-like particles (VLPs) in yeast. These VLPs could act as antigen carriers during vaccine development. Therefore, the information about PARV4 VP2 VLP antigenic sites could advance further research in this area. In this work, human parvovirus 4 VLPs obtained from yeast were used to generate monoclonal antibodies (mAbs) in mice. Epitope mapping of the obtained mAbs showed at least three distinct antigenic sites of the VP2 protein. On top of that, molecular cloning was used to replace PARV4 VP2 antigenic sites with heterologous peptides. The chimeric PARV4 VLPs bearing polyhistidine inserts obtained from yeast were observed using electron microscopy while polyhistidine-specific antibodies detected heterologous peptides of the chimeric VP2 proteins.

Complete genome sequence of Buttiauxella phage vB_ButM_GuL6.

We present here the results of the analysis of the complete genome sequence of a lytic bacteriophage, vB_ButM_GuL6, which is the first virus isolated from Buttiauxella. Electron microscopy revealed that vB_ButM_GuL6 belongs to the family Myoviridae, order Caudovirales. The genome of vB_ButM_GuL6 is a linear, circularly permuted 178,039-bp dsDNA molecule with a GC content of 43.4%. It has been predicted to contain 282 protein-coding genes and two tRNA genes, tRNA-Met and tRNA-Gly. Using bioinformatics approaches, 99 (36%) of the vB_ButM_GuL6 genes were assigned a putative function. Genome-wide comparisons and phylogenetic analysis indicated that vB_ButM_GuL6 represents a new species of the subfamily Tevenvirinae and is most closely related to Escherichia virus RB43. These phages, together with Cronobacter phages Miller, CfP1, and IME-CF2, likely form a new genus within the subfamily Tevenvirinae.

Saliva as an alternative specimen for detection of Schmallenberg virus-specific antibodies in bovines.

BACKGROUND: Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. Enzyme-linked immunosorbent assays (ELISA) are commercially available for detection of SBV-specific antibodies in bovine sera and milk. Here we describe the development and evaluation of an indirect ELISA based on a yeast derived recombinant SBV nucleocapsid protein (N) for the detection of SBV-specific antibodies in bovine saliva. Development of a non-invasive test to detect antibodies in individual bovine saliva samples could potentially provide a test suitable for calves and adult cattle. The aim of this study was to investigate the agreement between the levels of antibodies (IgG) measured in milk and sera, and the level of antibodies (IgG and IgA) in saliva, in comparison with the antibody levels detected in sera and milk with commercially available test. RESULTS: Serum, milk and saliva samples from 58 cows were collected from three dairy herds in Lithuania and tested for the presence of SBV-specific antibodies. The presence of IgG antibodies was tested in parallel serum and milk samples, while the presence of IgA and IgG antibodies was tested in saliva samples. The presence of SBV-specific IgG and IgA in saliva was tested using an indirect ELISA based on a yeast-derived recombinant N protein. The presence of SBV-specific IgG in milk and sera was tested in parallel using a commercial recombinant protein based test. The sensitivities of the newly developed tests were as follows: 96 % for the IgG serum assay and 94 % for the IgG milk assay and 85 % and 98 % for IgG and IgA in saliva tests, when compared with data generated by a commercial IgG assay. CONCLUSIONS: Data from testing the saliva IgG and IgA and also the milk and serum IgG with indirect SBV-specific ELISAs showed close agreement with the commercial serum and milk IgG assay data. The level of IgG in saliva was notably lower in comparison to IgA. The newly developed method exhibits the potential to serve as an easily transferable tool for epidemiological studies.

Generation of recombinant Schmallenberg virus nucleocapsid protein in yeast and development of virus-specific monoclonal antibodies.

Schmallenberg virus (SBV), discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N) protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs). Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.