Regulation of Mannitol Metabolism in Enterococcus faecalis and Association with parEF0409 Toxin-Antitoxin Locus Function.

The parEF0409 type I toxin-antitoxin locus is situated between genes for two paralogous mannitol family phosphoenolpyruvate phosphotransferase systems (PTSs). In order to address the possibility that parEF0409 function was associated with sugar metabolism, genetic and phenotypic analyses were performed on the flanking genes. It was found that the genes were transcribed as two operons: the downstream operon essential for mannitol transport and metabolism and the upstream operon performing a regulatory function. In addition to genes for the PTS components, the upstream operon harbors a gene similar to mtlR, the key regulator of mannitol metabolism in other Gram-positive bacteria. We confirmed that this gene is essential for the regulation of the downstream operon and identified putative phosphorylation sites required for carbon catabolite repression and mannitol-specific regulation. Genomic comparisons revealed that this dual-operon organization of mannitol utilization genes is uncommon in enterococci and that the association with a toxin-antitoxin system is unique to Enterococcus faecalis. Finally, we consider possible links between parEF0409 function and mannitol utilization. IMPORTANCE Enterococcus faecalis is both a common member of the human gut microbiota and an opportunistic pathogen. Its evolutionary success is partially due to its metabolic flexibility, in particular its ability to import and metabolize a wide variety of sugars. While a large number of phosphoenolpyruvate phosphotransferase sugar transport systems have been identified in the E. faecalis genome bioinformatically, the specificity and regulation of most of these systems remain undetermined. Here, we characterize a complex system of two operons flanking a type I toxin-antitoxin system required for the transport and metabolism of the common dietary sugar mannitol. We also determine the phylogenetic distribution of mannitol utilization genes in the enterococcal genus and discuss the significance of the association with toxin-antitoxin systems.

Genetic diversity and prevalence of Atypical Porcine Pestivirus in the Midwest of US swine herds during 2016-2018.

Atypical porcine pestivirus (APPV), a highly divergent pestivirus, has a wide geographical distribution around the world. APPV is known to cause type A-II congenital tremors in newborn piglets. The main objective of this study is to access APPV prevalence in the US swine herds utilizing a newly developed quantitative real-time RT-PCR assay. Retrospective analysis of 1,785 samples revealed a 19.0% prevalence in Midwest swine herds over a period of three years (2016-2018). Among all clinical and field samples that were APPV positive, 82 samples (24.19%) were also positive for one or more swine viral pathogens. Two APPV US strains identified in this study demonstrated significant sequence diversity (~12% in full genome) compared to the first reported APPV strain from the United States in 2014. Of the two strains identified in this study, USA/023005/2016 is closer to two strains identified in Germany, and USA/047310/2017 shares more similarities with two US strains including Minnesota-1 and ISDVDL2014016573. Partial NS5B sequences (9127-9836 nt of the polyprotein gene) obtained from 54 APPV-positive samples revealed considerable sequence diversity, ranging from 85.8% to 100% nucleotide identity, within the US strains in samples from different geographic regions. Analysis of all US samples indicates high prevalence of APPV in Minnesota (37.35%), followed by Illinois (32.86%), Iowa (30.60%) and Kansas (21.89%). APPV was detected in 15.48% of samples assayed from 2017, slightly higher than that in 2016 (13.08%), but much lower than 2018 (28.77%). Among the various sample types tested, oral fluid samples had the highest prevalence and lowest average Ct value suggesting their suitability as a reliable diagnostic specimen for APPV detection. Overall, sequence variation among APPV strains and prevalence of the pathogen within the United States provides a basis for understanding the genetic diversity and molecular epidemiology of APPV in the US swine herds.

Detection and Whole-Genome Characterization of a G8P[1] Group A Rotavirus Strain from Deer.

Rotavirus A strain 14-02218-2, with genome constellation G8P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3, was isolated from newborn fawns. The 14-02218-2 rotavirus strain is related to bovine and bovine-like rotavirus strains. To our knowledge, this is the first report on whole-genome-based characterization of a deer rotavirus G8P[1] strain.

An inactivated vaccine made from a U.S. field isolate of porcine epidemic disease virus is immunogenic in pigs as demonstrated by a dose-titration.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), a highly pathogenic and transmissible virus in swine, was first detected in the U.S. in May, 2013, and has caused tremendous losses to the swine industry. Due to the difficulty in isolating and growing this virus in cell culture, few vaccine studies using cell culture propagated PEDV have been performed on U.S. strains in pigs. Therefore, the objective of this study was to evaluate the humoral immune response to the selected inactivated PEDV vaccine candidate in a dose-titration manner. RESULTS: PEDV was isolated from a pig with diarrhea and complete genome sequencing found >99% nucleotide identity to other U.S. PEDV. Inactivated adjuvanted monovalent vaccines were administered intramuscularly to five week old pigs in a dose titration experimental design, ranging from 6.0-8.0 log10 tissue culture infective dose (TCID50/mL), to evaluate immunogenicity using a fluorescent foci neutralization assay (FFN), fluorescent microsphere immunoassay (FMIA), and enzyme-linked immunosorbent assay (ELISA) on sera. Pigs vaccinated with 8.0 log10 TCID50/mL inactivated virus showed significantly higher FFN titers as well as FMIA and ELISA values than 6.0 log10 TCID50/mL vaccinates and the negative controls. CONCLUSIONS: These results demonstrate the immunogenicity of a PEDV inactivated viral vaccine with a U.S. strain via dose-titration. A future vaccination-challenge study would illustrate the efficacy of an inactivated vaccine and help evaluate protective FFN titers and ELISA and FMIA responses.

Genome sequence of torovirus identified from a pig with porcine epidemic diarrhea virus from the United States.

Porcine torovirus (PToV) strain PToV-NPL/2013 was identified from a pig that tested positive for porcine epidemic diarrhea virus (PEDV). The spike protein-encoding gene from PToV-NPL/2013 had 92% identity with PToV-SH1, suggesting that PToV circulating in the United States is slightly different from the isolates circulating in China. To our knowledge, this is the first report of PToV in the United States.

Inactivation of the Rgg2 transcriptional regulator ablates the virulence of Streptococcus pyogenes.

Streptococcus pyogenes adapts to different niches encountered in the human host via the activity of numerous regulatory proteins including the Rgg family of transcriptional regulators. The S. pyogenes chromosome encodes four Rgg paralogues designated Rgg1 (RopB), Rgg2 (MutR), Rgg3, and Rgg4 (ComR). In order to understand the role of the Rgg2 protein in the regulation of metabolic and virulence-associated properties of S. pyogenes, the rgg2 gene was inactivated in the M1 serotype strain SF370. Inactivation of rgg2 increased the growth yield of S. pyogenes in THY broth, increased biofilm formation, and increased production of SIC, which is an important virulence factor that inhibits complement mediated lysis. To identify Rgg2-regulated genes, the transcriptomes of SF370 and the rgg2 mutant strains were compared in the middle-exponential and post-exponential phases of growth. Rgg2 was found to control the expression of dozens of genes primarily in the exponential phase of growth, including genes associated with virulence (sse, scpA, slo, nga, mf-3), DNA transformation, and nucleotide metabolism. Inactivation of rgg2 decreased the ability of S. pyogenes to adhere to epithelial cells. In addition, the mutant strain was more sensitive to killing when incubated with human blood and avirulent in a murine bacteremia model. Finally, inoculation of mice with the avirulent rgg2 mutant of S. pyogenes SF370 conferred complete protection to mice subsequently challenged with the wild-type strain. Restoration of an intact rgg2 gene in mutant strain restored the wild-type phenotypes. Overall, the results demonstrate that Rgg2 is an important regulatory protein in S. pyogenes involved in controlling genes associated with both metabolism and virulence.

First identification and characterization of porcine enterovirus G in the United States.

Porcine enterovirus G (EV-G) is a member of the family Picornavirdae, genus Enterovirus. To date, eleven EV-G types (EV-G1 through EV-G11) have been identified in pigs from Asia and Europe however they have never been reported in North America. In this study, we isolated and characterized the complete genome of NP/2013/USA, an EV-G from a porcine diarrhea sample from the United States. The complete genome consists of 7,390 nucleotides excluding the 3' poly(A) tail, and has an open reading frame that encodes a 2,169 amino acid polyprotein. NP/2013/USA was most similar at the nucleotide (84%) and amino acid (95%) level to the HM131607, an EV-G1 type isolated from China in 2012.

Characterization of epizootic hemorrhagic disease virus from a bovine with clinical disease with high nucleotide sequence identity to white-tailed deer isolates.

Epizootic hemorrhagic disease virus (EHDV) was isolated from a pregnant cow in Indiana, USA, exhibiting excessive salivation, pyrexia and abortion. VP2, VP5, and VP7 sequences of the isolated bovine EHDV showed 97.7, 97.4, and 97.9 % identity to a serotype 2 reference virus. Bovine EHDV was closely related (>99.9 %) to white tailed deer (WTD) EHDV collected from Iowa in 2013 and showed less than 2.1 % divergence from EHDV collected from WTD across the USA in 2013. The high degree of sequence identity between bovine and WTD EHDV isolates demonstrates that similar viruses concurrently circulate in both species and suggests possible further incursions into bovines.

Genome Sequence of the Novel Reassortant Mammalian Orthoreovirus Strain MRV00304/13, Isolated from a Calf with Diarrhea from the United States.

Mammalian orthoreovirus (MRV) strain MRV00304/13 was isolated from diarrheic calves. The serotype-specific antigen σ1 was found to be 95% identical to that of bovine MRV1. All predicted viral proteins had >92% identity to those of MRV except µ2 and σ1s (80 and 72% identities, respectively), suggesting that MRV00304/13 is a novel reassortant MRV1.

Whole genome analysis of epizootic hemorrhagic disease virus identified limited genome constellations and preferential reassortment.

Epizootic hemorrhagic disease virus (EHDV) is a Culicoides transmitted orbivirus that causes haemorrhagic disease in wild and domestic ruminants. A collection of 44 EHDV isolated from 2008 to 2012 was fully sequenced and analysed phylogenetically. Serotype 2 viruses were the dominant serotype all years except 2012 when serotype 6 viruses represented 63 % of the isolates. High genetic similarity (>94 % identity) between serotype 1 and 2 virus VP1, VP3, VP4, VP6, NS1, NS2 and NS3 segments prevented identification of reassortment events for these segments. Additionally, there was little genetic diversity (>96 % identity) within serotypes for VP2, VP5 and VP7. Preferential reassortment within the homologous serotype was observed for VP2, VP5 and VP7 segments for type 1 and type 2 viruses. In contrast, type 6 viruses were all reassortants containing VP2 and VP5 derived from an exotic type 6 with the remaining segments most similar to type 2 viruses. These results suggest that reassortment between type 1 and type 2 viruses requires conservation of the VP2, VP5 and VP7 segment constellation while type 6 viruses only require VP2 and VP5 and are restricted to type 2-lineage VP7. As type 6 VP2 and VP5 segments were exclusively identified in viruses with type 2-derived VP7, these results suggest functional complementation between type 2 and type 6 VP7 proteins.

Mobuck virus genome sequence and phylogenetic analysis: identification of a novel Orbivirus isolated from a white-tailed deer in Missouri, USA.

The genus Orbivirus includes a diverse group of segmented dsRNA viruses that are transmitted via arthropods, have a global distribution and affect a wide range of hosts. A novel orbivirus was co-isolated with epizootic haemorrhagic disease virus (EHDV) from a white-tailed deer (Odocoileus virginianus) exhibiting clinical signs characteristic of EHDV. Using antiserum generated against EHDV, a pure isolate of the novel non-cytopathic orbivirus was obtained in Aedes albopictus cell culture. Genomic sequencing and phylogenetic analysis of predicted ORFs showed that eight of the ten ORFs were most homologous to Peruvian horse sickness virus (PHSV), with amino acid identities of 44.3-73.7 %. The remaining two ORFs, VP3 and VP5, were most similar to Middle Point orbivirus (35.9 %) and Yunnan orbivirus (59.8 %), respectively. Taxonomic classification of orbiviruses is largely based on homology of the major subcore structural protein VP2(T2), encoded by segment 2 for mobuck virus. With only 69.1 % amino acid identity to PHSV, we propose mobuck virus as the prototype of a new species of Orbivirus.

Transcriptional regulation of a bacteriophage encoded extracellular DNase (Spd-3) by Rgg in Streptococcus pyogenes.

The Streptococcus pyogenes transcriptional regulator Rgg controls the expression of virulence-associated genes encoded both within the core genome and within horizontally transmissible DNA such as temperate bacteriophage. Previously, we showed that Rgg binds to the non-coding DNA upstream of the bacteriophage gene encoding an extracellular DNase Spd-3. In the current study, we further characterized Rgg-mediated regulation of spd-3 expression. Two spd-3 transcripts were identified by northern blotting. The 5' ends were 27 and 594 nucleotides upstream of the start codon as determined with primer extension analysis and 5' RACE (rapid amplification of c-DNA ends), respectively. Results obtained with gel shift assays showed that purified Rgg bound specifically to non-coding DNA containing the promoters of both transcripts. Transcriptional fusion analyses confirmed the presence of Rgg-repressible promoters within these DNA regions. In addition, repression was associated with direct DNA binding by Rgg as determined with chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (qPCR). The results show that the chromosomally encoded transcriptional regulator, Rgg, directly represses both bacteriophage promoters controlling the expression of Spd-3. The results provide new information regarding the regulation of prophage encoded virulence factors of S. pyogenes and highlight the complex evolutionary history of S. pyogenes and temperate bacteriophage.

Growth phase-dependent modulation of Rgg binding specificity in Streptococcus pyogenes.

Streptococcus pyogenes Rgg is a transcriptional regulator that interacts with the cofactor LacD.1 to control growth phase-dependent expression of genes, including speB, which encodes a secreted cysteine protease. LacD.1 is thought to interact with Rgg when glycolytic intermediates are abundant in a manner that prevents Rgg-mediated activation of speB expression via binding to the promoter region. When the intermediates diminish, LacD.1 dissociates from Rgg and binds to the speB promoter to activate expression. The purpose of this study was to determine if Rgg bound to chromatin during the exponential phase of growth and, if so, to identify the binding sites. Rgg bound to 62 chromosomal sites, as determined by chromatin immunoprecipitation coupled with DNA microarrays. Thirty-eight were within noncoding DNA, including sites upstream of the genes encoding the M protein (M49), serum opacity factor (SOF), fibronectin-binding protein (SfbX49), and a prophage-encoded superantigen, SpeH. Each of these sites contained a promoter that was regulated by Rgg, as determined with transcriptional fusion assays. Purified Rgg also bound to the promoter regions of emm49, sof, and sfbX49 in vitro. Results obtained with a lacD.1 mutant showed that both LacD.1 and Rgg were necessary for the repression of emm49, sof, sfbX49, and speH expression. Overall, the results indicated that the DNA binding specificity of Rgg is responsive to environmental changes in a LacD.1-dependent manner and that Rgg and LacD.1 directly control virulence gene expression in the exponential phase of growth.

Identification of Rgg binding sites in the Streptococcus pyogenes chromosome.

Streptococcus pyogenes Rgg is a regulatory protein that controls the transcription of 588 genes in strain NZ131 during the post-exponential phase of growth, including the virulence-associated genes encoding the extracellular SpeB protease, pullulanase A (PulA), and two extracellular nucleases (SdaB and Spd-3). Rgg binds to DNA proximally to the speB promoter (PspeB) to activate transcription; however, it is not known if Rgg binds to the promoters of other genes to influence expression, or if the perturbation of other global regulons accounts for the genome-wide changes in expression associated with the mutant. To address this issue, chromatin immunoprecipitation followed by DNA microarray analysis (ChIP-chip) was used to identify the DNA binding sites of Rgg. Rgg bound to 65 sites in the chromosome. Thirty-five were within noncoding DNA, and 43% of these were adjacent to genes previously identified as regulated by Rgg. Electrophoretic mobility shift assays were used to assess the binding of Rgg to a subset of sites bound in vivo, including the noncoding DNA upstream of speB, the genes encoding PulA, Spd-3, and a transcriptional regulator (SPY49_1113), and prophage-associated genes encoding a putative integrase (SPY49_0746) and a surface antigen (SPY49_0396). Rgg bound to all target DNAs in vitro, consistent with the in vivo results. Finally, analyses with a transcriptional reporter system showed that the DNA bound by Rgg contained an active promoter that was regulated by Rgg. Overall, the results indicate that Rgg binds specifically to multiple sites in the chromosome, including prophage DNA, to influence gene expression.

A naturally occurring Rgg variant in serotype M3 Streptococcus pyogenes does not activate speB expression due to altered specificity of DNA binding.

The transcriptional regulator Rgg of Streptococcus pyogenes is essential for expression of the secreted cysteine protease SpeB. Although all isolates of S. pyogenes possess the speB gene, not all of them produce the protein in vitro. In a murine model of infection, the absence of SpeB production is associated with invasive disease. We speculated that naturally occurring mutations in rgg, which would also abrogate SpeB production, may be present in invasive isolates of S. pyogenes. Examination of the inferred Rgg sequences available in public databases revealed that the rgg gene in strain MGAS315 (a serotype M3 strain associated with invasive disease) encodes a proline at amino acid position 103 (Rgg(103P)); in contrast, all other strains encode a serine at this position (Rgg(103S)). A caseinolytic assay and Western blotting indicated that strain MGAS315 does not produce SpeB in vitro. Gene-swapping experiments showed that the rgg gene of MGAS315 is solely responsible for the lack of SpeB expression. In contrast to Rgg(103S), Rgg(103P) does not bind to the speB promoter in gel shift assays, which correlates with a lack of speB expression. Despite its inability to activate speB expression, Rgg(103P) retains the ability to bind to DNA upstream of norA and to influence its expression. Overall, this study illustrates how variation at the rgg locus may contribute to the phenotypic diversity of S. pyogenes.