Complete genome sequence of bacteriophage MrAaronian isolated from an Arthrobacter globiformis culture.

Arthrobacteriophage MrAaronian contains a 54,509 bp DNA genome with 87 predicted protein-coding genes. MrAaronian has siphovirus morphology and was collected from a flowerbed soil sample in Poughkeepsie, NY, and isolated on an Arthrobacter globiformis B-2979 culture. MrAaronian has > 99% nucleotide identity with cluster AW arthrobacteriophages Michelle, Stayer, Sloopyjoe, and StarLord.

Molecular Surveillance for Lymphoproliferative Disease Virus and Reticuloendotheliosis Virus in Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) in Texas, USA.

Reticuloendotheliosis virus (REV) and lymphoproliferative disease virus (LPDV) are avian retroviruses that can cause neoplastic disease and present with similar pathologies. Lymphoproliferative disease virus has been reported in the Eastern US and states bordering Texas, USA, but has not been previously detected within the state. In a prior study, we detected REV in native Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) and an Eastern Wild Turkey (Meleagris gallopavo silvestris) originating from West Virginia. Given LPDV detection in states bordering Texas and our finding of an REV-positive Eastern Wild Turkey imported from a LPDV endemic region, we sought to determine LPDV prevalence in Texas and continue surveillance for REV. During 2018-20, dried blood spots from 373 individual Rio Grande Wild Turkeys from 20 different counties were tested for the presence of proviral REV or LPDV DNA. In affected counties, approximately 4% of individuals were infected with REV (7/197) or LPDV (10/273) and one bird was coinfected with both viruses. Phylogenetic analysis indicated a close relationship of the LPDV isolates to variants from other Southern and Central states. This study provides molecular evidence of LPDV in Texas, and continued surveillance is necessary to determine the potential effects of the virus on reproductive success, coinfections, and overall health of Wild Turkey populations.

Complete Genome Sequence of Bacteriophage Loca, Isolated on a Microbacterium foliorum Culture.

Microbacteriophage Loca was extracted from a shopping cart handle swab sample in Stephenville, TX, and isolated on a Microbacterium foliorum NRRL-24224 culture. The 17,475-bp double-stranded DNA genome contains 25 predicted protein-coding genes and has >96% nucleotide identity to bacteriophages Quaker and Livingwater.

Genome Sequence of Fowlpox Virus-Integrated Reticuloendotheliosis Virus from a Rio Grande Wild Turkey (Meleagris gallopavo intermedia).

We report the genome sequence of a nearly intact reticuloendotheliosis virus (REV) insertion within a field strain of fowlpox virus from a Rio Grande wild turkey in Gillespie County, TX. The proviral REV genome comprises 7,943 bp and contains partial long terminal repeats.

Near-Complete Proviral Genome Sequence of Reticuloendotheliosis Virus Isolated from an Attwater's Prairie Chicken (Tympanuchus cupido attwateri).

We report the near-complete proviral genome sequence of a reticuloendotheliosis virus isolated and propagated from an endangered Attwater's prairie chicken (Tympanuchus cupido attwateri) during a 2016-2017 outbreak at a captive breeding facility.

Complete Genome Sequence of Bacteriophage Fizzles, Isolated from Microbacterium foliorum.

Microbacteriophage Fizzles has a 62,078-bp linear double-stranded DNA genome sequence, predicted to contain 104 protein-coding genes. Fizzles is a Siphoviridae actinobacteriophage isolated from an ant hill soil sample collected in Stephenville, TX. Microbacteriophage Fizzles has >83.6% nucleotide identity with microbacteriophages Squash and Nike.

Instructional Models for Course-Based Research Experience (CRE) Teaching.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Complete Genome Sequence of Bacteriophage IndyLu, Isolated from a Microbacterium foliorum Culture.

Microbacteriophage IndyLu was isolated from Microbacterium foliorum NRRL B-24224. The 41,958-bp double-stranded DNA genome has 71 predicted protein coding genes and 1 tRNA. The lytic actinobacteriophage was extracted from soil samples collected in Stephenville, TX, and is related to cluster EB bacteriophages Didgeridoo and Lahqtemish.

Complete Genome Sequences of Mycobacterium Phages Tripl3t and Zeuska.

Tripl3t and Zeuska are siphoviral bacteriophages that were isolated from Mycobacterium smegmatis mc2 155 and contain double-stranded DNA genomes 53,565 bp and 53,598 bp in length, respectively. Tripl3t and Zeuska were annotated by students at Bluff Dale High School (Bluff Dale, TX) and Tolar High School (Tolar, TX) in community engagement with Tarleton State University.

Complete Genome Sequence of Mycobacteriophage Joy99.

Joy99 is a siphoviral mycobacteriophage with a 59,837-base pair double-stranded DNA genome and is predicted to contain 97 protein-coding genes and a single tRNA gene. Joy99 was isolated in Saint Louis, MO, and annotated by students at Bluff Dale High School in community engagement with Tarleton State University.

Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild Turkeys, and Chickens in Brazil.

Reticuloendotheliosis viruses (REVs) are known to cause immunosuppressive and oncogenic disease that affects numerous avian species. Reticuloendotheliosis viruses are present worldwide and recently have been reported in South America with cases of infected commercial flocks in Argentina. We surveyed for the presence of REV in birds from a state in the northern region of Brazil using real-time PCR. We report here the presence of REV in Brazil, detected in Muscovy Ducks (Cairina moschata), Wild Turkeys (Meleagris gallopavo), and chickens (Gallus gallus) at a relatively high prevalence (16.8%). Phylogenetic analysis indicated a close relationship of these strains to variants in the US. This study provides evidence of REV in the Amazon biome and provides a baseline for future surveillance of the virus in the region and throughout Brazil.

Complete Genome Sequence of Bacteriophage Finny, Isolated from a Microbacterium foliorum Culture.

Actinobacteriophage Finny contains a circularly permuted 40,313-bp double-stranded DNA genome with 63 predicted protein-coding genes. Finny was directly isolated from a soil sample collected in New Braunfels, Texas, that was incubated with Microbacterium foliorum SEA B-24224. Finny is closely related to bacteriophages MCubed, Andromedas, ColaCorta, Eleri, and Sansa.

Complete Genome Sequence of Cluster O Mycobacterium smegmatis Bacteriophage Ryadel.

Mycobacteriophage Ryadel is a newly isolated cluster O Siphoviridae bacteriophage, characterized by an unusual prolate capsid, containing a 72,658-base-pair double-stranded DNA genome with 132 predicted protein-coding genes. Conserved among cluster O bacteriophages, the Ryadel genome contains 31 copies of a unique 17-bp sequence with dyad symmetry.

Survey of Reticuloendotheliosis Virus in Wild Turkeys (Meleagris gallopavo) in Texas, USA.

Reticuloendotheliosis virus (REV) is an immunosuppressive and sometimes oncogenic avian retrovirus that establishes lifelong infection in a wide range of avian species. REV-infected wild birds roaming near at-risk captive flocks, such as is the case for the highly endangered Attwater's Prairie Chicken (APC; Tympanuchus cupido attwateri), could act as a reservoir for viral transmission. In wild birds, prevalence rates of REV are low and appearance of associated disease is uncommon. During 2016-17, nearly half of all captive adult APC mortality at Fossil Rim Wildlife Center captive breeding facility in Glen Rose, Texas, US was attributed to REV infection. The unusually high REV prevalence rate prompted us to survey for this virus in wild galliforms throughout the region. From 2016-17, 393 blood samples collected from two subspecies of Wild Turkeys (Meleagris gallopavo) were tested for REV proviral DNA through amplification of the viral 3' long terminal repeat and segments of the viral pol gene. In REV-affected counties, 5% (5/98) of native Rio Grande Wild Turkeys (Meleagris gallopavo intermedia) were identified as REV-positive. In addition, we detected REV in one of 62 Eastern Wild Turkeys (Meleagris gallopavo silvestris) that had been imported during conservation efforts. To better determine protective measures, continued surveillance, including collection and genetic analysis of REV-infected samples, is necessary to identify sources of REV outbreaks in captive APC flocks.

Complete Genome Sequence of Cluster A1 Mycobacterium smegmatis Bacteriophage Arlo.

Mycobacteriophage Arlo is a newly isolated Siphoviridae bacteriophage isolated from soil samples collected in Bluff Dale, Texas. Mycobacteriophage Arlo has a 52,960 base-pair double-stranded DNA genome that is predicted to contain 96 protein-coding genes. Mycobacteriophage Arlo is related to mycobacteriophage DD5 and other cluster A1 bacteriophages.

Discovery and characterization of auxiliary proteins encoded by type 3 simian T-cell lymphotropic viruses.

UNLABELLED: Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE: Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Co-dependence of HTLV-1 p12 and p8 functions in virus persistence.

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8.

Human T-cell leukemia/lymphoma virus type 1 p30, but not p12/p8, counteracts toll-like receptor 3 (TLR3) and TLR4 signaling in human monocytes and dendritic cells.

The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p30 protein, essential for virus infectivity in vivo, is required for efficient infection of human dendritic cells (DCs) but not B and T cells in vitro. We used a human monocytic cell line, THP-1, and dendritic cells to study the mechanism of p30 and p12/p8 requirements in these cell types. p30 inhibited the expression of interferon (IFN)-responsive genes (ISG) following stimulation by lipopolysaccharide (LPS) of Toll-like receptor 4 (TLR4) and by poly(I·C) of TLR3 but not of TLR7/8 with imiquimod. Results with THP-1 mirrored those for ex vivo human primary monocytes and monocyte-derived dendritic cells (Mo-mDC). The effect of p30 on TLR signaling was also demonstrated by ablating its expression within a molecular clone of HTLV-1. HTLV-1 infection of monocytes inhibited TLR3- and TLR4-induced ISG expression by 50 to 90% depending on the genes, whereas the isogenic clone p30 knockout virus was less effective at inhibiting TLR3 and TRL4 signaling and displayed lower infectivity. Viral expression and inhibition of ISG transcription was, however, rescued by restoration of p30 expression. A chromatin immunoprecipitation assay demonstrated that p30 inhibits initiation and elongation of PU.1-dependent transcription of IFN-α1, IFN-ß, and TLR4 genes upon TLR stimulation. In contrast, experiments conducted with p12/p8 did not demonstrate an effect on ISG expression. These results provide a mechanistic explanation of the requirement of p30 for HTLV-1 infectivity in vivo, suggest that dampening interferon responses in monocytes and DCs is specific for p30, and represent an essential early step for permissive HTLV-1 infection and persistence.

Palmitoylation and p8-mediated human T-cell leukemia virus type 1 transmission.

The orf-I gene of human T-cell leukemia type 1 (HTLV-1) encodes p8 and p12 and has a conserved cysteine at position 39. p8 and p12 form disulfide-linked dimers, and only the monomeric forms of p8 and p12 are palmitoylated. Mutation of cysteine 39 to alanine (C39A) abrogated dimerization and palmitoylation of both proteins. However, the ability of p8 to localize to the cell surface and to increase cell adhesion and viral transmission was not affected by the C39A mutation.

A public HTLV-1 molecular epidemiology database for sequence management and data mining.

BACKGROUND: It is estimated that 15 to 20 million people are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). At present, there are more than 2,000 unique HTLV-1 isolate sequences published. A central database to aggregate sequence information from a range of epidemiological aspects including HTLV-1 infections, pathogenesis, origins, and evolutionary dynamics would be useful to scientists and physicians worldwide. Described here, we have developed a database that collects and annotates sequence data and can be accessed through a user-friendly search interface. The HTLV-1 Molecular Epidemiology Database website is available at http://htlv1db.bahia.fiocruz.br/. METHODOLOGY/PRINCIPAL FINDINGS: All data was obtained from publications available at GenBank or through contact with the authors. The database was developed using Apache Webserver 2.1.6 and SGBD MySQL. The webpage interfaces were developed in HTML and sever-side scripting written in PHP. The HTLV-1 Molecular Epidemiology Database is hosted on the Gonçalo Moniz/FIOCRUZ Research Center server. There are currently 2,457 registered sequences with 2,024 (82.37%) of those sequences representing unique isolates. Of these sequences, 803 (39.67%) contain information about clinical status (TSP/HAM, 17.19%; ATL, 7.41%; asymptomatic, 12.89%; other diseases, 2.17%; and no information, 60.32%). Further, 7.26% of sequences contain information on patient gender while 5.23% of sequences provide the age of the patient. CONCLUSIONS/SIGNIFICANCE: The HTLV-1 Molecular Epidemiology Database retrieves and stores annotated HTLV-1 proviral sequences from clinical, epidemiological, and geographical studies. The collected sequences and related information are now accessible on a publically available and user-friendly website. This open-access database will support clinical research and vaccine development related to viral genotype.