Studying the transmission of simian retroviruses to humans can help define the importance of these infections to public health. We identified a substantial prevalence (4/231, 1.8%) of infection with simian foamy viruses (SFV) among humans occupationally exposed to nonhuman primates. Evidence of SFV infection included seropositivity, proviral DNA detection and isolation of foamy virus. The infecting SFV originated from an African green monkey (one person) and baboons (three people). These infections have not as yet resulted in either disease or sexual transmission, and may represent benign endpoint infections.

The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing ( IMPORTANCEThe Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms, de novo assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results.

Detection of distantly related viruses by high-throughput sequencing (HTS) is bioinformatically challenging because of the lack of a public database containing all viral sequences, without abundant nonviral sequences, which can extend runtime and obscure viral hits. Our reference viral database (RVDB) includes all viral, virus-related, and virus-like nucleotide sequences (excluding bacterial viruses), regardless of length, and with overall reduced cellular sequences. Semantic selection criteria (SEM-I) were used to select viral sequences from GenBank, resulting in a first-generation viral database (VDB). This database was manually and computationally reviewed, resulting in refined, semantic selection criteria (SEM-R), which were applied to a new download of updated GenBank sequences to create a second-generation VDB. Viral entries in the latter were clustered at 98% by CD-HIT-EST to reduce redundancy while retaining high viral sequence diversity. The viral identity of the clustered representative sequences (creps) was confirmed by BLAST searches in NCBI databases and HMMER searches in PFAM and DFAM databases. The resulting RVDB contained a broad representation of viral families, sequence diversity, and a reduced cellular content; it includes full-length and partial sequences and endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Testing of RVDBv10.2, with an in-house HTS transcriptomic data set indicated a significantly faster run for virus detection than interrogating the entirety of the NCBI nonredundant nucleotide database, which contains all viral sequences but also nonviral sequences. RVDB is publically available for facilitating HTS analysis, particularly for novel virus detection. It is meant to be updated on a regular basis to include new viral sequences added to GenBank. To facilitate bioinformatics analysis of high-throughput sequencing (HTS) data for the detection of both known and novel viruses, we have developed a new reference viral database (RVDB) that provides a broad representation of different virus species from eukaryotes by including all viral, virus-like, and virus-related sequences (excluding bacteriophages), regardless of their size. In particular, RVDB contains endogenous nonretroviral elements, endogenous retroviruses, and retrotransposons. Sequences were clustered to reduce redundancy while retaining high viral sequence diversity. A particularly useful feature of RVDB is the reduction of cellular sequences, which can enhance the run efficiency of large transcriptomic and genomic data analysis and increase the specificity of virus detection.

Using nonstringent annealing conditions and a 2.75-kilobase segment of cloned African green monkey DNA that specifically hybridizes to the proviruses of AKR'ecotropic murine leukemia virus (MuLV) and baboon endogenous virus (BaEV) as a probe, we detected related sequences in three different preparations of human brain DNA fragments. The blot-hybridization pattern obtained with cleaved human DNA was similar to that previously reported for the interaction of MuLV cDNA and cleaved mouse DNA and suggested the presence ofnumerous copies of retrovirus-related sequences in the human genome. The labeled 2.75-kilobase fragment derived from cloned monkey DNA was used to screen a human DNA library in Charon 4A. One clone obtained hybridized to three contiguous MuLV-and BaEV-reactive fragments of the cloned monkey DNA and to multiple fragments of human DNA including a prominent 1.0-kilobase EcoRI fragment also present in the clone.In the mouse, certain endogenous type C proviruses have been shown to be vertically transmitted (1-3), have been mapped to specific chromosomal loci (4-6), and have the potential ofbeing expressed as infectious ecotropic and xenotropic (7) murine leukemia viruses (MuLV). Comparison ofresults from nucleic acid hybridization experiments, which indicate the existence of numerous copies of endogenous mouse proviruses (8-10), with those derived from genetic andvirus isolation studies, which point to a few defined inducible loci in the genomes of certain inbred mouse strains (4-6, 11), strongly suggests that the majority of the MuLV-reactive sequences in mouse DNA consist ofincomplete viral DNA segments whose function is unknown.Type C endogenous retroviruses have also been recovered from several different Old World monkeys including the baboon (12), stump-tail macaque (13), rhesus (14), and colobus (15 We recently. reported the molecular cloning of a 17-kilobase (kb) segment from African green monkey (AGM) DNA which had nearly 5 kb of homology with AKR ecotropic MuLV DNA (21). In the present manuscript we show that the internal organization of the endogenous type C AGM and baboon provi:-ruses, including 8 of 10 restriction enzyme cleavage sites, hasbeen highly conserved. Furthermore, by using a radiolabeled. 2.75-kb BamHI fragment ofthe cloned ACM-DNA, which specifically hybridizes to a similarly sized BamHI fragment (22) of the BaEV provirus, hybridization to several bands in three different restricted preparations of human DNA was observed. Based on our ability to detect type C virus-related sequences in human DNA, we cloned an 11-kb segment from human DNA which hybridizes to AGM and mouse proviral DNAs.MATERIALS AND METHODS Preparation and Cleavage ofCellular DNA. High molecular weight AGM and rhesus monkey liver DNAs were purified from fresh tissue as described (23). Baboon cellular-DNA was prepared from a cell line (CP 21) established from primary skin fibroblasts. DNA was isolated from three human brain specimens as outlined (24). Cellular DNAs were digested with restriction enzymes, ele...

We have determined the nucleotide sequence in the U3-R regions of the long terminal repeat (LTR) associated with NFS-Th-I xenotropic murine leukemia virus (MuLV) DNA The RNA genome of type C retroviruses contains terminally redundant sequences (R) that are located adjacent to segments unique to the 5' (U5) and 3' (U3) termini (1). After infection, the viral RNA is reverse transcribed into double-stranded DNA, which subsequently becomes integrated into cellular chromosomal DNA (2, 3). Both the unintegrated and integrated copies of proviral DNA contain terminally duplicated sequences derived from each end of the viral genome forming a structure (U3-R-U5) referred to as the long terminal repeat (LTR). LTRs contain important regulatory signals for the promotion, initiation, and processing of viral mRNAs and may play a role in the integration of proviral DNA into the host chromosome (3). Furthermore, nucleotide sequence analyses show that LTRs possess structural features characteristic of transposable elements found in bacteria, yeast, and Drosophila (4)(5)(6)(7)(8)(9)(10)(11).In several vertebrate species, DNA copies of type C retroviruses have been identified as genetically stable integral components of chromosomal DNA (12). Such endogenous proviruses have been shown to be vertically transmitted, can be mapped to specific chromosomal loci, and, in some cases, may be expressed as infectious retroviruses (12). We Because we had previously shown that the LTRs associated with endogenous MuLV proviruses could be distinguished from the LTR segments associated with known infectious viruses (13) and since the numerous copies of these elements present in mammalian chromosomal DNA could potentially regulate the expression of adjacent cellular genes, we determined the nucleotide sequences in the U3 R regions of five endogenous LTRs. The results of these sequence analyses and comparisons with LTRs associated with infectious xenotropic, ecotropic, and MCF proviruses are presented.MATERIALS AND METHODS Recombinant DNA Clones. The Pst I/Sma I segment derived from the U3-R region of the LTRs associated with the endogenous BALB/c MuLV proviral DNA clones B-56, B-73, B-14 (5' LTRs), and B-34 (3' LTR) (13) and with NFS-Th-1 xenotropic DNA cloned from MuLV-infected cells (14) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Recombinant phages containing murine leukemia virus (MuLV)-reactive DNA sequences were isolated after screening of a BALB/c mouse embryo DNA library and from shotgun cloning of EcoRI-restricted AKR/J mouse liver DNA. Twelve different clones were isolated which contained incomplete MuLV proviral DNA sequences extending various distances from either the 5' or 3' long terminal repeat (LTR) into the viral genome. Restriction maps indicated that the endogenous MuLV DNAs were related to xenotropic MuLVs, but they shared several unique restriction sites among themselves which were not present in known MuLV proviral DNAs. Analyses of internal restriction fragments of the endogenous LTRs suggested the existence of at least two size classes, both of which were larger than the LTRs of known ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. Five of the six cloned endogenous MuLV proviral DNAs which contained envelope (env) DNA sequences annealed to a xenotropic MuLV env-specific DNA probe; in addition, four of these five also hybridized to an ecotropic MuLV-specific env DNA probe. Cloned MCF 247 proviral DNA also contained such dual-reactive env sequences. One of the dual-reactive cloned endogenous MuLV DNAs contained an env region that was indistinguishable by AluI and HpaII digestion from the analogous segment in MCF 247 proviral DNA and may therefore represent a progenitor for the env gene of this recombinant MuLV. In addition, the endogenous MuLV DNAs were highly related by AluI cleavage to the Moloney MuLV provirus in the gag and pol regions.Based upon their host range, murine leukemia viruses (MuLVs) isolated from inbred mice have been divided into three classes: ecotropic, which infect only mouse cells; xenotropic, which grow mostly in heterologous cells; and mink cell focus-forming (MCF) viruses (19, 23), which are dual-tropic and replicate both in murine and nonmurine cells. Tryptic peptide (16-18), RNase T1 oligonucleotide fingerprinting (41,43), as well as heteroduplex mapping analyses (14,15) have demonstrated that MCF MuLVs are recombinants which contain both ecotropic and xenotropic determinants in their envelope (env) region.The MCF MuLVs have been associated with the development of AKR thymic lymphomas based upon their appearance in late preleukemic thymuses and tumor tissues (23,27) and their ability to accelerate the onset of lymphomas in AKR mice (16,45).Biological and biochemical studies have shown that MuLV-related sequences are present in mouse chromosomal DNA. Hybridization analyses indicate that there are at least 20 to 50 copies of endogenous MuLV-related sequences per haploid genome (1,2,7,13,20,26,46,48). In general, inbred mouse strains carry no more than two or three loci for inducible ecotropic MuLVs (42) and no more than one or two for inducible xenotropic MuLVs (29). To study the relationship between the endogenous MuLV DNA sequences and the proviruses of infectious MuLVs, we have analyzed MuLV-reactive recombinant phages isolated from a BALB/c mouse embryo DNA library a...

The feline oncornavirus-associated cell membrane antigen (FOCMA)

Simian foamy viruses (SFVs) are highly prevalent in all nonhuman primate species and can infect humans following occupational and non-occupational exposure to infected animals and their tissues, blood or body fluids. Virus transmission results in a stable, persistent infection that seems to be latent. SFV infections are thus far nonpathogenic, with no evidence of adverse clinical outcome in their natural nonhuman primate hosts or by experimental injection in animals and upon cross-species transmission in humans. Since the emergence of pathogenic viruses from nonpathogenic viruses upon cross-species infection is well-documented for several retroviruses, it is prudent to take necessary precautions to deter SFV infections in humans. These steps will help prevent the emergence of a novel pathogen and reduce the risk of transmission of another potential pathogenic human retrovirus.