A New Approach to Establish a Cell Line with Reduced Risk of Endogenous Retroviruses

Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV.     

Primate gammaretroviruses require an ancillary factor not required for murine gammaretroviruses to infect BHK cells

BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice.     

Endogenous RD-114 virus genome expression in malignant tissues of domestic cats.

Endogenous xenotropic cat type C virus (RD-114)- and infectious feline leukemia virus (FeLV)-specific gene expressions were measured in spontaneous sarcomas carcinomas, and nonmalignant cat tissues by molecular hybridization for virus-specific RNA and competition radio-immunoassays for the major internal protein (p30) of these two viruses. The results indicate that RD-114 gene expression in sarcomas and carcinomas at both RNA and p30 levels is significantly higher than histologically normal tissues from cats free of cancer. In contrast, the levels of FeLV viral RNA and p30 are fount to be low or undetectable in the majority of these tumored and normal tissues examined. Whereas variability in the amounts of RD-114 OR FeLV RNA and p30 expressed is found in tissues from different cats, their expression is fairly uniform in multiple malignant tissues of the same cat. The finding of widespread occurrence of elevated RD-114 gene expression in sarcomas and carcinomas is consistent with our similar observation with natural lymphomas of domestic cats and suggests that expression of certain functions of this endogenous virus may be etiologically involved in the development of many different spontaneous neoplasms of cats.     

Infectious endogenous retroviruses in cats and emergence of recombinant viruses

Endogenous retroviruses (ERVs) comprise a significant percentage of the mammalian genome, and it is poorly understood whether they will remain as inactive genomes or emerge as infectious retroviruses. Although several types of ERVs are present in domestic cats, infectious ERVs have not been demonstrated. Here, we report a previously uncharacterized class of endogenous gammaretroviruses, termed ERV-DCs, that is present and hereditary in the domestic cat genome. We have characterized a subset of ERV-DC proviral clones, which are numbered according to their genomic insertions. One of these, ERV-DC10, located in the q12-q21 region on chromosome C1, is an infectious gammaretrovirus capable of infecting a broad range of cells, including human. Our studies indicate that ERV-DC10 entered the genome of domestic cats in the recent past and appeared to translocate to or reintegrate at a distinct locus as infectious ERV-DC18. Insertional polymorphism analysis revealed that 92 of 244 domestic cats had ERV-DC10 on a homozygous or heterozygous locus. ERV-DC-like sequences were found in primate and rodent genomes, suggesting that these ERVs, and recombinant viruses such as RD-114 and BaEV, originated from an ancestor of ERV-DC. We also found that a novel recombinant virus, feline leukemia virus subgroup D (FeLV-D), was generated by ERV-DC env transduction into feline leukemia virus in domestic cats. Our results indicate that ERV-DCs behave as donors and/or acceptors in the generation of infectious, recombinant viruses. The presence of such infectious endogenous retroviruses, which could be harmful or beneficial to the host, may affect veterinary medicine and public health.     

Local replication of simian immunodeficiency virus in the breast milk compartment of chronically-infected, lactating rhesus monkeys

Retroviruses have been linked to a variety of diseases such as neoplastic and immunodeficiency disorders and neurologic and respiratory diseases. Recently, a novel infectious human retrovirus, the xenotropic murine leukemia virus-related virus (XMRV), has been identified in cohorts of patients with either a familial type of prostate cancer or chronic fatigue syndrome. The apparent unrelatedness of these diseases raised the question of the potential involvement of XMRV in other diseases. Here, we investigated the presence of XMRV in a selection of pediatric idiopathic infectious diseases with symptoms that are suggestive of a retroviral infection, as well as in children with respiratory diseases and in adult patients with spondyloarthritis (SpA). Using a XMRV env-nested PCR, we screened 72 DNA samples obtained from 62 children hospitalized in the Montpellier university hospital (France) for hematological, neurological or inflammatory pathologies, 80 DNA samples from nasopharyngeal aspirates from children with respiratory diseases and 19 DNA samples from SpA. None of the samples tested was positive for XMRV or MLV-like env sequences, indicating that XMRV is not involved in these pathologies.     

No evidence for XMRV nucleic acids, infectious virus or anti-XMRV antibodies in Canadian patients with chronic fatigue syndrome

The gammaretroviruses xenotropic murine leukemia virus (MLV)-related virus (XMRV) and MLV have been reported to be more prevalent in plasma and peripheral blood mononuclear cells of chronic fatigue syndrome (CFS) patients than in healthy controls. Here, we report the complex analysis of whole blood and plasma samples from 58 CFS patients and 57 controls from Canada for the presence of XMRV/MLV nucleic acids, infectious virus, and XMRV/MLV-specific antibodies. Multiple techniques were employed, including nested and qRT-PCR, cell culture, and immunoblotting. We found no evidence of XMRV or MLV in humans and conclude that CFS is not associated with these gammaretroviruses.     

Long-term infection and vertical transmission of a gammaretrovirus in a foreign host species

Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari.     

Baboon Virus Isolate M-7 with Properties Similar to Feline Virus RD-114

A virus (M-7) isolated from baboon placental tissue demonstrates many similarities to endogenous feline virus RD-114. Immunodiffusion analysis shows a group-specific antigen (gs-1) line of identity between M-7 and RD-114. Anti-RD-114 DNA polymerase IgG inhibits M-7 polymerase by 57% compared to 97% for RD-114. M-7 virus has helper activity as demonstrated by rescue of murine sarcoma virus (MSV) from sarcoma-positive leukemia-negative human amnion cells. The host range of the rescued M-7 pseudotype of MSV, MSV (M-7), is similar to that of RD-114 virus. MSV (M-7) is also able to transform baboon cells and causes no detectable transformation of feline cells without addition of helper feline leukemia virus. Interference properties of M-7 and RD-114 virus are identical. Virus-specific neutralizing antisera, although partially cross-reacting, can distinguish MSV (M-7) from MSV (RD-114). These similarities and differences between RD-114 and M-7 viruses are best explained as type-specific differences between two viruses within the same strain.     

Isolation of an RD-114-Like Oncornavirus from a Cat Cell Line

A clone of cells derived from a continuous line of cat cells (CCC) spontaneously produced an RNA C-type virus (CCC virus) which did not have the group-specific antigen of the standard strains of feline leukemia viruses but did have that of the RD-114 virus. Single-hit infection of a virus yielding CCC cell with only the feline leukemia virus pseudotype of murine sarcoma virus [MSV(FeLV)] resulted in the release of a pseudotype of MSV coated with the CCC virus envelope. Host range, transmission of virus, helper functions, interference properties, and specific neutralization showed that the CCC and the RD-114 isolates as well as their respective MSV pseudotypes are closely similar if not identical. Parental, virus-negative cells frozen before the existence of RD-114 were chemically induced to yield CCC-like virus de novo. Infection of susceptible human cells with the chemically induced virus resulted in interference with the CCC virus pseudotype of MSV but not with the FeLV pseudotype of MSV.     

Expression of Middle-Component RNA of Cowpea Mosaic Virus: In Vitro Generation of a Precursor to Both Capsid Proteins by a Bottom-Component RNA-Encoded Protease from Infected Cells

The internal organization of endogenous xenotropic murine leukemia virus proviruses was determined in a series of blot hybridization experiments in which DNA from several different inbred mouse strains, digested with restriction enzymes known to cleave xenotropic proviral DNAs at least twice, was annealed to generalized murine leukemia virus or xenotropic env-specific DNA probes. Comigrating bands of variable intensity which hybridized to the xenotropic env probe were identified in all inbred mouse DNA preparations. At least seven classes of endogenous xenotropic proviral DNA with respect to SacI cleavage maps were detected in mouse DNA. Two of the seven classes were indistinguishable from proviruses associated with known infectious xenotropic murine leukemia viruses. These results are consistent with the existence of related but organizationally distinct families of endogenous xenotropic proviral DNA that are present in different relative abundances in mouse genomic DNA.     

Molecular genetic characterization of the RD-114 gene family of endogenous feline retroviral sequences.

RD-114 is a replication-competent, xenotropic retrovirus which is homologous to a family of moderately repetitive DNA sequences present at ca. 20 copies in the normal cellular genome of domestic cats. To examine the extent and character of genomic divergence of the RD-114 gene family as well as to assess their positional association within the cat genome, we have prepared a series of molecular clones of endogenous RD-114 DNA segments from a genomic library of cat cellular DNA. Their restriction endonuclease maps were compared with each other as well as to that of the prototype-inducible RD-114 which was molecularly cloned from a chronically infected human cell line. The endogenous sequences analyzed were similar to each other in that they were colinear with RD-114 proviral DNA, were bounded by long terminal redundancies, and conserved many restriction sites in the gag and pol regions. However, the env regions of many of the sequences examined were substantially deleted. Several of the endogenous RD-114 genomes contained a novel envelope sequence which was unrelated to the env gene of the prototype RD-114 env gene but which, like RD-114 and endogenous feline leukemia virus provirus, was found only in species of the genus Felis, and not in other closely related Felidae genera. The endogenous RD-114 sequences each had a distinct cellular flank which indicates that these sequences are not tandem but dispersed nonspecifically throughout the genome. Southern analysis of cat cellular DNA confirmed the conclusions about conserved restriction sites in endogenous sequences and indicated that a single locus may be responsible for the production of the major inducible form of RD-114.     

Isolation from cats of an endogenous type C virus with a novel envelope glycoprotein.

A search for variant endogenous cat viruses led to a novel isolate. Although the major envelope glycoprotein of this virus was similar in size to that of an RD-114-like virus that was coisolated, it was unrelated to RD-114 or feline leukemia virus by immunological and biological criteria. This degree of dissimilarity suggests a different evolutionary progenitor from that for the RD-114 and feline leukemia virus viral envelopes. The novel virus did, however, code for gag gene polypeptides which are closely related to RD-114 virus. Neither the novel isolate nor the RD-114-like coisolate induced foci in S+L- cat cells which restrict focus induction by RD-114 virus. This suggests that the two viruses share a common genomic target of restriction which resides outside of the env region.     

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10⁵ cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.     

No evidence of murine leukemia virus-related viruses in live attenuated human vaccines

Background

The association of xenotropic murine leukemia virus (MLV)-related virus (XMRV) in prostate cancer and chronic fatigue syndrome reported in previous studies remains controversial as these results have been questioned by recent data. Nonetheless, concerns have been raised regarding contamination of human vaccines as a possible source of introduction of XMRV and MLV into human populations. To address this possibility, we tested eight live attenuated human vaccines using generic PCR for XMRV and MLV sequences. Viral metagenomics using deep sequencing was also done to identify the possibility of other adventitious agents.

Results

All eight live attenuated vaccines, including Japanese encephalitis virus (JEV) (SA-14-14-2), varicella (Varivax), measles, mumps, and rubella (MMR-II), measles (Attenuvax), rubella (Meruvax-II), rotavirus (Rotateq and Rotarix), and yellow fever virus were negative for XMRV and highly related MLV sequences. However, residual hamster DNA, but not RNA, containing novel endogenous gammaretrovirus sequences was detected in the JEV vaccine using PCR. Metagenomics analysis did not detect any adventitious viral sequences of public health concern. Intracisternal A particle sequences closest to those present in Syrian hamsters and not mice were also detected in the JEV SA-14-14-2 vaccine. Combined, these results are consistent with the production of the JEV vaccine in Syrian hamster cells.

Conclusions

We found no evidence of XMRV and MLV in eight live attenuated human vaccines further supporting the safety of these vaccines. Our findings suggest that vaccines are an unlikely source of XMRV and MLV exposure in humans and are consistent with the mounting evidence on the absence of these viruses in humans.     

Characterization, mapping, and distribution of the two XMRV parental proviruses

Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1(n) receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.     

Lack of infection with XMRV or other MLV-related viruses in blood, post-mortem brains and paternal gametes of autistic individuals

Background

Autistic spectrum disorder (ASD) is characterized by impaired language, communication and social skills, as well as by repetitive and stereotypic patterns of behavior. Many autistic subjects display a dysregulation of the immune system which is compatible with an unresolved viral infection with prenatal onset, potentially due to vertical viral transmission. Recently, the xenotropic murine leukemia virus-related virus (XMRV) has been implicated in chronic fatigue syndrome (CFS) and in prostate cancer by several, though not all studies.

Methodology/Principal Findings

We assessed whether XMRV or other murine leukemia virus (MLV)-related viruses are involved in autistic disorder. Using nested PCR targeted to gag genomic sequences, we screened DNA samples from: (i) peripheral blood of 102 ASD patients and 97 controls, (ii) post-mortem brain samples of 20 ASD patients and 17 sex- and age-matched controls, (iii) semen samples of 11 fathers of ASD children, 25 infertile individuals and 7 fertile controls. No XMRV gag DNA sequences were detected, whereas peripheral blood samples of 3/97 (3.1%) controls were positive for MLV.

Conclusions|Significance

No MLV-related virus was detected in blood, brain, and semen samples of ASD patients or fathers. Hence infection with XMRV or other MLV-related viruses is unlikely to contribute to autism pathogenesis.     

In-Depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer

XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.     

DNA extraction columns contaminated with murine sequences

Sequences of the novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV) have been described in human prostate cancer tissue, although the amounts of DNA are low. Furthermore, XMRV sequences and polytropic (p) murine leukemia viruses (MLVs) have been reported in patients with chronic fatigue syndrome (CFS). In assessing the prevalence of XMRV in prostate cancer tissue samples we discovered that eluates from naïve DNA purification columns, when subjected to PCR with primers designed to detect genomic mouse DNA contamination, occasionally gave rise to amplification products. Further PCR analysis, using primers to detect XMRV, revealed sequences derived from XMRV and pMLVs from mouse and human DNA and DNA of unspecified origin. Thus, DNA purification columns can present problems when used to detect minute amounts of DNA targets by highly sensitive amplification techniques.     

Innate immunity against HIV: a priority target for HIV prevention research

The xenotropic/polytropic subgroup of mouse leukemia viruses (MLVs) all rely on the XPR1 receptor for entry, but these viruses vary in tropism, distribution among wild and laboratory mice, pathogenicity, strategies used for transmission, and sensitivity to host restriction factors. Most, but not all, isolates have typical xenotropic or polytropic host range, and these two MLV tropism types have now been detected in humans as viral sequences or as infectious virus, termed XMRV, or xenotropic murine leukemia virus-related virus. The mouse xenotropic MLVs (X-MLVs) were originally defined by their inability to infect cells of their natural mouse hosts. It is now clear, however, that X-MLVs actually have the broadest host range of the MLVs. Nearly all nonrodent mammals are susceptible to X-MLVs, and all species of wild mice and several common strains of laboratory mice are X-MLV susceptible. The polytropic MLVs, named for their apparent broad host range, show a more limited host range than the X-MLVs in that they fail to infect cells of many mouse species as well as many nonrodent mammals. The co-evolution of these viruses with their receptor and other host factors that affect their replication has produced a heterogeneous group of viruses capable of inducing various diseases, as well as endogenized viral genomes, some of which have been domesticated by their hosts to serve in antiviral defense.     

Differential association of transfer RNAs with the genomes of murine, feline and primate retroviruses

The tRNAs that are bound to the genomic RNAs of several murine, feline, and primate retroviruses have been identified. Transfer RNAs were divided into those loosely bound and those tightly bound by stepwise thermal dissociation of the 70 S RNA. They were then identified and semiquantitated by aminoacylation. Proline tRNA is the most tenaciously bound tRNA in several strains of murine leukemia virus, two strains of feline leukemia virus, and the primate viruses simian sarcoma, baboon endogenous, and gibbon ape lymphoma. In the feline xenotropic virus, RD-114, tRNAGly is enriched in the most tightly bound fraction. In Mason-Pfizer monkey virus, as in the murine mammary tumor virus, tRNALys is the tRNA most tenaciously bound to its genomic RNA. Besides the most tightly associated tRNA, one or more different tRNAs are found in relatively large amounts in association with the 70 S RNA. (For convenience, we refer to the largest RNA ccomplex (50-70 S) isolated from any of the retroviruses studies as '70 S' RNA.) These tRNAs can be distinguished from the most tightly bound tRNA by the fact that they can be dissociated at lower temperatures. However, they occur in the same relative abundance as the tightly bound tRNA.