MCF Viruses Research Articles

Subclinical Ovine Gammaherpesvirus 2-Related Infections in Free-Ranging Wild Boars (Sus scrofa) from Southern Brazil.

Ovine gammaherpesvirus 2 (OvGHV2), is a Macavirus and the cause of sheep-associated malignant catarrhal fever (SA-MCF), in which sheep are the asymptomatic reservoir hosts. Susceptible mammalian populations infected by OvGHV2 may develop clinical SA-MCF or subclinical infections. All members of the Macavirus genus known to be associated with MCF are collectively referred to as the MCF virus (MCFV) complex. This report describes the occurrence of subclinical OvGHV2-related infections in free-ranging wild boars (Sus scrofa) from southern Brazil. Specific body organs (n = 14) and biological samples (nasal and oral swabs; n = 17) were collected from 24 asymptomatic wild boars from a conservation unit located within the Central-eastern mesoregion of Paraná State. Organs were processed to observe histopathological patterns suggestive of diseases of domestic animals; only pulmonary samples were used in an immunohistochemical assay designed to detect MCFV tissue antigens. Furthermore, all samples were submitted to molecular assays designed to detect the OvGHV2 tegument protein gene. Viral-induced pneumonia was diagnosed in two wild boars; one of these contained OvGHV2 DNA, with MCFV antigens identified in the other. Additionally, MCFV tissue antigens were detected within pulmonary epithelial cells of the lungs with and without pulmonary disease. Collectively, OvGHV2 was detected in 37.5% (9/24) of all wild boars, with detection occurring in the organs of 57.1% (8/14) wild boars and the oral cavity of one animal. These results demonstrated that these wild boars were subclinically infected by OvGHV2, and that infection produced typical pulmonary alterations. In addition, the detection of OvGHV2 within the oral cavity of one wild boar may suggest that this animal may be a potential disseminator of this pathogen to susceptible animal populations, including livestock and wildlife, acting as a possible bridge host for OvGHV2. Furthermore, infection by OvGHV2 probably occurred due to incidental contact with asymptomatic sheep maintained within the surrounding rural areas and not within the conservation units.

Ivermectin inhibits replication of the malignant catarrhal fever virus alcelaphine herpesvirus 1

Malignant catarrhal fever is a lymphoproliferative disease of cattle and other ungulates that is caused by genetically and antigenically related gamma herpesviruses of the genus Macavirus. Infection of the natural host species is efficient and asymptomatic but spread to susceptible hosts is often fatal with clinical signs including fever, depression, nasal and ocular discharge. There is no recognised treatment for MCF but a vaccine for one MCF virus, alcelaphine herpesvirus 1 (AlHV-1), has been described. In this paper we describe the inhibition of AlHV-1 replication and propagation by the anthelminthic drug ivermectin. Concentrations of 10 μM or greater led to significant reductions in both copy number and viable titre of virus tested in culture medium, with little replication detected at over 20 μM ivermectin. In the absence of alternative treatments, further testing of ivermectin as a candidate antiviral treatment for MCF may therefore be justified.

Ovine gammaherpesvirus 2 infections in cattle without typical manifestations of sheep-associated malignant catarrhal fever and concomitantly infected with bovine coronavirus.

Sheep-associated malignant catarrhal fever (SA-MCF) is a severe, frequently fatal, lymphoproliferative disease that affects a wide variety of ruminants and is caused by ovine gammaherpesvirus 2 (OvHV-2), a member of the MCF virus (MCFV) complex. The typical clinical manifestations of SA-MCF are well known and easily recognized by veterinarians, resulting in clinical diagnosis of MCF when characteristic clinical signs are present. This article describes the findings observed in cattle infected with OvHV-2 but without typical clinical manifestations of SA-MCF. Three calves with episodes of diarrhea before death and a yearling that died suddenly were investigated. Gross alterations were not suggestive of SA-MCF. Histopathology revealed a combination of proliferating vascular lesions (PVLs) and necrotizing vasculitis in three animals (two calves and the yearling); with PVLs being identified only at the carotid rete mirabile of two calves infected with OvHV-2. Additional significant histopathologic lesions included atrophic enteritis, portal lymphocytic hepatitis, interstitial pneumonia, suppurative bacterial bronchopneumonia, and pulmonary hemorrhage. An immunohistochemical assay designed to identify only antigens of MCFV revealed, positive, intralesional, intracytoplasmic immunoreactivity within epithelial cells of multiple tissues of all animals with PVLs. PCR assays amplified OvHV-2 DNA from multiple tissues of the animals that contained MCFV proteins, confirming the MCFV identified as OvHV-2. Additionally, bovine coronavirus (BCoV) nucleic acids were amplified from tissues of all animals, including the animal not infected by OvHV-2. Collectively, these findings confirmed the participation of OvHV-2 in the development of the disease patterns observed in these animals that were concomitantly infected by BCoV and provide additional confirmation that cattle can be subclinically infected with OvHV-2. Consequently, the real occurrence of OvHV-2-related disease may be more elevated than reported, since asymptomatic or subclinically infected animals are not likely to be investigated for OvHV-2. Furthermore, PVLs should be included as possible histologic indicators of OvHV-2-related diseases in ruminants.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42770-021-00653-6.

Ovine gammaherpesvirus-2 infection associated with chronic interstitial pneumonia in a sheep

Sheep Associated-Malignant Catarrhal Fever (SA-MCF) is severe, frequently lethal, lymphoproliferative disease predominantly of ruminants, that is caused by ovine gammaherpesvirus-2 (OvHV-2), a member of the MCF virus (MCFV) complex. However, SA-MCF in sheep is a rare entity with few demonstrations of natural diseases worldwide. This report documents the clinical, radiographical, pathological, immunohistochemical, and molecular findings of SA-MCF in a sheep. A 4-year-old, female, mixed-breed sheep with progressive emaciation for at least one month was humanely euthanized due to poor prognosis. Clinically, the animal had tachypnea, ruminal hypomotility, productive coughing with bilateral muffling sounds during pulmonary auscultation. Radiographical evaluation revealed alveolar opacity of the cranioventral pulmonary region. Grossly, there were distinct rib impressions on the pleural surface of the lungs, suggestive of interstitial pneumonia. Histopathologic evaluation of the lungs revealed several disease patterns including 1) chronic interstitial pneumonia with vasculitis and proliferating vascular lesions, and thrombosis; 2) pulmonary abscesses associated with embolic dissemination of Corynebacterium pseudotuberculosis from superficial lymph node due to caseous lymphadenitis, CLA; 3) granulomatous pneumonia associated with pulmonary nematodes; and 4) chronic pleuritis, probably due to caseous lymphadenitis. Additional significant histologic findings included widespread lymphocytic vasculitis and proliferating vascular lesions in multiple tissues, atrophic enteritis, segmental degeneration of myocardial fibers with lymphocytic pericarditis, lymphocytic interstitial nephritis, and non-suppurative encephalitis. An immunohistochemistry (IHC) assay, based on the monoclonal antibody 15A (MAb-15A), that is specific to all MCFV known to cause MCF, revealed positive, intracytoplasmic, intralesional immunoreactivity, predominantly within bronchial and bronchiolar epithelial cells of the lungs and cryptal epithelial cells of the small intestine, followed by the renal tubular epithelium, cardiomyocytes, and with patchy immunolabelling within neurons of the cerebral cortex. Molecular testing done to detect a wide range of bacterial and viral agents of ruminant diseases, only amplified OvHV-2 DNA from fresh tissue fragments of the lungs, kidney, liver, spleen, and cerebrum. Direct sequencing confirmed that the PCR amplicon derived from the pulmonary fragments had 99.2–99.7% nucleotide sequence identity with OvHV-2 reference strains and strains of OvHV-2 from Brazil. The clinical, radiographical, gross, histopathologic, IHC, and molecular findings in the lungs are consistent with chronic interstitial pneumonia associated with infection by OvHV-2. Furthermore, the non-detection of other viral agents associated with pulmonary diseases in ruminants suggest that OvHV-2 was directly associated with the development of chronic pneumonia in this sheep. Additionally, the dental alterations, CLA, and the pulmonary nematode may have contributed towards the reduced immunological statue of the animal and facilitated the occurrence of SA-MCF. These findings may indicate that OvHV-2 may be a major participant in the pathogenesis of pulmonary disease of sheep under special conditions. Moreover, the proliferating vascular lesions identified in multiple tissues are additional evidence of chronic manifestations of OvHV-2 infections as described in chronic SA-MCF of cattle, while the widespread vasculitis is consistent with SA-MCF. Additionally, the IHC findings using the MAb-15A confirmed that this diagnostic approach is efficient to identify intralesional antigens of OvHV-2.

A review of the epidemiological, clinical, and pathological aspects of malignant catarrhal fever in Brazil.

Sheep-associated malignant catarrhal fever (SA-MCF), the form of MCF that occurs in Brazil, is a severe, frequently fatal, infectious disease caused by ovine gammaherpesvirus-2 (OvHV-2), in which sheep are the asymptomatic hosts and cattle and other cloven-hoofed animals are the accidental hosts. This review provides a critical analysis of the historical, epidemiological aspects and the estimated economic impacts associated with SA-MCF in Brazil. Moreover, the clinical manifestations and pathological lesions associated with SA-MCF in cattle are reviewed and discussed and the phylogenetic distribution of OvHV-2 in Brazil is presented. OvHV-2 is the only MCF virus identified in animals from Brazil.It is recommended that a histopathologic diagnosis of SA-MCF be based on all aspects of vascular disease in the affected animal and not only lymphocytic/necrotizing vasculitis and/or fibrinoid change. Conformation of the intralesional participation of OvHV-2 in these alterations can be achieved by immunohistochemistry and/or in situ hybridization assays. Additionally, it is proposed that OvHV-2 should be considered as a possible infectious disease agent associated with the development of bovine respiratory disease in cattle. Furthermore, the possible role of the small intestine in the dissemination of OvHV-2 is discussed.

Immunohistochemical Detection of Intralesional Antigens of Ovine Gammaherpesvirus-2 in Cattle with Sheep-associated Malignant Catarrhal Fever

Sheep-associated malignant catarrhal fever (SA-MCF) is a severe lymphoproliferative disease of ruminants caused by ovine gammaherpesvirus-2 (OvHV-2). Since the initial identification of SA-MCF there has been extensive research related to the pathogenesis of OvHV-2, based primarily on serological and molecular assays associated with typical histopathological findings. The monoclonal antibody (MAb-15A) binds to a common epitope in MCF viruses and is used frequently in serological investigations. However, the utilization of this antibody to detect antigens of OvHV-2 in tissues has not been examined. Accordingly, this study standardized an immunohistochemical assay using MAb-15A to identify antigens of OvHV-2 in tissues of cattle (n=5) with SA-MCF. All animals developed acute neurological signs, without ocular and nasal manifestations, and had nucleic acids of OvHV-2 in brain tissue detected by polymerase chain reaction. The principal histopathological findings were lymphocytic nephritis (n=5), widespread arterial proliferation and vasculitis (n=5), lymphocytic portal hepatitis (n=3), non-suppurative meningoencephalitis (n=2) and atrophic enteritis with cryptal necrosis and dilation (n=2). Intralesional intracytoplasmic antigens of OvHV-2 were identified within multiple epithelial cells of the kidneys of all animals, the intestines of animals with and without atrophic enteritis, and within epithelial cells of bile ducts in animals with lymphocytic hepatitis. Additionally, there was positive intracytoplasmic immunoreactivity within histiocytes and lymphocytes in several tissues. These findings suggest that the MAb-15A detects antigens of OvHV-2 within epithelial cells and leucocytes in several organs. Moreover, this assay would contribute significantly towards understanding of the pathogenesis of SA-MCF and may be used for retrospective studies. Additionally, angiopathy in SA-MCF may be a progressive lesion, which may terminate in luminal occlusion and probably occurs irrespectively of the eye and head form of MCF.

Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles.

Alcelaphine herpesvirus–1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF.

A geographic cluster of malignant catarrhal fever in moose (Alces alces) in Norway.

Three cases of lethal sheep-associated malignant catarrhal fever (SA-MCF) in free-ranging moose (Alces alces) were diagnosed in Lesja, Norway, December 2008-February 2010. The diagnosis was based on PCR identification of ovine herpesvirus 2 (OvHV-2) DNA (n = 3) and typical histopathologic lesions (n = 1). To study the possibility of subclinical or latent MCF virus (MCFV) infection in this moose population and in red deer (Cervus elaphus), we examined clinically normal animals sampled during hunting in Lesja 2010 by serology and PCR. Sera from 63 moose and 33 red deer were tested for antibodies against MCFV by competitive-inhibition enzyme-linked immunosorbent assay. To test for MCFVs, a consensus PCR for herpesviral DNA was run on spleen samples from 23 moose and 17 red deer. All samples were antibody and PCR negative. Thus, there is no evidence of previous exposure, subclinical infection, or latent infection in this sample. This seasonal cluster of SA-MCF cases (2008-10) may be attributable to exposure of moose to lambs when OvHV-2 shedding is presumed to be maximal, compounded by an unusual extended grazing period by sheep in the autumn.

Development of a Management Program for a Mixed Species Wildlife Park Following an Occurrence of Malignant Catarrhal Fever

During late 2001 and early 2002, a mixed species wildlife park in North Carolina experienced an acute outbreak of morbidity and mortality in Pere David's deer (Elaphurus davidianus), axis deer (Axis axis), blackbuck antelope (Antelope cervicapra), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus). Clinical signs varied from fulminant disease, progressing from depression to bloody scours to death in fewer than 4 days in Pere David's deer, to a more protracted form of disease, ranging from 2 wk to 3 mo, in axis deer. In moribund axis deer, high levels of anti-malignant catarrhal fever (MCF) virus antibody by competitive-inhibition enzyme-linked immunosorbent assay were detected. Ovine herpesvirus 2 (OvHV-2) DNA was detected in peripheral blood leukocytes of the affected axis deer. No other MCF viruses were detected. Retrospective examination of frozen tissue samples from the affected Pere David's deer and blackbuck antelope also confirmed the presence of OvHV-2 DNA. Initial control efforts were directed at preventing further deaths of clinically susceptible animals by removing MCF virus reservoir species, particularly ovine species. The most prevalent ovine species in the wildlife park was mouflon sheep (Ovis musimon). All sheep were removed from the park by June 2002, and the last MCF death occurred in October 2002. Since mouflon sheep had been a prominent attraction in the wildlife park, the owner wanted a means to reintroduce this species to the park. Derivation of OvHV-2-uninfected mouflon lambs was undertaken using the previously described program for production of OvHV-2-free sheep (Ovis ovis). The rederived MCF virus-negative mouflon sheep were introduced into the park in approximately January 2004. As of December 2007, no further cases of MCF have occurred since the removal of OvHV-2-positive mouflon sheep and reintroduction of the virus-free lambs. This paper describes the successful management and control of MCF in a densely populated mixed species animal park.

Observations on rinderpest and rinderpest-like diseases throughout West and Central African countries during rinderpest eradication projects

Between 1998 and 2005, the Regional Reference Laboratory at Bingerville (Ivory-Coast) received samples for analysis from Western and Central African countries. From a total of 606 sera; 65 tissue samples and 75 swabs received, no rinderpest virus or specific gene products or antibodies against rinderpest were detected. Use of the PCR on the tissue and swabs (total of 140 samples) identified the genomic presence of BVD (4/140), MCF (2/140), IBR (1/140) and FMD (6/140) viruses. These cause diseases that produce similar clinical signs to rinderpest. The quality of many samples sent to the reference laboratory did not meet the laboratory requirements and this compromised analysis of some specimens.

Reexamination of Amphotropic Murine Leukemia Virus Neurovirulence: Neural Stem Cell-Mediated Microglial Infection Fails to Induce Acute Neurodegeneration

The 4070A amphotropic murine leukemia virus (A-MuLV) has been variably reported to harbor neurovirulence determinants within its env gene. In this report we reexamined this issue by applying two approaches previously demonstrated to amplify murine leukemia virus neurovirulence. The first approach involved introducing the 4070A env gene into the background of Friend virus clone FB29 to enhance peripheral virus replication kinetics and central nervous system entry. The resulting chimeric virus, FrAmE, exhibited widespread vascular infection throughout the central nervous system (CNS); however, parenchymal infection was quite limited. Neither clinical neurological signs nor spongiform neurological changes accompanied FrAmE CNS infection. To overcome this CNS entry limitation, 4070A and FrAmE were delivered directly into the CNS via transplantation of infected C17.2 neural stem cells (NSCs). Significantly, NSC dissemination of either 4070A or FrAmE resulted in widespread, high-level amphotropic virus expression within the CNS parenchyma, including the infection of microglia, the critical target required for inducing neurodegeneration. Despite the extensive CNS infection, no associated clinical neurological signs or acute neuropathological changes were observed. Interestingly, we observed the frequent appearance of circulating polytropic (MCF) virus in the serum of amphotropic virus-infected animals. However, neither peripheral inoculation of an amphotropic/MCF virus mixture nor transplantation of NSCs expressing both amphotropic and MCF viruses induced acute clinical neurological signs or spongiform neuropathology. Thus, the results generated in this study suggest that the 4070A env gene is not inherently neurovirulent. However, the frequent appearance of endogenous MCF viruses suggests the possibility that the interactions of amphotropic viruses with endogenous retroviral elements could contribute to the development of retrovirus-induced neurodegenerative disease.

Newly Recognized Herpesvirus Causing Malignant Catarrhal Fever in White-Tailed Deer ( Odocoileus virginianus )

Malignant catarrhal fever (MCF) was diagnosed by clinical signs and lesions in five out of six white-tailed deer (Odocoileus virginianus) in a North American zoo. The clinical signs and histopathological lesions in these deer were typical of MCF. Antibody to an epitope conserved among the MCF viruses was detected in the sera collected from the deer. PCR failed to amplify viral sequences from DNA extracted from peripheral blood leukocytes (PBL) and/or spleens of the deer with primers specific for ovine herpesvirus 2 (OHV-2) or specific for alcelaphine herpesvirus 1 (AHV-1). By using degenerate primers targeting a conserved region of a herpesviral DNA polymerase gene, a DNA fragment was amplified from the PBL or spleens of all six deer and sequenced. Alignment of the sequences demonstrated that the virus in the deer belongs to the Gammaherpesvirinae subfamily, exhibiting 82% identity to OHV-2, 71% to AHV-1, and 60% to a newly identified bovine lymphotropic herpesvirus. This virus, which causes classical MCF in white-tailed deer, is a newly recognized agent belonging to the MCF group of gammaherpesviruses. It is the third reported pathogenic MCF virus, genetically distinct but closely related to OHV-2 and AHV-1. The reservoir for the virus has not been identified.

Differential Behavior of the Mo+PyF101 Enhancer Variant of Moloney Murine Leukemia Virus in Rats and Mice

The Mo+PyF101 enhancer variant of Moloney murine leukemia virus (M-MuLV) has been very useful in investigating M-MuLV leukemogenesis. When inoculated subcutaneously (sc) into neonatal mice, Mo+PyF101 M-MuLV is attenuated for development of disease. Previous studies in mice infected with wild-type M-MuLV have revealed several important preleukemic events, including development of splenic hyperplasia, defects in bone marrow hematopoiesis, andin vivogeneration of MCF viruses that arise by recombination in the uninfected mouse. Mo+PyF101 M-MuLV is defective in inducing these effects after sc inoculation. In the experiments reported here, a study of Mo+PyF101 M-MuLV infection in rats was carried out. Wild-type M-MuLV is leukemogenic in rats, but infected rats do not form MCF recombinants since they lack the necessary endogenous polytropic envelope sequences. Since Mo+PyF101 M-MuLV's leukemogenic defect is correlated with a failure to generate MCF recombinants, it seemed possible that wild-type M-MuLV might not have a leukemogenic advantage over Mo+PyF101 M-MuLV in rats, where MCF recombinants cannot form. Neonatal Fisher F344 rats were inoculated sc or intraperitoneally by wild-type and Mo+PyF101 M-MuLVs. Surprisingly, Mo+PyF101 M-MuLV was completely deficient in leukemogenesis in rats when inoculated by either route while wild-type M-MuLV induced lymphoma with the predicted time course. The leukemogenic defect for Mo+PyF101 M-MuLV resulted from a pronounced defect for establishing infection in rats. Further studies of wild-type M-MuLV in rats indicated that infection was confined almost exclusively to the thymus at early times. In mice wild-type M-MuLV establishes substantial infection in other hematopoietic organs such as spleen and bone marrow as well. Thymic infection was also correlated with a decrease in thymic cellularity at early times.

Single Amino Acid Changes in the Murine Leukemia Virus Capsid Protein Gene Define the Target ofFv1Resistance

The mouseFv1genetic locus controls resistance to subgroups of ecotropic, MCF, and amphotropic murine leukemia viruses (MuLVs). In addition to the four previously defined alleles ofFv1(Fv1n, Fv1b, Fv1nr, Fv10), we present evidence that the novel restriction pattern characteristic of DBA/2J mice maps to theFv1locus and therefore represents a novel allele, here designatedFv1d.Previous studies had demonstrated that theFv1-mediated viral tropism is determined within the capsid protein gene, and that N- and B-tropic virus capsids differ only in two adjacent amino acids. We introduced various amino acid substitutions at these two sites in the N-tropic AKV MLV capsid gene, and typed resulting viruses for host range on cells carrying all fiveFv1alleles as well as on cells from additional wild mouse species withFv1-like differences in virus susceptibility. Results indicate that alteration of the first of the two amino acids does not alter tropism, but alteration of the second alone is sufficient to convert the N-tropic AKV MLV to a B-tropic virus. Substitution of leucine for arginine at this site produced a virus with an unusual tropism not characteristic of any of the naturally occurring or laboratory strains of MuLV.

Recombinant mink cell focus-inducing virus and long terminal repeat alterations accompany the increased leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus after intraperitoneal inoculation

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.

Infection by mink cell focus-forming viruses confers interleukin 2 (IL-2) independence to an IL-2-dependent rat T-cell lymphoma line.

The development of T-cell lymphomas in rodents infected with type C retroviruses has been linked to the generation of a class of envelope (env) recombinant viruses called mink cell focus-forming viruses (MCF viruses) in the preleukemic thymus. To determine whether infection by MCF viruses altered the growth phenotype of retrovirus-induced T-cell lymphomas, a Moloney murine leukemia virus-induced interleukin-2 (IL-2)-dependent rat T-cell lymphoma line (4437A) was infected with MCF-247, modified MCF-V33 (mMCF-V33), or NZB-xenotropic (NZB-X) virus. The effects of virus infection on the IL-2 dependence of these cells was examined by cultivating them in the absence of IL-2. After IL-2 withdrawal, the uninfected and NZB-X-infected cells went through a crisis period characterized by massive death. All the independently maintained cultures of MCF- and mMCF-V33-infected cells, on the other hand, became IL-2 independent without a crisis. All the polytropic virus-infected IL-2-independent cultures contained a population of cells that was polyclonal with regard to polytropic provirus integration. Over this polyclonal background each culture produced multiple clones of cells that were selected rapidly after IL-2 withdrawal. Furthermore, the resulting MCF- or mMCF-V33-infected IL-2-independent cells retained the expression of IL-2 receptor. These data show that MCF and mMCF-V33 viruses may alter the growth phenotype of a T-cell lymphoma line and suggest that their effect on cell growth may be due to the direct interaction of the MCF envelope glycoprotein with cellular components, perhaps the IL-2 receptor.

A new leukemogenic retrovirus isolated from tumor cells derived from a radio-induced lymphoma of C57BL/6 mice: analysis of the env and LTR sequences

We report the cloning and characterization of a new ecotropic provirus encountered in a radio-induced thymic lymphoma of the C57BL/6 mouse. The provirus with an abnormally long LTR was inserted in the chromosomal DNA within the Pvt-1/MLVi-1/Mis-1 region which is a common integration site for MCF virus in mice and for Mo-MuLV in rats. This new ecotropic provirus was molecularly cloned and found to be infectious and competent for replication after transfection of murine cells. The recovered virus termed T3651/B was B-ecotropic, T-lymphotropic (in vivo) and highly leukemogenic for newborn C57BL/6 mice and for adult mice provided they were submitted to a subleukemogenic dose of irradiation. As compared to the AKV prototype N-ecotropic endogenous retrovirus, the T3651/B env proteins are only affected by few scattered point mutations. In contrast, the LTR has five repeats of enhancer sequences containing consensus motifs specific of the nuclear factors NF1-like, LVa, LVb and SEF1. Since a virus with such properties was encountered only once in 31 radio-induced tumors and isolated at a fourth tumor passage, a direct role of T3651/B virus in tumor genesis after irradiation is uncertain. Nevertheless, it is clear that T3651/B virus is a new leukemogenic retrovirus with a particular LTR structure which fits well with the model proposed by Rassart et al. (J. Virol. 58, 96–106, 1986) for the emergence of a thymotropic highly leukemogenic RadLV.

Isolation of a friend recombinant polytropic virus with a T-cell-restricted leukemogenicity

Hematopoietic malignant cells of various types were isolated from ICFW mice inoculated as newborn or adult with F-MuLV and grafted into recipient mice. After repeated in vivo cell transplants, several recombinant polytropic viruses (also termed MCF or dualtropic viruses) were isolated from tumors by limiting dilution. Two virus isolates designated RA1-17 and EA1-17 were recovered from the spleen and the omentum, respectively, of the same grafted animal. When inoculated into newborn mice, RA1-17 induced erythroblastosis similar to that induced by other Friend recombinant polytropic viruses. Remarkably, EA1-17 induced T-cell leukemia after a short latency. To our knowledge, this is the first description of a polytropic recombinant virus with a T-cell tropism isolated after inoculation of F-MuLV.

H-2-associated and background genes influence the development of a murine retrovirus-induced immunodeficiency syndrome.

Host genetic determinants of resistance or susceptibility to a retrovirus-induced immunodeficiency syndrome, termed MAIDS, were evaluated in Fv-1b mice infected with the mixture of ecotropic, MCF, and defective murine leukemia viruses designated LP-BM5 murine leukemia viruses. Genes of the MHC were shown to exert a major influence on the development of disease and the extent of virus spread in mice infected as adults. Strains bearing the b, f, k, q, r, and s haplotypes were moderately to highly susceptible to MAIDS whereas mice of the d haplotype were the most resistant. Resistance to disease was strongly associated with inhibition of mink cell focus-inducing virus spread and, to a lesser extent, with inhibition of ecotropic virus expression. Mapping studies localizing resistance associated with the d haplotype to H-2Dd were confirmed by the demonstration that B6 mice carrying this gene as a transgene or by recombination were resistant to disease. Penetrance of resistance to disease associated with expression of H-2Dd was markedly influenced by MHC genes mapping to the left of H-2D and by non-MHC loci such that some strains bearing this gene were highly susceptible to MAIDS. The combined effects of MHC and background genes among 40 strains examined yielded a remarkably wide spectrum of disease phenotypes with the onset of advanced disease ranging from 10 wk to 72 wk postinfection. Resistance to disease in moderately to highly resistant strains was shown to develop with age. Unexpectedly, the disease resistant phenotype was found to be a recessive trait.

Genetics of Endogenous Murine Leukemia Virusesa

Inbred strains of mice contain in the genome 40-60 endogenous proviruses related to murine leukemia virus. To assess the genetic and pathogenic consequences of these to the host, we have developed a strategy to distinguish among the three different host-range subgroups--xenotropic, polytropic and modified polytropic--by using oligonucleotide probes specific for a polymorphic region in env. Each of these proteins detects a relatively small number of bands in a Southern blot, thus permitting us to enumerate all individual proviruses of this group. Using this approach, we have determined the distribution of different proviruses among inbred and recombinant inbred (RI) strains congenic or coisogenic for specific mutants. Using the RI results, we have been able to place over 100 proviruses on the mouse genetic map. A number of these are closely linked to well-characterized mutations, and we have been able to establish that at least one mutation, hr (hairless), was caused by a proviral insertion. If the other close linkages also prove to reflect causality, we estimate that up to 5% of recessive mutations in the mouse might be caused by insertion of proviruses of this group. Using a similar probe strategy, we have followed the evolution of murine leukemia viruses during spontaneous leukemogenesis in AKR mice. We have found that the final leukemogenic (MCF) virus is a recombinant of three different endogenous parents; an ecotropic virus, a polytropic virus that directs the gp70 region of env, and a xenotropic virus (identified as the inducible element Bxv-1) that directs the LTR. In addition to the recombinations, all such viruses also have a reduplication of the enhancer region of the LTR, compared to the endogenous parent. MCF viruses are created by these three genetic changes, which occur in a reproducible fashion and appear in the thymus between 10 and 14 weeks of age.