The primary neutralization epitope of porcine respiratory and reproductive syndrome virus strain VR-2332 is located in the middle of the GP5 ectodomain.

Pigs infected with porcine respiratory and reproductive syndrome virus (PRRSV) strain VR-2332 were found to generate high levels of antibodies (Abs) that bound in an indirect ELISA to synthetic peptides representing segments of the primary envelope glycoprotein (GP5) ectodomain of this virus. Use of overlapping GP5 ectodomain peptides of various length indicated that the epitope recognized by the Abs was located in the middle of the ectodomain (amino acids 36-52), in the same relative segment that contains the single linear neutralization epitope of the closely related mouse arterivirus, lactate dehydrogenase-elevating virus (LDV). The VR-2332 GP5 segment exhibits 77% amino acid homology with the corresponding GP5 ectodomain segments of both the European PRRSV strain Lelystad virus (LV) and LDV. This explains some observed crossreaction between the pig Abs and neutralizing anti-LDV monoclonal Abs with peptides representing the GP5 ectodomains of VR-2332, LV and LDV. The GP5 binding Abs of pigs seem to be the primary PRRSV neutralizing Abs, since the well timed appearance in sera of all VR-2332 infected pigs of GP5 peptide binding Abs correlated 100% with the appearance of neutralizing Abs and earlier studies indicated that GP5 of PRRSV, like that of other arteriviruses, contains the main neutralization epitope of PRRSV. In addition, one neutralizing anti-LDV monoclonal Ab that is specific for the GP5 ectodomain epitope of LDV also strongly neutralized both PRRSV strains, VR-2332 and LV. The PRRSV GP5 epitope is associated with an N-glycan that is conserved in both PRRSV genotypes and all LDV isolates. This N-glycan may impede the humoral immune control of PRRSV in infected pigs and might be responsible for the low immunogenicity of PRRSV when injected into mice.

Replication competition between lactate dehydrogenase-elevating virus quasispecies in mice. Implications for quasispecies selection and evolution.

The common quasispecies of lactate dehydrogenase-elevating virus (LDV), LDV-P and LDV-vx, are highly resistant to the humoral host immune response because the single neutralization epitope on the ectodomain of the primary envelope glycoprotein, VP-3P, carries three large N-glycans. Two laboratory mutants, LDV-C and LDV-v, have lost two of the N-glycans on the VP-3P ectodomain, thereby gaining neuropathogenicity for AKR/C58 mice but at the same time, becoming susceptible to the humoral immune response of the host. In attempts to further assess the origins and evolution of these LDVs we have determined their competitiveness by monitoring their fate in mixed infections of wild type, SCID, nude, and cyclophosphamide-treated mice by reverse transcription/polymerase chain reaction assays that distinguish between them. In mixed infections with LDV-P and LDV-vx, LDV-C and LDV-v became rapidly lost even when present initially in large excess over the former. In mixed infections of mice unable to generate neutralizing antibodies, LDV-C and LDV-v also became replaced by LDV-P and LDV-vx as predominant quasispecies but more slowly than in immunocompetent mice. The results indicate that the humoral immune response plays an important role in the displacement of LDV-C and LDV-v by LDV-P and LDV-vx but that in addition, LDV-C and LDV-v possess an impaired ability to compete with LDV-P and LDV-vx in the productive infection of the subpopulation of macrophages that represents the host for all these LDVs. In addition, LDV-v outcompeted LDV-C in mixed infections and the same was the case for neutralization escape mutants of LDV-v and LDV-C which had regained all three N-glycosylation sites on the VP-3P ectodomain. Thus a hierarchy exists in replication fitness: LDV-P/LDV-vx>LDV-v>LDV-C, which is unrelated to the number of N-glycans on the VP-3P ectodomain. The implications of the results in relation to the evolution and selection of the LDV-quasispecies is discussed. LDV-P and LDV-vx are genetically highly stable and thus seem to have achieved evolutionary stasis with optimum ability to establish viremic persistent infections of mice that are unimpeded by the host immune responses.

An ORF-2a protein is not present at a significant level in virions of the arterivirus lactate dehydrogenase-elevating virus.

MRNA2 of the arteriviruses lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV) encodes two proteins that are read in different frames, an about 26 kDa minor envelope glycoprotein and an about 8 kDa protein that lacks N-glycosylation sites and a signal peptide, but possesses a central hydrophobic segment. Recent studies have shown that both proteins of EAV are translated from mRNA 2 in EAV infected BHK cells, that the 8 kDa protein is membrane associated and that small amounts of it are recovered in purified virions (Snijder, E.J., van Tol, H., Pederson, K.W., Raamsman, M.J.B., de Vries, A.A.F., 1999. Identification of a novel structural protein of arteriviruses. J. Virol. 73, 6335-6345). The authors concluded that the 8 kDa protein is another arterivirus envelope protein and designated it E protein. However, we have not detected a significant level of an 8 kDa protein in LDV virions and thus conclude that it is not a structural virion component.

Complexity of the single linear neutralization epitope of the mouse arterivirus lactate dehydrogenase-elevating virus.

Results from indirect ELISAs using synthetic peptides of various length that represent segments of the ectodomain of the envelope glycoprotein, VP-3P, of lactate dehydrogenase-elevating virus (LDV) showed that the primary neutralization epitope of LDV is located in a short linear hydrophilic segment in the center of the ectodomain. The epitope becomes slightly altered by amino acid substitutions in the ectodomain and inactivation of virions by various treatments. Neutralizing anti-VP-3P antibodies (Abs) to the epitope interact with the synthetic peptides only if they possess a certain conformation. When the peptides were immobilized on ELISA plates, neutralizing mAbs elicited to inactivated LDV and neutralizing Abs from infected mice bound best to the peptides that consisted of the full-length, 30-amino-acid-long ectodomain. The Abs bound poorly, if at all, to most of the shorter peptides when immobilized, whether truncated at the N- or C-end, but when in solution the same peptides strongly inhibited the binding of the Abs to immobilized full-length peptides. Thus, a conformation of the epitope required for Ab binding and (or) its steric accessibility were lost upon immobilization of the shorter peptides on ELISA plates. Abs raised in mice to peptide-bovine serum albumin conjugates reacted only with immobilized peptides in the indirect ELISA and failed to neutralize LDV. The neutralization epitope of the common LDV quasispecies, LDV-P and LDV-vx, is flanked by N-glycans that block the immunogenicity of the epitope and the neutralization of these LDVs. Abs to a second weakly immunogenic and probably discontinuous epitope appear in LDV infected mice about 1 month postinfection.

Isolation of lactate dehydrogenase-elevating viruses from wild house mice and their biological and molecular characterization.

Lactate dehydrogenase-elevating virus (LDV) was first identified as a contaminant of transplantable mouse tumors that were passaged in laboratory mice. It has been assumed that these LDVs originated from LDVs endemic in wild house mouse populations. In order to test this hypothesis and to explore the relationships between LDVs from wild house mice among each other and to those isolated from laboratory mice, we have isolated LDVs from wild house mice and determined their biological and molecular properties. We have screened for LDV tissues of 243 wild house mice that had been caught in various regions of North, Central and South America between 1985 and 1994. We were able to isolate LDVs from the tissues of four mice, three had been caught in Baltimore, MD and one in Montana. We demonstrate that the phenotypic properties (ability to establish a long-term viremic infection, low immunogenicity of the neutralization epitope, high resistance to antibody neutralization and lack of neuropathogenicity) of the four wild house mouse LDVs are identical to those of the primary LDVs isolated from transplantable tumors (LDV-P and LDV-vx), which are distinct from those of the neuropathogenic LDV-C. Furthermore, ORF 5 and ORF 2 and their protein products (the primary envelope glycoprotein VP-3P, and the minor envelope glycoprotein, respectively) of the wild house mouse LDVs were found to be closely related to those of LDV-P and LDV-vx. The LDVs caught in Baltimore, MD were especially closely related to each other, whereas the LDV isolated in Montana was more distantly related, indicating that it had evolved independently. The ectodomain of VP-3P of all four wild house mouse LDVs, like those of LDV-P and LDV-vx, possess the same three polylactosaminoglycan chains, two of which are lacking in the VP-3P ectodomain of LDV-C. These results further strengthen the conclusion that the three polylactosaminoglycan chains are the primary determinants of the phenotypic properties of LDV-P/vx.

Neuropathogenicity and sensitivity to antibody neutralization of lactate dehydrogenase-elevating virus are determined by polylactosaminoglycan chains on the primary envelope glycoprotein.

Common strains of lactate dehydrogenase-elevating virus (LDV, an arterivirus), such as LDV-P and LDV-vx, are highly resistant to antibody neutralization and invariably establish a viremic, persistent, yet asymptomatic, infection in mice. Other LDV strains, LDV-C and LDV-v, have been identified that, in contrast, are highly susceptible to antibody neutralization and are incapable of a high viremic persistent infection, but at the same time have gained the ability to cause paralytic disease in immunosuppressed C58 and AKR mice. Our present results further indicate that these phenotypic differences represent linked properties that correlate with the number of N-glycosylation sites associated with the single neutralization epitope on the short ectodomain of the primary envelope glycoprotein, VP-3P. The VP-3P ectodomains of LDV-P/vx possess three N-glycosylation sites, whereas those of LDV-C/v lack the two N-terminal sites. We have now isolated four independent neutralization escape variants of neuropathogenic LDV-C and LDV-v on the basis of their ability to establish a high viremic persistent infection in mice. The VP-3P ectodomains of all four variants had specifically regained two N-glycosylation sites concomitant with decreased immunogenicity of the neutralization eptitope and decreased sensitivity to antibody neutralization as well as loss of neuropathogenicity.

Selective antibody neutralization prevents neuropathogenic lactate dehydrogenase-elevating virus from causing paralytic disease in immunocompetent mice.

Neuropathogenic lactate dehydrogenase-elevating viruses (LDV) cytocidally infect anterior horn neurons in C58 and AKR mice via interaction with endogenous murine retroviruses to cause a paralytic disease, age-dependent poliomyelitis (ADPM). The induction of ADPM requires a suppressed host immune system as a result of old age, genetic defects (such as nude mice) or any immunosuppressive treatment. Previous results have shown that the infection of anterior horn neurons by neuropathogenic LDV isolates and the subsequent development of ADPM are prevented by anti-LDV antibodies either induced actively during infection or when passively administered. However, the mechanism of protection was unclear since both neutralizing and non-neutralizing polyclonal antibodies seemed protective, whereas only neutralizing monoclonal antibodies were protective. Furthermore, the protection of motor neurons from infection occurred in the absence of any apparent effect on LDV replication in a subpopulation of macrophages known to be the primary permissive host cells. These paradoxes have now been resolved. We have recently reported that the neuropathogenic LDV isolates contain both neuropathogenic and non-neuropathogenic quasispecies that differ in their ability to establish a high viremia persistent infection. Using biological clones of both neuropathogenic and non-neuropathogenic quasispecies, we now demonstrate that both replicate in the same subpopulation of permissive macrophages, but that the neuropathogenic quasispecies are about 100 times more susceptible to in vitro antibody neutralization than the non-neuropathogenic ones, and that antibodies that neutralize the neuropathogenic but not the non-neuropathogenic quasispecies develop as soon as 7 days after infection with neuropathogenic LDVs and selectively suppress the replication of the neuropathogenic LDVs in vivo in FVB, BALB/c, C57 BL/6 and C58 mice. The previously observed lack of neutralizing effect of early polyclonal anti-LDV antibodies and the apparent ineffective antibody control of LDV replication in macrophages were due to outgrowth of the non-neuropathogenic quasispecies that are also present in the neuropathogenic LDV inoculum and are highly resistant to antibody neutralization. Using cloned neuropathogenic LDV quasispecies, we demonstrate a clear relationship in the development of neutralizing antibodies, replication suppression of the neuropathogenic LDVs and the prevention of ADPM in C58 mice. Our results therefore establish an inseparable relationship between the neuron-protective effect of an antibody and its neutralization of the neuropathogenic LDV quasispecies and explain why neuropathogenic LDVs cause paralytic disease only in immunosuppressed mice.

Lactate dehydrogenase-elevating virus variants: cosegregation of neuropathogenicity and impaired capability for high viremic persistent infection.

Neuropathogenic isolates of lactate dehydrogenase virus (LDV) differ from non-neuropathogenic isolates in their unique ability to cause a paralytic disease (age-dependent poliomyelitis, ADPM) in immunosuppressed C58 and AKR mice by cytocidally infecting their anterior horn neurons. We have recently reported that an original neuropathogenic LDV isolate, LDV-C-BR, contained a low level of a coexisting non-neuropathogenic LDV which, in a mixed infection of mice, rapidly outcompeted the former resulting in apparent loss of neuropathogenicity of the reisolated LDV. This correlated with an impaired ability of the neuropathogenic LDV to establish a viremic persistent infection. In the present study we identified the presence of three different quasispecies in another original neuropathogenic LDV by sequence analysis of cDNA clones of ORF 5 (encoding the primary envelope glycoprotein VP-3P) obtained from the isolate. Successful development of differential reverse transcription-polymerase chain reaction assays allowed us to biologically clone all three quasispecies through repeated end point dilutions. Only one of the quasispecies (LDV-v) was neuropathogenic. The other two, LDV-vP (probably the same as LDV-P) and LDV-vx (a novel LDV quasispecies that had not been previously identified), were non-neuropathogenic and found to be the common LDV quasispecies associated with almost all LDVs originally isolated from mice carrying various other transplantable tumors. The neuropathogenic LDV-v became selectively amplified in the spinal cords of paralyzed mice, but possessed an impaired ability to establish a persistent viremic infection and was rapidly out-competed by LDV-vP and LDV-vx in mixed infections, just as reported previously for LDV-C-BR. The results further support our hypothesis that neuropathogenicity and impaired capability for viremic persistence of LDV are determined by the same molecular feature. The only consistent and biologically relevant molecular difference we have observed between neuropathogenic and non-neuropathogenic LDVs is the number of polylactosaminoglycan chains associated with the ectodomain of VP-3P.

Neuropathogenicity and susceptibility to immune response are interdependent properties of lactate dehydrogenase-elevating virus (LDV) and correlate with the number of N-linked polylactosaminoglycan chains on the ectodomain of the primary envelope glycoprotein.

We have developed differential RT-PCR methods to distinguish different isolates of LDV and have purified several quasispecies by repeated end point dilution in mice. They fall into two groups, each possessing two or more members. Group A viruses are non-neuropathogenic, highly resistant to in vitro neutralization by antibodies and efficient in establishment of a life-long, persistently viremic infection in mice despite a detectable immune response. Group B viruses, on the other hand, are neuropathogenic, much more sensitive to antibody neutralization and have an impaired ability to establish a high viremia persistent infection in immune competent mice. These properties seem to be interdependent and correlate with the number of N-glycosylation sites on the short (about 30 amino acid long) ectodomain of the primary envelope glycoprotein, VP-3P, which probably is part of the attachment site for the LDV receptor on permissive cells and harbors an epitope(s) reacting with neutralizing antibodies. Group A viruses possess three closely spaced N-linked polylactosaminoglycan chains, whereas group B viruses lack the two N-terminal ones. We postulate that lack of these polylactosaminoglycan chains endows group B viruses with the ability to interact with a receptor on anterior horn neurons resulting in neuropathogenesis. At the same time, it increases an interaction with neutralizing antibodies thus impeding the infection of macrophages newly generated during the persistent phase of infection which is essential for the continued rounds of replication of the virus.

Lactate dehydrogenase-elevating virus (LDV): lifelong coexistence of virus and LDV-specific immunity.

Viruses have developed various strategies to coexist with vertebrate hosts. Lactate dehydrogenase-elevating virus (LDV) is a highly cytopathic virus exhibiting an extraordinary rate of replication; LDV nevertheless establishes a persistent infection without harming the host. The cytotoxic and helper T cell responses to LDV were monitored in mice with different genetic backgrounds. LDV-specific cytotoxic and helper T cells were found in all strains tested. These responses persisted for at least up to 250 days despite high levels of LDV in the blood. Thus, the cytopathic LDV induces and maintains an inefficient immune response that is not exhausted. LDV infection in mice reveals a special type of host-virus equilibrium where LDV quickly establishes persistence despite continuously induced LDV-specific helper and cytotoxic T cell responses, which apparently are too slow to control the highly cytopathic and extremely fast replicating virus.

Detection of lactate dehydrogenase-elevating virus in transplantable mouse tumors by biological assay and RT-PCR assays and its removal from the tumor cell.

It is known that lactate dehydrogenase-elevating virus (LDV) of mice is a common contaminant of transplantable tumors of both murine and human origin. It is imperative that tumors that are maintained by transplantation in mice are examined for LDV and freed of the virus, when present, before use in experimental studies, because an LDV infection of mice exerts considerable effects on lymphoid cell populations and cytokine production and other effects. Methods for LDV detection are described using a biological assay and reverse transcription (RT)-polymerase chain reaction (PCR) technology and their application is illustrated. A differential RT-PCR method that distinguishes between three quasispecies of LDV is also described and applied to an examination of LDVs isolated from a number of different tumors. Each of the LDV isolates was found to contain at least two different quasispecies, generally in different concentrations.

Coexistence in lactate dehydrogenase-elevating virus pools of variants that differ in neuropathogenicity and ability to establish a persistent infection.

Neuropathogenic isolates of lactate dehydrogenase-elevating virus (LDV) differ from nonneuropathogenic isolates in their unique ability to infect anterior horn neurons of immunosuppressed C58 and AKR mice and cause paralytic disease (age-dependent poliomyelitis [ADPM]). However, we and others have found that neuropathogenic LDVs fail to retain their neuropathogenicity during persistent infections of both ADPM-susceptible and nonsusceptible mice. On the basis of a segment in open reading frame 2 that differs about 60% between the neuropathogenic LDV-C and the nonneuropathogenic LDV-P, we have developed a reverse transcription-PCR assay that distinguishes between the genomes of the two LDVs and detects as little as 10 50% infectious doses (ID50) of LDV. With this assay, we found that LDV-P and LDV-C coexist in most available pools of LDV-C and LDV-P. For example, various plasma pools of 10(9.5) ID50 of LDV-C/ml contained about 10(5) ID50 of LDV-P/ml. Injection of such an LDV-C pool into mice of various strains resulted in the rapid displacement in the circulation of LDV-C by LDV-P as the predominant LDV, but LDV-C also persisted in the mice at a low level along with LDV-P. We have freed LDV-C of LDV-P by endpoint dilution (LDV-C-EPD). LDV-C-EPD infected mice as efficiently as did LDV-P, but its level of viremia during the persistent phase was only 1/10,000 that observed for LDV-P. LDV-permissive macrophages accumulated and supported the efficient replication of superinfecting LDV-P. Therefore, although neuropathogenic LDVs possess the unique ability to infect anterior horn neurons of ADPM-susceptible mice, they exhibit a reduced ability to establish a persistent infection in peripheral tissues of mice regardless of the strain. The specific suppression of LDV-C replication in persistently infected mice is probably due in part to a more efficient neutralization of LDV-C than LDV-P by antibodies to the primary envelope glycoprotein, VP-3P. Both neuropathogenicity and the higher sensitivity to antibody neutralization correlated with the absence of two of three N-linked polylactosaminoglycan chains on the ca. 30-amino-acid ectodomain of VP-3P, which seems to carry the neutralization epitope(s) and forms part of the virus receptor attachment site.

ORF 3 of lactate dehydrogenase-elevating virus encodes a soluble, nonstructural, highly glycosylated, and antigenic protein.

Open reading frame (ORF) 3 of the genome of lactate dehydrogenase-elevating virus (LDV), strain P, was cloned into the plasmid pcDNAI/Amp and in vitro transcribed and translated. Translation of ORF 3 yielded a soluble protein of the expected size (about 21 kDa). When synthesized in the presence of endoplasmic reticulum (ER) membranes the resulting glycoprotein of about 36 kDa became associated with the membranes. However, disruption of the ER vesicles by incubation in carbonate buffer, pH 11.5, resulted in the release of the protein from the membranes. Hydrophobic moment analysis of the ORF 3 protein indicated the absence of any potential transmembrane segments, except for a N-terminal signal peptide, but no cleavage of the signal peptide was observed during membrane-associated in vitro synthesis. The ORF 3 protein elicited a strong antibody response in infected mice. The antibodies from infected mice as well as a monoclonal antibody specifically precipitated the in vitro-synthesized ORF 3 protein, but no protein from LDV virions. The overall results suggest that the ORF 3 protein is a nonstructural, highly glycosylated, and antigenic glycoprotein that is probably soluble and secreted or at most only weakly associated with membranes via the signal peptide.

Regulation of transplacental virus infection by developmental and immunological factors: studies with lactate dehydrogenase-elevating virus.

Placental and fetal infections with lactate dehydrogenase-elevating virus (LDV) were determined by virus titration, indirect fluorescence antibody (IFA), and in situ hybridization with cDNA probes. Experiments were designed to determine the effects of gestational age, timing of maternal LDV infection, and immunological (antibody and cytokine) factors on mouse placental and fetal LDV infection. Virus infection of the placenta was detected at high levels (almost all placentas infected) within 24 h post-maternal infection (p.m.i.), whereas fetal LDV infection was detected only at a low level by 24 h p.m.i. The percentage of fetuses becoming LDV infected progressively increased between 24 and 72 h p.m.i. When fetal infection was studied at 72 h p.m.i., earlier gestational ages (9-11 days) were associated with fetal resistance to infection, whereas between 12.5 and 15 days of gestation, virus infection was detected in 50-71% of fetuses. Maternal treatment with interferon-gamma (IFN-gamma) or anti-LDV monoclonal antibodies was associated with reduced rates of fetal, but not placental, LDV infection. These results demonstrate that both developmental and immunological factors are important in the regulation of transplacental LDV infection.

Membrane association of the C-terminal half of the open reading frame 1a protein of lactate dehydrogenase-elevating virus.

ORF 1a of lactate dehydrogenase-elevating virus, strain P (LDV-P), encodes a protein of 2206 amino acids. Eisenberg hydrophobic moment analysis of the protein predicted the presence of eleven transmembrane segments in the C-terminal half of the molecule (amino acids 980-1852) that flank the serine protease domain. cDNAs encoding ORF 1a protein segments encompassing transmembrane segments 5 to 11 and its amphipathic C-terminal end as well as the N-terminal 80 amino acids of the downstream ORF 1b protein were transcribed and the transcripts in vitro translated in the absence and presence of microsomal membranes. The synthesis of the protein products with putative transmembrane segments was enhanced by the presence of the microsomal membranes and the proteins became membrane associated. When synthesized in the absence of membranes they were recovered in the supernatant upon ultracentrifugation of the translation reaction mixtures, whereas they were recovered in the membrane pellet when synthesized in the presence of membranes. Furthermore, the latter proteins were not released from the membranes by disruption of the membrane vesicles in carbonate buffer, pH 11.5, and large portions of the proteins were resistant to digestion by trypsin, chymotrypsin and proteinase K. No N-glycosylation was observed and only little, if any, processing of the protein by the putative serine protease. The results indicate that the C-terminal half of the ORF 1a protein represents a non-glycosylated integral membrane protein. Potential modes of synthesis and function of the protein are discussed. In addition, the results showed that the synthesis of the ORF 1a protein was generally terminated at its termination codon, but that read-through into the ORF 1b gene occurred with low frequency.

Differential glycosylation of the ectodomain of the primary envelope glycoprotein of two strains of lactate dehydrogenase-elevating virus that differ in neuropathogenicity.

ORF 5 encoding the primary envelope glycoprotein, VP-3P, of a highly neuropathogenic isolate of lactate dehydrogenase-elevating virus (LDV-v) has been sequenced. It exhibits 92% nucleotide identity with the ORF 5 of an LDV isolate that lacks neuropathogenicity, LDV-P, and the amino acid identities of the predicted VP-3Ps of the two strains is 90%. Most striking, however, is the absence in the ectodomain of LDV-v VP-3P of two out of three potential N-glycosylation sites present in the ectodomain of VP-3P of LDV-P. The ectodomain of VP-3P has been implicated to play an important role in host receptor interaction. VP-3P of another neuropathogenic LDV strain, LDV-C, lacks the same two N-glycosylation sites (Godeny et al., 1993). In vitro transcription/translation of the ORFs 5 of LDV-P and LDV-v indicated that all three N-glycosylation sites in the ectodomain of LDV-P VP-3P became glycosylated when synthesized in the presence of microsomal membranes, whereas the glycosylation of the ORF 5 proteins of LDV-v and LDV-C was consistent with glycosylation at a single site. No other biological differences between the neuropathogenic and non-neuropathogenic strains have been detected. They replicate with equal efficiency in mice and in primary macrophage cultures.

Mouse hepatitis virus infection of mice causes long-term depletion of lactate dehydrogenase-elevating virus-permissive macrophages and T lymphocyte alterations.

Intraperitoneal injection of pathogen-free B10.A mice with mouse hepatitis virus (MHV)-A59 resulted in a short subclinical infection which was terminated by a rapid antiviral immune response. The infection resulted in a rapid, but transient, about 10-fold increase in the number of macrophages and total cells in the peritoneum of the mice. This increase was preceded by a complete depletion of the peritoneum of the subpopulation of macrophages that supports a productive infection by lactate dehydrogenase-elevating virus (LDV). The depletion of LDV-permissive macrophages was a long-term effect; at 50 days post-infection with MHV, the proportion of LDV-permissive macrophages in the peritoneum had reached only 20% of that observed in the peritoneum of uninfected mice, whereas the total number of macrophages in the peritoneum had returned to normal. Furthermore, MHV infection resulted in a long-term alteration in the proliferative response of spleen T cells to concanavalin A (ConA) and in their ability to produce interferon gamma; several times higher concentrations of ConA were required to induce a maximum proliferative response in spleen T cell populations from 5-week MHV-infected B10.A mice than in spleen T cell populations from infected companion mice but the former produced 5 times more interferon gamma than the T cells from uninfected mice.

Detection of related positive-strand RNA virus genomes by reverse transcription/polymerase chain reaction using degenerate primers for common replicase sequences.

A set of degenerate sense and antisense primers were designed on the basis of short segments with identical amino acids in the predicted ORF 1b replicase proteins of lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus, strain Lelystad virus (PRRSV-LV), which are members of a new group of positive-strand RNA viruses. Reverse transcription/polymerase chain reaction amplification using this set of degenerate primers yielded products of the expected size from the genomes of all three viruses. It also yielded a product of appropriate size from the genome of another strain of PRRSV (VR2332), the ORF 1b sequence of which is unknown, but the 3' end of the genome of which differs from that of the PRRSV-LV genome by about 50%. No products were generated from the genome of simian hemorrhagic fever virus (SHFV), another member of this virus group. However, an appropriate product was generated with a second set of degenerate primers which was designed from the same ORF 1b segments of LDV, EAV and PRRSV-LV as the first set but on the basis of human codon preferences. Sequence analysis showed that the amplified SHFV ORF 1b segment exhibited about 50% nucleotide identity with the corresponding segments of ORF 1b of LDV, EAV and PRRSV. The results show that these and other degenerate primer sets might be useful for the search of related viruses in other mammalian species.

Expression of ecotropic murine leukemia virus in the brains of C58/M, DBA2/J, and in utero-infected CE/J mice.

In C58 and AKR mice, endogenous N-tropic, ecotropic murine leukemia virus (MuLV) proviruses become activated in rare cells during embryogenesis. Resultant replication-competent progeny viruses then actively infect a large number of cells throughout the fetus, including cells in the developing central nervous system. By in situ hybridization analyses, we have assessed the presence of ecotropic MuLV RNA in the brains of C58 mice as a function of age. Only a few ecotropic MuLV-positive cells were observed in weanling mice, but the number of positive cells in the brain increased progressively with increasing age of the mice. Throughout the lives of the mice, the ecotropic MuLV RNA-positive cells were primarily located in well-defined white-matter tracts of the brain (commissura anterior, corpus callosum, fimbria hippocampi, optical tract, and striatum) and of the spinal cord. Cells of the subventricular zone also expressed ecotropic MuLV RNA, and in older mice a small number of positive cells were present in the grey matter. Infection of endogenous ecotropic MuLV provirus-less CE/J mice in utero with ecotropic MuLV clone AKR-623 resulted in the extensive infection of brain cells. The regional distribution of ecotropic MuLV RNA-containing cells was the same as observed in the brains of C58 mice, in which cells became infected by endogenously activated virus, but the number of positive cells was higher.