Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

The lactate dehydrogenase-elevating virus capsid protein is a nuclear-cytoplasmic protein.

Arteriviruses replicate in the cytoplasm and do not require the nucleus function for virus multiplication in vitro. However, nucleocapsid (N) protein of two arteriviruses, porcine reproductive respiratory syndrome virus and equine arteritis virus, has been observed to localize in the nucleus and nucleolus of virus-infected and N-gene-transfected cells in addition to their normal cytoplasmic distribution. In the present study, the N protein of lactate dehydrogenase-elevating virus (LDV) of mice was examined for nuclear localization. The subcellular localization of LDV-N was determined by tagging N with enhanced green fluorescence protein (EGFP) at the N- and C-terminus. Both N-EGFP and EGFP-N fusion proteins localized to the nucleus and nucleolus of gene-transfected cells. Labeled N also accumulated in the perinuclear region, the site of virus replication. The LDV-N sequence contains a putative 'pat4'-type nuclear localization signal (NLS) consisting of 38-KKKK. To determine its functional significance, a series of deletion constructs of N were generated and individually expressed in cells. The results showed that the 'pat4' NLS was essential for nuclear translocation. In addition, the LDV-N interacted with the importin-alpha and -beta proteins, suggesting that the LDV-N nuclear localization may occur via the importin-mediated nuclear transport pathway. These results provide further evidence for the nuclear localization of N as a common feature within the arteriviruses.

Characterization of lactate dehydrogenase-elevating virus ORF6 protein expressed by recombinant baculoviruses.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity and can replicate only in a subset of mouse primary macrophages in vitro. Because it is difficult to grow and purify sufficient quantities of LDV virions from the primary macrophages, it has been difficult to further characterize LDV envelope proteins. A few expression systems have been reported for structural analysis of the nonglycosylated envelope protein M/VP-2, however, very few studies of the antigenicity of M/VP-2 have been reported. We cloned and expressed the ORF6 gene, which encodes the M/VP-2, as a fusion protein with a polyhistidine metal-binding tag (6 x His-tag) in Autographa californica nuclear polyhedrosis virus (baculovirus) under the control of the polyhedrin promoter. In Western blotting analysis, the expressed protein was similar in size to the native M/VP-2 plus 6 x His-tag. The usefulness of the baculovirus-expressed LDV ORF6 protein for analysis of the immunogenicity of LDV M/VP-2 was discussed.

Detection of lactate dehydrogenase-elevating virus by use of a fluorogenic nuclease reverse transcriptase-polymerase chain reaction assay.

Lactate dehydrogenase-elevating virus (LDEV) induces persistent infections in laboratory mice, alters in vivo physiology, and is a common contaminant of biological materials such as transplantable tumor cell lines. The fluorogenic nuclease reverse transcriptase polymerase chain reaction (fnRT-PCR) assay combines RT-PCR analysis with an internal fluorogenic hybridization probe, thereby eliminating post-PCR processing and potentially enhancing specificity. An fnRT-PCR assay specific for LDEV was therefore developed by targeting primer and probe sequences to a unique region of the LDEV nucleocapsid (VP1) gene. Using the LDEV fnRT-PCR assay, we detected only LDEV and did not detect other RNA viruses that are capable of naturally infecting rodents. Using this assay, we detected as little as 10 fg of LDEV RNA; the assay was 10-fold less sensitive when directly compared with the mouse bioassay (measurement of serum LD after inoculation), without the problematic false-positive serum LD enzyme elevations associated with the mouse bioassay. Using the fnRT-PCR assay, we also were able to detect viral RNA in numerous tissues and in feces collected from experimentally inoculated C3H/HeN mice, but we did not detect any viral RNA in similar samples collected from age- and strain-matched mock-infected mice. Finally, using the fnRT-PCR assay, we were able to detect LDEV RNA in biological samples that had previously been determined to be contaminated with LDEV by use of the mouse bioassay and an RT-PCR assay at another laboratory. In conclusion, the LDEV fnRT-PCR assay is a potentially high-throughput diagnostic assay for detection of LDEV in mice and contaminated biological materials.

Production of virus-specific antiserum corresponding to sequences in the lactate dehydrogenase-elevating virus (LDV) ORF6 protein.

The elucidation of the antigenic structure of the envelope proteins of Arteriviridae which includes lactate dehydrogenase-elevating virus (LDV) will provide further understanding of a mechanism of strict host cell specificity. To analyze the linkage between LDV envelope proteins, M/VP-2 and VP-3, which may play an important role in viral infectivity, we generated specific antibody against M/VP-2 that has not been reported in previous studies. A synthetic polypeptide corresponding to the C-terminal region of LDV strain C (LDV-C) ORF6, which encodes M/VP-2, was chemically synthesized and coupled to keyhole limpet hemocyanin (KLH). The peptide was immunogenic in rabbits and induced antibody specific for viral protein. Western blotting and immunofluorescence analysis of virion M/VP-2 in infected macrophages showed that the antibody was able to react specifically with authentic virion protein. The immunoreactive antibody against LDV M/VP-2 described in this study will be useful for further studies of the specific roles of the envelope proteins in arterivirus assembly and infectivity.

Analysis of protein expression by mammalian cell lines stably expressing lactate dehydrogenase-elevating virus ORF 5 and ORF 6 proteins.

Lactate dehydrogenase-elevating virus (LDV) has a strict species-specificity. Because only a subset of mouse primary macrophages have been identified that can support LDV replication in vitro, the precise molecular mechanism of viral entry and replication remains unclear. To analyze the LDV envelope proteins, which probably mediate viral attachment to the host cell, we developed a mammalian system for stable co-expression of LDV open reading frame (ORF) 5- and ORF 6-encoded proteins (ORF 5 and ORF 6 proteins), which correspond to envelope VP-3 and M/VP-2, respectively, and compared these expressed proteins to the native ones. Western blotting analysis combined with N-glycanase digestion revealed that ORF 5 and ORF 6 proteins were similar in size to native VP-3 and M/VP-2, and that ORF 5 protein was N-glycosylated, like the native VP-3. Immunofluorescence microscopy revealed that both ORF 5 and ORF 6 proteins were distributed throughout the cytoplasm and were colocalized in most cells. Moreover, ORF 5 protein was localized both in the perinuclear region and the Golgi complex and transported to the cell surface. This mammalian expression system in which the exogenously expressed proteins closely resemble the native proteins will provide the experimental basis for further studies of the interactions between LDV envelope proteins and host cells.

Porcine reproductive and respiratory syndrome virus: origin hypothesis.

Porcine reproductive and respiratory syndrome is a serious swine disease that appeared suddenly in the midwestern United States and central Europe approximately 14 years ago; the disease has now spread worldwide. In North America and Europe, the syndrome is caused by two genotypes of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus whose genomes diverge by approximately 40%. My hypothesis, which explains the origin and evolution of the two distinct PRRSV genotypes, is that a mutant of a closely related arterivirus of mice (lactate dehydrogenase-elevating virus) infected wild boars in central Europe. These wild boars functioned as intermediate hosts and spread the virus to North Carolina in imported, infected European wild boars in 1912; the virus then evolved independently on the two continents in the prevalent wild hog populations for approximately 70 years until independently entering the domestic pig population.

Construction of chimeric arteriviruses reveals that the ectodomain of the major glycoprotein is not the main determinant of equine arteritis virus tropism in cell culture.

The recent development of arterivirus full-length cDNA clones makes possible the construction of chimeric arteriviruses for fundamental and applied studies. Using an equine arteritis virus (EAV) infectious cDNA clone, we have engineered chimeras in which the ectodomains of the two major envelope proteins, the glycoprotein GP(5) and the membrane protein M, were replaced by sequences from envelope proteins of related and unrelated RNA viruses. Using immunofluorescence microscopy, we monitored the transport of the hybrid GP(5) and M proteins to the Golgi complex, which depends on their heterodimerization and is a prerequisite for virus assembly. The only viable chimeras were those containing the GP(5) ectodomain from the porcine (PRRSV) or mouse (LDV) arteriviruses, which are both considerably smaller than the corresponding sequence of EAV. Although the two viable GP(5) chimeras were attenuated, they were still able to infect baby hamster kidney (BHK-21) and rabbit kidney (RK-13) cells. These cells can be infected by EAV, but not by either PRRSV or LDV. This implies that the ectodomain of the major glycoprotein GP(5), which has been postulated to be involved in receptor recognition, is not the main determinant of EAV tropism in cell culture.

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.

Analysis of the Fv1 alleles involved in the susceptibility of mice to lactate dehydrogenase-elevating virus-induced polioencephalomyelitis.

Development of polioencephalomyelitis in mice infected with lactate dehydrogenase-elevating virus (LDV) requires expression of N-tropic ecotropic MuLV retroviruses. 129/Sv mice are resistant to N-tropic MuLV expression and therefore do not develop LDV-induced polioencephalomyelitis. The Fv1 gene determines the susceptibility to retrovirus replication. We sequenced the open reading frame of the Fv1nr allele of 129/Sv mice. It differs by only one nucleotide, modifying one amino acid in the encoded protein, from the Fv1n allele of susceptible AKR and C58 animals. We excluded that the resistance of 129/Sv mice to LDV-induced polioencephalomyelitis resulted from the absence of endogenous N-tropic retrovirus, by infecting (129/Sv x C58/J) F1 animals. Therefore it is possible that the amino acid that defines the Fv1nr allele is responsible for resistance of 129/Sv mice to N-tropic MuLV expression and to LDV-induced polioencephalomyelitis.

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.

Regulation of immune complexes during infection of mice with lactate dehydrogenase-elevating virus: studies with interferon-gamma gene knockout and tolerant mice.

Mice persistently infected with lactate dehydrogenase-elevating virus (LDV) develop circulating IgG-containing hydrophobic immune complexes, with a molecular mass of 150 to 300 kd, which bind to the surfaces of high-capacity enzyme-linked immunosorbent assay (ELISA) plates. LDV infection also stimulates polyclonal B-cell activation and autoimmunity. For this study, interferon-gamma gene knockout (GKO) mice were utilized to study circulating immune complexes and other parameters of LDV infection. The kinetics of LDV viremia, formation of plasma IgG anti-LDV antibodies, and LDV replication in the spleen and liver were essentially normal in GKO mice. Polyclonal activation of B cells, as reflected by increased total plasma IgG concentration during LDV infection, was found to be intact in GKO mice, although at a lower magnitude than in control mice. The plasma concentration of IgG-containing hydrophobic immune complexes was reduced about 75% in LDV-infected GKO mice relative to normal LDV-infected controls. Allogeneic tissue responses were also found to be reduced in LDV-infected GKO mice relative to those in normal LDV-infected controls. These results dissociate specific anti-LDV immunity from formation of hydrophobic immune complexes, show that the IgG anti-LDV response as well as LDV replication in the spleen and liver are insensitive to physiological levels of interferon (IFN)-gamma, and suggest that IgG-containing immune complexes stimulated by LDV infection are a marker for autoimmunity.

High-frequency homologous genetic recombination of an arterivirus, lactate dehydrogenase-elevating virus, in mice and evolution of neuropathogenic variants.

On the basis of genome nucleotide differences between a nonneuropathogenic and a neuropathogenic lactate dehydrogenase-elevating virus (LDV) quasispecies (LDV-P and LDV-C, respectively), we have designed sets of primers for polymerase chain reaction (PCR) amplification that can detect recombinants between them in a 1276-nt-long segment ranging from ORF 5 to ORF 7. Mice were infected with large amounts of both LDVs and bled at various times postinfection (p.i.). RNA was extracted from plasma samples and reverse transcribed and the first-strand products were PCR amplified with four sets of sense and antisense primers that discriminate between parental (P/P and C/C) and recombinant (P/C and C/P) genomic segments. Both P/C and C/P recombinants were detected in plasma from six different mice at 1 day p.i. No recombinant products were generated with in vitro mixtures of LDV-P and LDV-C. End-point dilution experiments indicated that the generation of P/C and C/P recombinants varied between mice but that in some mice the frequency of recombination in the 1276-nt-long genome segment was as high as 5%. Sequence analyses of clones of some recombinants indicated that recombination had occurred at 26- to 43-nt-long stretches of homology between the LDV-P and the LDV-C genomes. Sequence analyses of the 3157-nt-long 3' end of the genomes of the neuropathogenic LDV-v and of a newly discovered nonneuropathogenic quasispecies, LDV-vx, showed that LDV-v is a natural recombinant of LDV-vx that has specifically acquired by a double recombination about 400 nt of the 5' end of ORF 5 of the neuropathogenic LDV-C and thereby the unique properties of LDV-C, neuropathogenicity and high sensitivity to antibody neutralization. In dual infections of mice with LDV-P and LDV-C all genetic recombinants, like the LDV-C parent itself, had been lost by 7 days p.i., and only LDV-P persisted. The results further support the view that LDV-P and LDV-vx have evolved to a highly stable relationship with their host, the mouse.

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.

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.