Zongyu Chen

Neonatal Infection of Mice With Lactate Dehydrogenase-elevating Virus Results in Suppression of Humoral Antiviral Immune Response but does not Alter the Course of Viraemia or the Polyclonal Activation of B Cells and Immune Complex Formation

Neonatal infection of FVB mice with lactate dehydrogenase-elevating virus (LDV) prevented the normal formation of anti-LDV antibodies observed in mice infected at 5 days of age or older. Even 22 weeks post-infection, the concentration of circulating anti-LDV antibodies in neonatally infected mice was insignificant. However, the time course and level of persistent viraemia were the same in neonatally infected mice lacking anti-LDV antibodies as in mice infected at 5 or 15 days of age which developed normal antiviral immune responses. The results support the view that LDV replication in mice is unaffected by antiviral immune responses and instead is primarily dependent on the rate of regeneration of LDV-permissive macrophages. This view is further supported by the following findings. Treatment of mice with cyclophosphamide or dexamethasone, which are known to increase plasma LDV levels, increased the proportion of LDV-permissive macrophages in the peritoneum. Injection of mice with interleukin-3, which is known to stimulate macrophage development, increased plasma LDV levels in persistently infected mice 10- to 100-fold. During the first month of age when mice possess a higher proportion of LDV-permissive macrophages than older mice and peritoneal macrophages exhibit self-sustained growth, the persistent plasma LDV titres were also 10- to 100-fold higher than in older mice. The polyclonal activation of B cells induced by LDV that results in a permanent elevation of IgG2a or IgG2b in the circulation, and the formation of 180K to 300K immune complexes containing IgG2a or IgG2b were also the same in neonatally infected mice and mice infected 5 or 15 days after birth. Thus, the polyclonal activation of B cells occurs in the absence of an antiviral humoral immune response and the immune complexes do not contain anti-LDV antibodies. The immune complexes probably consist of autoantibodies formed in the course of the polyclonal activation of B cells and their cellular antigens.

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.

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.

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.

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.

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

Summary. 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.

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

Abstract 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 hemorhagic 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.

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.

Mammalian mRNAs encoding protein closely related to ubiquitin-conjugating enzyme encoded by yeast DNA repair gene RAD6

A clone of about 1 kb has been isolated from a human brain cDNA library. The clone possesses a 151 amino acid open reading frame that exhibits 72% amino acid identity with the E2 ubiquitin-conjugating enzyme encoded by the RAD6 gene of Saccharomyces cerevisiae. A 90% amino acid identity was observed in a central sequence surrounding a cysteine, which most likely contributes the sulfhydryl group involved in the formation of the ubiquitin-E2 thiolester linkage. Northern hybridization analyses have identified a poly(A)-containing mRNA of about 1 kb encoding the E2-like sequence in human CEM lymphoblastoid and HeLa cells, Novikoff rat hepatoma cells and S49 mouse leukemia cells. Southern hybridization analyses indicate the presence of a single gene encoding this sequence in both human cell lines, but of two or more related genes in the rodent cell lines.