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.

Complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (LDV).

The apparently complete sequence of the RNA genome of the neurovirulent isolate of lactate dehydrogenase-elevating virus (LDV-C) has been determined. The LDV-C genome is at least 14,222 nucleotides in length and contains eight open reading frames (ORFs). ORF 1a, which encodes a protein of 242.8 kDa and is located at the 5' end of the genome, contains at least two putative papain-like cysteine protease domains, and one putative chymotrypsin-like serine protease domain. This ORF terminates with a UAG stop codon that can be bypassed if a -1 frameshift occurs. The frameshift region consists of a heptanucleotide "slippery" sequence, 5'-UUUAAAC-3', followed by a putative pseudoknot. ORF 1b encodes a protein of 155.4 kDa containing, in its N-terminal portion, an RNA-dependent RNA polymerase and an RNA helicase domain separated by a Zn finger domain. Another domain of unknown function that is also conserved in coronaviruses and toroviruses is located at the C-terminus of the ORF 1b product. Three cleavage sites in the ORF 1a polyprotein and three in the ORF 1b polyprotein were predicted for the chymotrypsin-like protease and tentatively delimit the mature nonstructural proteins of LDV. Six small, overlapping 3' ORFs (ORFs 2 through 7) encode proteins with calculated sizes of 25.8, 21.6, 19.8, 23.9, 18.9, and 12.3 kDa. ORF 7 encodes the virion nucleocapsid protein Vp-1, while ORF 6 encodes the nonglycosylated envelope protein Vp2. ORFs 5, 4, 3, and 2 each encode glycoproteins which may be virion envelope proteins. LDV is closely related to equine arteritis virus, Lelystad virus (LV), and simian hemorrhagic fever virus. These four viruses belong to a new group of positive-strand RNA viruses and are related to coronaviruses and toroviruses.

Genotypic variation among six isolates of lactate dehydrogenase-elevating virus.

In the present study, six lactate dehydrogenase-elevating virus (LDV) isolates obtained independently from inbred mice were compared by RNA oligonucleotide fingerprint analysis. The genome RNAs of four of the isolates gave unique fingerprint patterns. The patterns obtained for the other two isolates were similar, but not identical to one of the four unique patterns. These results indicate that more than one genotype of LDV exists and that virus isolates can be grouped by genotype. We have also demonstrated the presence of a 3'-terminal poly(A) tract by direct sequencing of 3'-32P-labelled LDV genome RNA. The presence of a 3'-terminal poly(A) tract distinguishes LDV from the members of the family Flaviviridae, which lack a 3'-poly(A), and justifies the current classification of LDV within the family Togaviridae.

Structure and chemical-physical characteristics of lactate dehydrogenase-elevating virus and its RNA.

Lactate dehydrogenase-elevating virus (LDV) was purified from culture fluid of infected primary cultures of various mouse tissues (peritoneal macrophage, bone marrow, spleen, and embryo) and from plasma of infected mice. Electron microscopy of negatively stained virus and positively stained sections of LDV revealed spherical particles of uniform size with a diameter of about 55 nm, containing an electron-dense core with a diameter of about 30 nm. During sample preparation the envelope had a tendency to slough off and disintegrate to form aggregates of various sizes and small hollow particles with a diameter of 8 to 14 nm. Two strains of LDV exhibited a density of 1.13 g/cm3 in isopycnic sucrose density gradient centrifugation whether propagated in primary cultures of the various mouse tissues or isolated from plasma of infected mice. A brief incubation of LDV in a solution containing 0.01% Nonidet P-40 or Triton X was sufficient to release the viral nucleocapsid, whereas a similar treatment had no effect on Sindbis virus. The nucleocapdis of LDV exhibited a density of 1.17 g/cm3, was devoid of phosphatidylcholine, and contained only the smallest of the viral proteins, VP-1, which had a molecular weight of about 15,000. The envelope contained two proteins. VP-2 with a molecular weight of 18,000 and a glycoprotein, VP-3, which migrated heterogenously (24,000 to 44,000 daltons) during polyacrylamide gel electrophoresis. When compared to the sedimentation rate of 29S rRNA, the RNAs of LDV and Sindbis virus sedimented at 48 and 45S, respectively, whether analyzed by zone sedimentation in sucrose density gradients containing low or high salt concentrations or denatured by treatment with formaldehyde. Our results indicate that LDV should be classified as a togavirus, but that LDV is sufficiently different from alpha and flaviviruses to be excluded from these groups.

Purification and electron microscopy of lactic dehydrogenase virus of mice.

Lactic dehydrogenase virus (LDV) was purified from infectious ascites fluid of mice bearing Ehrlich tumours using Sepharose gel filtration and rate zonal and isopycnic sedimentation. In glycerol gradients, a sedimentation coefficient of about 200S and a buoyant density of 1-14 g/ml was determined for the virus particle. Spherical particles with diam. between 62 and 80 nm, depending on the method of fixation and staining, have been identified electron microscopically. The virus particle consists of a spherical nucleocapsid wrapped into a double-layered envelope. The nucleocapsids, isolated by treatment with NP40 and purified by centrifuging on sucrose gradients had a sedimentation coefficient of 176S. Electron micrographs show spherical particles with a diam of 35 plus or minus nm. Classification of LDV as a member of the togaviridae family is discussed.