Persistence of lymphocytic choriomeningitis virus at very low levels in immune mice

Abstract

Lymphocytic choriomeningitis virus (LCMV), strain WE, is a non-cytopathic RNA virus that is highly adapted to its natural host, the mouse. Acute infection of adult mice leads to generalized virus spread, followed by cytotoxic T lymphocyte-mediated virus clearance below the detection levels of conventional assays within 2-3 weeks. Indirect evidence had suggested that virus or viral antigen might persist in the immune mouse. Here we demonstrate LCMV-WE persistence at low levels after infection with 10(2) or 10(6) plaque-forming units, shown as viral genome, viral antigen, and replicative virus using sensitive in vitro and in vivo assays. The finding that LCMV-WE persists in the face of apparently intact immune responses resembles the situation in some viral (hepatitis B and C, HIV) and bacterial (tuberculosis, leprosy) infections in humans; the results are relevant to the understanding not only of other murine and human persistent viral infections but also of protective immunological memory by "infection immunity."

Figure 1

Detection of LCMV-WE RNA in immune mice. (A) PCR amplification strategy with schematic representation of the S strand of LCMV, which encodes the glycoproteins (GP1 and GP2) and the nucleoprotein (NP) in opposite sense. The primers used are indicated. (B) RT-PCR on reverse-transcribed DNase-treated RNA extracted from spleen, kidney, and lung of two B6 mice (M7 and M12 in Table 3) infected 80 days previously with LCMV-WE (200 pfu i.v. for M7 and 2 × 10⁶ pfu i.v. for M12) using GP- and NP-specific primers as detailed in Material and Methods. Products were run on 1% agarose gels and were visualized with ethidium bromide. Product sizes are 907 bp for GP and 207 bp for NP. Controls in this experiment were no cDNA (lane 1) and RNA extracted from organs of an uninfected mouse (lanes 8–10). The positive control was cDNA template from MC57 cells infected with LCMV (multiplicity of infection 0.02) and was cultured for 48 h. (C) Partial sequence alignment (nucleotides 438–479) from amplified GP products (spleen isolates). Products were sequenced by using inner PCR primers (RC1/DM1) and were compared with LCMV-WE sequence derived from our viral stock (LCMV-WE-ZH) and from a plasmid frequently used in our laboratory, containing GP cDNA (GenBank accession number M22138). Positions of difference in nucleotides are indicated.

Figure 2

Detection of LCMV-antigen in different spleens and kidneys of LCMV-immune B6 mice by immunohistological analysis. Spleens and kidneys were harvested 80 days after infection with 2 × 10⁶ pfu LCMV-WE (C–E). Control sections from uninfected animals are shown (A and B). Frozen tissue sections were stained with VL-4, a rat anti-LCMV-NP monoclonal Ab (A–D). Paraffin sections were stained with a rabbit anti-LCMV serum [hyperimmune serum (His)] (E). (×400.)