Sequence analysis of the small RNA segment of guinea pig-passaged Pichinde virus variants.

Abstract

Abstract. The established animal model for Lassa fever is based on the new world arenavirus Pichinde (PIC). Natural isolates of PIC virus are attenuated in guinea pigs, but serial guinea pig passage renders them extremely virulent in that host. We have compared the nucleotide sequences of the small RNA segments of two attenuated, lowpassage variants of the PIC virus Munchique strain (CoAn 4763) and two virulent, high-passage derivatives. Missense mutations in the glycoprotein precursor (GPC) gene at codons GPC-119, GPC-140, and GPC-164 and the nucleoprotein gene (NP) codons NP-35 and NP-374 were most closely associated with virulence. Codon GPC-140 is predicted to represent a region of peak hydrophilicity of the glycoprotein 1 (GP1); it is conceivable that mutations at this site could influence virulence by altering B cell epitopes or virus attachment protein conformation.Of all the viral hemorrhagic fevers of humans, Lassa fever (caused by the arenavirus Lassa) is most amenable to pathogenesis studies because of the availability of a wellcharacterized, safe, and accurate animal model. This model uses the new world arenavirus Pichinde (PIC), 1 a member of the Tacaribe serogroup, which is non-pathogenic for humans. Serial passage of the Munchique isolate of PIC virus (CoAn 4763) in inbred guinea pigs dramatically increases the pathogenicity of the virus. 2 Guinea pigs infected with this adapted PIC virus develop fever, leukopenia, thrombocytopenia, platelet dysfunction, 3 and terminal vascular collapse 4 in the absence of significant hemorrhagic manifestations, resembling Lassa fever. 5 Like Lassa virus in humans, adapted PIC virus replicates in all extraneural tissues of guinea pigs, and induces minimal histopathologic changes in liver, spleen and other reticuloendothelial organs of the infected host. [6][7][8] In contrast, the parental, unpassaged virus is naturally attenuated, and causes a self-limited febrile illness with low mortality in inbred guinea pigs. 2 This system offers the opportunity to delineate molecular genetic determinants of virulence. The relatively short passage history of parent (attenuated) and passaged (virulent) PIC virus suggests that sequence analysis would uncover a relatively small number of mutations between the two; the disparate virulence phenotypes favors the correlation of pathogenicity with specific viral genetic alterations.Arenaviruses have bipartite, ambisense, single-stranded RNA genomes. The small (S) segment encodes the viral envelope glycoproteins and nucleoprotein. 9 The glycoprotein precursor (GPC) protein is translated from a virus sense mRNA derived from the 5Ј half of the S segment. The GPC polypeptide is post-translationally processed by host cell signal peptidase and furin-like proteases to yield GP1 and GP2. Biochemical studies suggest that tetramers of GP2, which have a transmembrane domain, form a coiled-coil stalk, atop which tetramers of the globular GP1 protein are bound by non-covalent interactions.9,10 The cytoplasmic tails of the GP2 molecules are believe...

Genome comparison of virulent and avirulent strains of the Pichinde arenavirus

“…In order to obtain accurate IGR consensus sequences for P2 and P18, we amplified short sequences spanning only the IGRs of the respective strains and cloned into the pGEM-T-easy vector. Multiple independent clones reveal identical sequences of the IGR for P2 and P18 as have previously been reported [51]. All in all, we believe that we have successfully assembled the consensus sequence for the full-length S segments of P2 and P18 strains that only show minor sequence variations from those that have been published previously [51].…”

“…Multiple independent clones reveal identical sequences of the IGR for P2 and P18 as have previously been reported [51]. All in all, we believe that we have successfully assembled the consensus sequence for the full-length S segments of P2 and P18 strains that only show minor sequence variations from those that have been published previously [51]. The P2 S segments differ at 17 positions (81, 135, 187, 405, 471, 567, 699, 1014, 1158, 1206, 1242, 1365, 2466, 2712, 3222, 3374, 3398), whereas the P18 S segments differ at 5 positions (1336, 1713, 1860, 3236, 3387) from the published sequences [51].…”

“…All in all, we believe that we have successfully assembled the consensus sequence for the full-length S segments of P2 and P18 strains that only show minor sequence variations from those that have been published previously [51]. The P2 S segments differ at 17 positions (81, 135, 187, 405, 471, 567, 699, 1014, 1158, 1206, 1242, 1365, 2466, 2712, 3222, 3374, 3398), whereas the P18 S segments differ at 5 positions (1336, 1713, 1860, 3236, 3387) from the published sequences [51]. The differences in sequence variations of the S segments obtained from the current and the previous study might result from the different sequencing methods employed.…”

“…A published report compared sequences of the S segments between the different guinea-pig passaged PICVs and found a total of 43 nt changes [51]. However, only 19 nt changes between the avirulent strains P1 and P2 and the virulent strains P17 and P18 appeared to be associated with the degree of viral virulence.…”

“…Published sequences of the S segment between the P2 and P18 strains have identified several sequence variations within the GPC and NP genes, suggesting that viral genomic sequence variations may dictate the differential behaviors of different strains of the virus in an infected host [51]. Reassortment experiments further suggest that genes encoded on both the S and L segments may contribute to the acquired virulence [52].…”