MOLECULAR CHARACTERIZATION OF A PATHOGENIC STRAIN OF JUNÍN VIRUS

THOMAS P; BACIGALUPO L; CALDERONE M; GOMEZ RM; FERRER MF

Otro; Reunión Conjunta SAIC. SAI. AAFE. NANOMED-AR; 2021

SAIC. SAI. AAFE. NANOMED-AR

The Junín virus (JUNV) is the etiological agent of Argentine hemorrhagic fever. The genomic sequence of JUNV pathogenic variant P3441 (P) is unknown.With the objective to characterize the P variant at a molecular level, BHK cells were infected with the P at MOI 1. At 3 days post-infection, cells were harvested and TransZol reagent (TransGen Biotech) was used to extract total RNA. Subsequently, 2 µg of viral RNA was incubated with 1 µl Reverse Transcriptase (Superscript IV, Invitrogen) and random primers (50 µM, Invitrogen) for 10 min at 25ºC followed by incubation at 50ºC for 50 min. Then, 1 µl of cDNA was used as a template in each subsequent PCR using 17 pair of primers to amplify the full genome. The amplified fragments were submitted to Macrogen (Korea) for capillary electrophoresis sequencing followed by analysis using the Ugene software. Results showed 59 genome differences between P and the vaccine attenuated Candid 1 (C#1) strain including some with potential biological functions such as the N protein R476, within a Z binding domain; D511 and L546, conserved in several pathogenic strains and found in the Z-interacting domain; and the Z protein V64 within Z RING domain. L protein showed more than 40 differences with C#1 but their relevance is uncertain since none is within functional domains. GPC proved to possess all the point mutations that have been studied by other groups. Then, specific primers were designed to amplify the 4 JUNV ORFs. Fragments of the expected size were obtained for ORFs codifying Z, N and GPC. In the case of L, four fragments were obtained. The fragments were subsequently cloned in the pGEM-T vector (Promega) following the manufacturer?s instructions. Up to the present time, all ORFs have been cloned except for one L fragment, which is still in progress. We conclude that our approach was useful to know the genome sequence of this viral variant as well as to make molecular tools for further studies.