Purifying selection constrains the evolution of Juquitiba virus in wild Oligoryzomys nigripes communities

Juquitiba virus (JUQV) is endemic in Oligoryzomys nigripes across several South American countries and causes hantavirus pulmonary syndrome when transmitted to humans via infectious saliva or excreta. We developed a next-generation sequencing (NGS) pipeline to generate the first complete reference genome for assessing the genetic diversity of JUQV in Oligoryzomys populations inhabiting the Mbaracayú Biosphere Reserve within the Atlantic Forest of Paraguay. From 32 additional Oligoryzomys specimens, we obtained 17 S- and M-segment viral RNA (vRNA) genomes from lungs with 94-100% sequence coverage and 101 additional vRNAs with ≥80% genome coverage and ≥500x sequence depth from saliva, urine, lungs, heart, kidney, liver, and spleen. Phylogenetic and phylogeographic analyses showed that the Paraguayan JUQV is genetically distinct from the Brazilian JUQV lineage. Shannon entropy calculations of genetic diversity revealed that saliva and lung samples had higher entropy values than urine, kidney, spleen, and heart samples. The greater genetic diversity was driven in part by greater nucleotide, but not amino acid, diversity in persistently infected rodent samples compared to acutely infected ones. Genetic diversity varied across collection sites, although, given the continuous habitat matrix, there was no apparent reason for these differences. Fixed Effects Likelihood analysis of lung, saliva, and urine sequences suggested that purifying selection was the primary driver of evolution, with no evidence of positive selection. Only three of the 29 codons in the N protein and the glycoprotein (GP) were under purifying selection, and only Gn harbored nonsynonymous mutations. We tested two of the nonsynonymous mutations within the Gn for their effect on entry into Vero cells using VSV-pseudotyped JUQV GP; however, only V504I resulted in a significant reduction in entry compared to wild-type Gn. In summary, tissue source, field locale, and persistent infection were clear drivers of virus evolution.