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

## TL;DR

This study examines how Juquitiba virus evolves in wild rodents in Paraguay, finding that purifying selection and persistent infections are key drivers of its genetic diversity.

## Contribution

The study presents the first complete reference genome and in-depth analysis of Juquitiba virus evolution in its natural rodent host.

## Key findings

- Saliva and lung samples showed higher genetic diversity than other tissues.
- Purifying selection, not positive selection, was the main driver of JUQV evolution.
- Persistent infections in rodents contributed to increased nucleotide diversity.

## Abstract

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.

The unpredictable nature of outbreaks of hantavirus pulmonary syndrome throughout the Americas has motivated field studies of the etiological agents of this disease (i.e., orthohantaviruses) in their reservoir hosts (rodents) to understand their maintenance within the host, potential spill-over into other wildlife or human populations, and the risk of outbreaks and pandemics. In contrast to other ecological biomes, such as the North American Southwest desert or Northern European forests, our group has been unable to find compelling evidence that climate, microhabitat, habitat disturbance, or predator removal affects the prevalence of orthohantaviruses endemic in sympatric rodents within the Atlantic Forest of South America. To gain further insight into the maintenance and shedding of orthohantaviruses, we developed and implemented a next-generation sequencing pipeline to characterize their genomes in wild rodent reservoir communities in the Atlantic Forest. We present the first in-depth examination of the intrinsic genetic diversity and evolution of the Juquitiba virus within its reservoir, Oligoryzomys nigripes. Our studies suggest that within-host interactions of virus and host are likely the most significant drivers of viral evolution.