# Co-Cultivation Assays for Detecting Infectious Human-Tropic Porcine Endogenous Retroviruses (PERVs)

## TL;DR

This paper compares different lab methods to detect if pig viruses can infect human cells, which is important for using pig organs in human transplants.

## Contribution

The study systematically evaluates three co-cultivation assay methods for detecting human-tropic PERVs and highlights their strengths and limitations.

## Key findings

- Gamma-irradiation of pig cells may alter gene expression and affect assay results.
- Double-chamber co-culture systems miss cell-to-cell transmission of retroviruses.
- Co-culture with resistance genes best simulates in vivo xenotransplantation conditions.

## Abstract

Porcine endogenous retroviruses (PERVs) are integrated into the genome of all pigs. As they can be released as infectious virus particles capable of infecting human cells in vitro, they pose a potential risk for xenotransplantation involving pig cells or organs. To assess whether pigs produce infectious human-tropic viruses, infection assays with human cells are required. There are three main types of assays. First is the incubation of human target cells with gamma-irradiated pig cells. This method ensures that viral transmission is assessed in the absence of replicating pig cells. However, gamma irradiation may alter gene expression in pig cells, potentially affecting the results. Second is the co-culture in a double-chamber system in which pig and human cells are separated by a porous membrane, preventing direct cell-to-cell contact. While this method allows for the detection of infection by free virus particles, it does not account for infection via cell-to-cell transmission, which is a common mode of retroviral infection. And third is the co-culture of pig cells with human cells expressing a resistance gene. The resistance gene allows selective elimination of pig cells upon the addition of a selection medium. This assay enables both free virus and cell-to-cell transmission as well as complete removal of pig cells, which may not be fully achieved in the first type of assay. The third assay best simulates the conditions of in vivo xenotransplantation. However, in all cases the selection of donor and recipient cells is crucial to the experimental outcome. Results only indicate whether a specific pig cell type releases PERVs and whether a specific human cell type is susceptible to infection. A negative infection result does not necessarily reflect the in vivo situation, in which a transplanted organ consists of multiple pig cell types interacting with a diverse range of human cells within a living organism. Knowledge of these limitations is important for authorities regulating clinical applications for xenotransplantation.