Project Summary Human cytomegalovirus (HCMV) is readily transmitted across the placenta during pregnancy and is the leading cause of congenital viral infections. Infected children often suffer from long-term central nervous system damage, including intellectual disability and sensorineural hearing loss. These morbidities could be avoided if transplacental transmission could be prevented. However, the species-specific nature of HCMV has precluded the study of the virus in small animal models and whether one or more viral genes enables transplacental transmission remains unknown. Because transplacental transmission is observed in many cytomegalo- and roseolovirus infections, we hypothesize that one or more conserved, nonessential ?-herpesvirus gene products facilitates transmission from infected mother to developing fetus. We will test this hypothesis using guinea pig cytomegalovirus (GPCMV), a small animal model of congenital HCMV infection in which the virus can naturally cross the placenta. In Aim 1, we will create a library of GPCMV strains with nonsense mutations in nonessential genes. To accomplish this, we have developed CRISPR/Cas9 genome editing as a methodology to generate CMV mutants. This new approach to engineer transgenic herpesviruses allows us to rapidly and efficiently introduce specific changes into the viral genome. Aim 2 will define which GPCMV genes are required for efficient transplacental transmission in a signature-tagged mutagenesis screen that utilizes the CRISPR/Cas9 mutants generated in Aim 1. By infecting pregnant guinea pigs with pools of GPCMV mutants, we will gain insights into which viral genes are required for replication in animals and what factors are specifically involved in transmission across the placenta. Mutants that are defective in transplacental spread in pooled infections will be subsequently studied in single-strain infections to elucidate how their gene products mediate viral transit across the placenta. This study will be the most comprehensive examination of the role of cytomegalovirus gene products in placental and congenital infections undertaken to date. Genes required for transplacental spread could be deleted to generate attenuated strains that cannot cause in utero infection, increasing the safety of HCMV- based vaccines. Additionally, these factors could be targeted by therapeutics with the aim of decreasing the risk of congenital HCMV after maternal primary infection or viral reactivation.