Key mutations in the envelope and NS1 proteins of Zika virus contribute to enhanced viral replication in mouse testis

Zika virus (ZIKV) belongs to the Flavivirus genus within the Flaviviridae family, which includes other important mosquito-borne human pathogens, including dengue (DENV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever viruses (YFV). ZIKV was initially isolated from a sentinel rhesus monkey in the Zika forest, Uganda in 1947. It received little attention until it caused the first notable human epidemic in the Pacific Ocean in 2007 followed by another major outbreak in French Polynesia in 2013. Recently, due to its association with Guillain-Barre syndrome (GBS) in adults and congenital Zika syndrome (CZS) in infants born to ZIKV-infected mothers, the biggest ZIKV outbreak that began in Brazil in 2015 was declared a Public Health Emergency of International Concern by the World Health Organization on 1 February 2016. During this epidemic, ZIKV infections were reported in over 80 countries worldwide with more than 1.3 million infections in the Americas alone. Currently, there are no antiviral drugs or vaccines available for ZIKV infection. ZIKV has also been shown to be transmitted among humans through male-to-female sexual contact. There is strong evidence that the male reproductive tract acts as a significant reservoir for ZIKV. Both infectious ZIKV and ZIKV RNA have been identified in the semen of ZIKV-infected men. The presence of viral RNA in human semen can persist for up to 370 days following the onset of symptoms. The relationship between the intensity and duration of viremia and the invasion of the genital tract by ZIKV, viral shedding in semen, and the subsequent risk of sexual transmission from males to females or males to males remains unclear. Exploring the changes of ZIKV genes during continuous replication in testes and identifying potential testicular adaptive mutations can provide an important reference for assessing the risk of ZIKV mutations. Here, the FSS13025 strain of ZIKV (named WT) was serially passaged in testes of A129 mice for 10 times. Analysis using qRT-PCR revealed that levels of viral RNA in testes of mice infected with passage 5 (P5) or P10 were significantly 4.6 and 3.8 times higher than those of WT, respectively. Deep sequencing analysis of the genomes of P5 and P10 identified mutations H401Y in the envelope protein and K146E in the NS1 protein. To further test the biological functions of the mutations, a recombinant virus carrying both H401Y and K146E mutations (named H401Y+K146E) was rescued using the FSS13025 strain as the genetic backbone by reverse genetics. H401Y+K146E showed an increased replication in multiple cell lines including testicular cell lines 15P-1 and TM3 compared to WT. Importantly, the replication of H401Y+K146E was significantly enhanced specifically in testes of A129 mice in comparison with WT. This study identifies two testis-adapted mutations of ZIKV, which confer enhanced viral replication in mouse testis. These two functional mutations have important implications for assessing the risk of ZIKV evolution in the future.