**1. Introduction**

Zika virus (ZIKV), discovered in the Ugandan Zika forest in 1947, is a single-stranded RNA arbovirus of the genus *Flavivirus* and the family Flaviviridae [1,2]. Prior to 2007, only 14 human cases of ZIKV infection had been reported. However, in 2007, the first major epidemic of ZIKV, with 185 confirmed cases, occurred in the Yap Islands of the Federated States of Micronesia [3,4]. Since then, ZIKV has spread to 30+ countries, with millions of suspected cases, and has gained international attention due to an association with microcephaly and Guillain-Barré Syndrome (GBS) [5–9]. Subsequently, ZIKV has been identified as a significant global health threat.

ZIKV is primarily transmitted by mosquitoes. However, it can also be transmitted sexually or by blood transfusion [10–12]. After inoculation from an infected mosquito, the virus replicates in tissues local to the bite, drains to local lymph nodes, and then spreads hematogenously to secondary replication sites [13]. In adults, most infections (~80%) are asymptomatic, with only about 20% of infections developing a self-limiting illness. Symptoms vary in severity, and may include fever, headache, maculopapular rash, arthralgia, myalgia, fatigue, and conjunctivitis [14]. Additionally, ZIKV infection during pregnancy can cross the placenta, where it targets neural stem and progenitor cells in

the developing fetus, leading to microcephaly, lissencephaly, and cognitive deficits, as well as ocular impairments such as chorioretinal atrophy and optic nerve disorders [15–17].

ZIKV is the only arbovirus known to be transmitted sexually [18]. Sexual transmission has been reported from male to female, male to male, and female to male, indicating that infectious virus persists in both semen (up to four months [18]) and vaginal secretions (up to six months [19]) [18,20–28]. However, the site of ZIKV persistence, leading to viral shedding in the genital secretions of males and females, is not clear. Although ZIKV has been reported to persist in testes, evidence of viral shedding in semen of vasectomized males suggests an additional site of persistence [29–31]. In women, the site of persistence has not been determined. We recently showed that ZIKV persistently infects primary adult cultured sensory neurons of the lumbosacral dorsal root ganglia (LS-DRG), which innervate the genitourinary tract (GUT), suggesting a potential alternative reservoir for viral shedding in urine and genital secretions [32]. The pathogenesis of ZIKV after sexual transmission has not been studied extensively, but sexual transmission may result in di fferent routes of spread within the host and potentially alter tissue tropism when compared to mosquito-borne transmission.

Efforts to understand the pathogenesis of ZIKV following mosquito-borne and sexual transmission have led to the development of various animal models. Several studies have shown that immunocompetent adult wild-type mice have minimal susceptibility to ZIKV infection and demonstrate di fferent disease manifestations than humans [33]. Thus, more recent studies have primarily used immunocompromised animals, such as mice lacking interferon (IFN) or IFN receptors, or immunocompetent mice treated with IFN-blocking antibodies [34–40]. Neonatal wild-type mice are susceptible to ZIKV infection, but they are also immunocompromised since rodents do not develop a mature immune response until at least one month of age [36,41–43]. Non-human primate models have provided valuable information [44–48]. However, non-human primate studies are limited in statistical power since relatively few animals can be used in studies. Additionally, non-human primate studies are expensive to perform and are limited to facilities that have the necessary infrastructure to house these animals. More recently, several studies have explored the use of swine as a model of ZIKV infection; however, most infected swine do not exhibit clinical signs and have demonstrated only low levels of viremia [49,50]. Additionally, swine pose similar constraints as non-human primates, as they require more space and are more expensive than small animal models. Thus, an immunocompetent small animal model is needed to study ZIKV pathogenesis by di fferent routes of infection.

Guinea pigs (*Cavia porcellus*) have served as reliable models of flavivirus infection, and due to their physiologic similarities to human immune responses and symptoms, are also used as a genital infection model for several viruses [51–53]. Furthermore, guinea pigs are used as a model for cytomegalovirus (CMV) congenital syndrome, which causes similar fetal anomalies as ZIKV [54,55]. To date, five studies have reported the outcome of ZIKV infection in guinea pigs, with varying results. Subcutaneous (SQ) inoculation of strain PRVABC59 (Puerto Rico) resulted in fever, lethargy, hunching, ru ffled fur, and decreased mobility, correlating with viremia and viral replication in spleen and brain [56]. Similarly, SQ inoculation of male guinea pigs with strain GZ01 (Venezuela) or FSS13025 (Cambodia) resulted in viremia and robust viral secretion in saliva and tears, as well as transmission to naïve co-caged mates, but no overt signs of disease [57]. However, intracerebral infection with strain MR766 (Uganda) or intraperitoneal inoculation with strain ArD41525 (Senegal) or CPC-0740 (Philippines) failed to produce signs of infection or viremia [1,58]. An additional study assessed fetal impact of mid-gestation infection in guinea pigs, finding viremia and robust antibody response following SQ inoculation with strain H/PF/2013 (French Polynesia) without e ffects on pups [59]. Although these studies produced variable results, likely due to di fferences in ZIKV strain, inoculum size, and inoculation route, none of these studies assessed vaginal infection or directly compared di fferent routes of infection. Therefore, we evaluated the use of guinea pigs as an immunocompetent small animal model of ZIKV infection by both subcutaneous (SQ) and vaginal (VAG) inoculation routes, simulating mosquito-borne and sexual transmission, to compare pathogenesis, tissues sites of viral persistence and viral shedding in bodily secretions following di fferent routes of infection.
