Mouse Model of Persistent Zika Virus Infection through Intrasplenic Viral Inoculation

Persistent Zika virus infection and pathological features

Clinical Relevance and the Need of Disease Models

Generally, Zika virus (ZIKV) causes acute viral diseases in which viremia will be gone approximately 7 days. However, persistent ZIKV infection has been reported since 2017, characterized by prolonged viral clearance and nonspecific to mild clinical symptoms. In 2018, a case study reported persistent viremia lasting at least 31 days after disease onset in a pregnant woman. While spontaneous abortion and disease transmission have been documented, other clinical implications remain unclear. To address this issue, in vivo models of persistent ZIKV infection (PZI) are required; however, such models are currently lacking. Notably, these in vivo models may also help identify limiting factors of PZI and provide deeper insights into disease pathogenesis and management.

Proposed Disease Model and Preliminary Evidence

The goals of PZI in vivo models include: 1) long-term detectable viral load, and 2) survival of infected mice. Immunocompromised models, such as type I and II interferon receptor-deficient mice, are not suitable for establishing PZI, as I observed that ZIKV caused mortality in infected mice approximately 13 days after subcutaneous viral challenge. On the other hand, immunocompetent mice are not susceptible to ZIKV via subcutaneous viral challenge due to “robust-ready to fight” peripheral immune system. The key question, therefore, is how to bypass this robust peripheral immunity to allow ZIKV to establish replication and subsequent systemic infection. One potential approach is direct viral inoculation into secondary replication sites; however, it remains unclear which internal organs may serve as suitable reservoirs for persistent infection. Persistence of ZIKV in the testis and brain have been indicated. To target these organs, however, display biological and technicals challenges including sex-specific, the presence of specialized tissue barriers, and more invasive inoculation technique particularly in the brain. To find other candidate, I revisited my data from immunodeficient mice, which showed that viral load in the spleen was relatively steady and low compared to other organs. Intriguingly, the spleen may possess elements that restrict ZIKV replication to low levels, making it a potential site for establishing viral persistence. These elements may include immature or precursor immune cells, similar to those involved in persistent splenic infections caused by other pathogens.

Additionally, the spleen has a vascular architecture that supports systemic viral dissemination, including tight connections to the liver via hepatic portal vein

Briefly, I infected 7–8-week-old immunocompetent C57BL/6 mice with ZIKV through intrasplenic inoculation. No interferon receptor-blocking antibodies were used in this experiment. ZIKV infected mice remained active at 21 days after infection (video not shown), with detectable NS3 viral protein not only in the spleen (Fig. A) but also in the liver (Fig. B). In addition, ZIKV RNA was detected in the brain (Data not shown), suggesting systemic viral spread. Spleen histopathology showed preserved splenic architecture without necrosis or fibrosis. However, I observed pale germinal centers in some nodules, suggesting an active immune response attempting to limit viral replication (Fig. C). In the liver, prominent inflammatory infiltrates were observed around the central vein, while overall hepatic architecture remained well preserved, with no obvious steatosis of hepatocytes or other pathological features (Fig. D). In the ovary, cystic dilatation of the ovarian bursa, a reduced number of developing follicles, and atretic follicles (characterized by pyknotic nuclei) were observed in mice infected via intrasplenic inoculation (Fig. E; ZIKV_IS). In contrast, mice infected via subcutaneous inoculation (ZIKV_SC) showed normal ovarian histology comparable to control mice (Mock_IS) (Fig. E). Previous studies have shown that ovarian bursa cysts may be associated with infection, miscarriage, or spontaneous abortion. Notably, ZIKV_IS resulted in intrauterine growth restriction, whereas ZIKV_SC showed normal pregnancy development comparable to Mock_IS control at gestational day E12.5 (Fig. F).

Take-Home Hypothesis & Clinical Implication

Mouse model of persistent Zika virus infection can be established in immunocompetent mice via intrasplenic viral inoculation, through which systemic viral infection can also be achieved. If the spleen truly plays a role in ZIKV persistence, then closer pathological examination (an aspect often overlooked in Zika disease) should be considered in clinical practice.

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Dr. Yogy Simanjuntak-HyVir

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