Zika Virus pathogenesis in adult mice: comparison to SINV ?

Zika Virus (ZIKV) is a mosquitoe born member of the Flaviviridae which was (initially) isolated in 1947 from a sentinel rhesus macaque monkey in Uganda. Although being reported in humans in Uganda and Tanzania, ZIKV was reported only to cause sporadic infections in Africa and Southeast Asia. Since 2007 however major outbreaks have been reported in Yap/Micronesia (2007), French Polynesia (2013-2014) and most recently in the Caribbean and South America, where ZIKV was introduced as early as 2014.

Until recently, clinical manifestations of ZIKV infection ranged from asymptomatic infections to mild dengue-like symptoms characterised by mild fever, rash, muscle/joint pain and headache. Following the 2007 epidemic however neurological complications following ZIKV infection including Guillan-Barre Syndrome (GBS) have been reported and during the current outbreak, ZIKV has been implicating to cause microcephaly and ocular malformations in foetuses born to ZIKV positive mothers. In the absence of a suitable animal model however an association between ZIKV infection and (foetal) neurological anomalies has not been definitively proven although the infection of both brain organoids and human neural precursor cells (hNPC) with ZIKV induces apoptosis, thus suggesting that ZIKV might cause neuronal anomalies in the foetus by inducing apoptosis. Since placental cells however probably do notsupport viral replication the mode of transmission is still elusive. The connection between ZIKV infection and neuronal anomalies is further complicated by the co-circulation of other viruses such Dengue Virus (DENV) that may enhance ZIKV replication or the severity of symptoms. Indeed, antibody-dependent enhancement (ADE) has been observed in humans as a result of a previous exposure to DENV after which antibodies against the structural precursor-membrane protein (prM) promote ADE following infection with a different DENV serotype. Whether this also increases the severity of ZIKV (particular of the Asian lineage) remains to be seen.

These and other questions however underline the necessity for an animal model.

                            ZIKV animal model: AG129 mice

As described before, early experiments dating back to the early 1950s suggested that in mice infected with ZIKV do not succumb to ZIKV if infected with a strain directly isolated from monkeys or passaged in C6/36 mosquitoe cells. In contrast, a mouse adapted strain causes severe disease including paralysis and subsequent death. In general, the severity of the disease is age dependent in so far as younger mice are more susceptible to ZIKV infection compared to older mice. Further experiments showed that ZIKV suspensions injected intracerebrally cause neuronal apoptosis both within the brain and CNS, suggesting that ZIKV induced paralysis might be due to apoptosis of neuronal cells. In principle, these results were confirmed by in vitro studies using brain organoids and hNPC.  

More recently, triple knockout (TKO) IRF3 -/- IRF5 -/- IRF7 -/- mice infected with ZIKV exhibited symptoms such as paralysis as early as 3 days p.i and up to 100% of infected TKO mice were dead 10 days p.i., suggesting that the antiviral interferon response induced by ZIKV is limiting the severity of the disease.

These findings are supported by observations in AG129 mice that are deficient for Interferon (IFN)-α /-β/- γ  and have been infected with a strain isolated from a traveller to French Polynesia (ZIKV H/PF/2013) either by foot pad (f.p.) or intraperitoneal (i.p.) injection. In this case, both young (3-4 week old) and adult (8 week old) mice exhibited symptoms as early as day 4 p.i. and had to be sacrificed at day 7 p.i. (young mice) and day 8 p.i. respectively. In contrast to earlier observations made in mice infected with a mouse adapted ZIKV 766 variant strain, infection of mice with ZIKV H/PF/2013 did not cause paralysis but only became weak, immobile and exhibited rapid weight loss. Highest viral titres were observed in the brain of infected mice which is in accordance with previously published results indicating that ZIKV MP1751 replicates well in astroglial and neuronal cells of infected mice. Both young and adult AG129 mice exhibited similar viral titres suggesting that in the absence of antiviral signalling, the age of mice is not relevant.

In accordance with previously published results high viral titres can also be detected in other tissues such as the heart, liver, spleen, kidney, and muscle suggesting that ZIKV can replicate in these tissues. In accordance with previous results, histopathological examination of brain tissue of infected mice revealed extensive cell death (pyknosis and necrosis) as well as infiltration of neutrophils, suggesting that ZIKV indeed causes severe brain pathology in AG129 mice. 

In causing a more severe disease in young mice compared to adult mice, mouse adapted ZIKV resembles other neuropathogenic viruses, such as Sindbis Virus (SINV). In the case of SINV, wt SINV is virulent only for neonatal mice but not weanling mice. Similar to ZIKV, SINV passaged in mouse brain increases neuroinvasiveness in adult mice which is due to two amino acid changes in the E2 protein (His55 to Gln and Glu70 to Lys in one isolate and Lys190 to Met and Glu260 to Lys in a second isolate); if mice adapted ZIKV strains also exhibit similar changes that increase viral entry or counteract the antiviral response has not been demonstrated yet. Age dependent neurovirulence of SINV however has been demonstrated to be due to apoptosis induced by the viral E2 protein, in particular dependent on the His55 to Gln amino acid change. Additionally, the expression of IRF-3 and IRF-7 in mature (differentiated) rat AP-7 neurons has been linked to decreased mortality in adult mice although other factors such as a better antibody response resulting in rapid clearance of SINV mediated by Interleukin-10 may also influence survival rates.

Again, in the case of ZIKV more studies using animal models are needed, involving the use of mice adapted ZIKV strains. Using a mouse model might also assist in determining the teratogenic effect of ZIKV, determine the role of co-infection with DENV and assist the development of a vaccine. 

Further reading

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