ZIKV and microcephaly: African and Asian isolates and infection of the vaginal mucosa

The recent emergence of Zika Virus (ZIKV) in the Americas has been associated with abnormal brain development of the foetal brain leading to microcephaly, other neurological abnormalities, severe depilation of neonates and foetal or neonatal death. it should be noted however that so far in the most affected countries -Brazil and Colombia- only a subset of all reported cases of microcephaly have been shown to be tested positive for ZIKV suggesting that other factors also may play a role in the observed increase of microcephaly cases. Nevertheless, as discussed before, studies in SJL mice infected with a ZIKV isolate from Brazil and from studies using the Asian ZIKV SZ01 confirm that the ZIKV infection can indeed cause microcephaly by inducing apoptosis of infected neural precursor cells but not mature neuronal cells which is supported by studies using hNPC and brain organoids. Interestingly, recent data indicate that the infection of both hNPC and human astrocytes with ZIKV ArB41644, a strain isolated from the Central African Republic, and ZIKV H/PF/2013, a strain isolated from French Polynesia in 2013, show different infectivity with ZIKV H/PF/2013 exhibiting less cells undergoing abnormal cell division as measured by CFSE positive cells using flow cytometry at  6 days p.i. as well as less cells displaying endoreplication/multi-nucleation and A decreased percentage of apoptotic cells as indicated by the absence of activated Caspase-3 4 days p.i.. The absence of apoptotic cells following the infection of neural stem cells (NSC) with ZIKV H/PF/2013 is in stark contrast with previously discussed results which were largely obtained from hNPC cells and brain organoids infected with ZIKV MR766 cells. Although the reason for the observed differences is not known, it might be that ZIKV MR766 -in contrast to ZIKV H/PF/2013- is a neurotrophic strain that has been extensively passaged in mice brain whereas the latter is a recent isolate with a relative low passage number and only been passaged in mosquitoe C6/36 cells that are derived from Aedes Albopictus  which might account for the higher percentage of apoptotic cells in hNPC infected with ZIKV MR766. If however viral induced apoptosis is also lower for ZIKV ArB41644 infected NSC cells when compared to ZIKV MR766 infected cells has not been determined.

In addition to the observed differences in viral titres and apoptosis, a qRT-PCR based array consisting 79 genes that are involved in antiviral signalling using mRNA collected at 4 days p.i. from ZIKV infected NSC indicate that 19 out of 20 genes are significantly upregulated in NSC infected with ZIKV ArB41644 but not in NSC infected with ZIKV H/PF/2013 the exception being CXCL8.

Table: Genes that are up-or downregulated in NSC, hNPC or organoids infected with different ZIKV isolates (nd= not determined; grey boxes= not induced)

Infection of NSC with ZIKV ArB41644 therefore might clear viral replication whereas the infection with ZIKV H/PF/2013 might lead to viral persistence that might contribute to the observed presence of ZIKV in the foetal CNS and foetal brain as well as in neonates infected with ZIKV BR since the current strain in the Americas derived from the Asian lineage. Unfortunately, the study did not include the original ZIKV MR766 nor an isolate from the current outbreak nor has it been established if the infection of immunocompetent and/or immunodeficient mice with ZIKV ArB41644 increases mortality and/or abnormalities in offspring of infected mice, although the infection of immunodeficient Ifnar1 -/- mice with ZIKV Dakar 41519 -similar to ZIKV H/PF/2013 decreases survival by 100% whereas ZIKV MR766 decreases the survival rate of infected Ifnar -/-  mice by 60%, whereas in wt mice, none of the isolates tested (Dakar 41519, MR766, H/PF/2013) induces mortality if they infected s.c. . 

More importantly, further studies are needed to asses if both strains from different lineages  also show different transmission with regard to the placenta and the infection of vaginal tissue such as the vaginal mucosa. As discussed in a previous post, the infection of primary human trophoblasts (PHTs), that prevent the infection of placental cells, with ZIKV FSS13025 (an isolate from Cambodia propagated in C6/36 cells) induces the expression of a variety of genes involved in antiviral signalling including a subset that are also induced by ZIKV ArB41644 (namely, CXCL10, ISG15, OAS2, STAT1 and TLR3), suggesting that both isolates exhibit lower viral titres due to the induction of common antiviral signalling pathways and both might also not readily be transmitted across the placental barrier. 

Table: Genes that exhibit a higher expression in primary placental cells compared to control cells 

In contrast to both isolates from Asia and the primary isolate from Africa, ZIKV MR766 downregulates the expression of ISG15, MyD88 and IFNAR-1 in infected hNPC (although ISG15 is upregulated in human epithelial cells from the skin), suggesting that ZIKV MR766 does inhibit antiviral signalling more efficiently.

Both ZIKV MR766 and a recently isolated strain from Nicaragua, ZIKV Nica-2/16, infect and replicate in primary human placental cells and in explants such as cytotrophoblasts, fibroblasts, Hofbauer cells in chorionic villi, amniotic epithelial cells as well as trophoblast progenitor cells but corresponding studies using primary or low passage isolates from the African lineage have not been conducted so it remains to be seen if primary isolates from Africa do indeed infect vaginal and/or placental tissue and thus potentially are transmitted to the embryo and foetus. In any case, the infection of placental cells results in high viral titres in amniotic epithelial cells mid gestation (22.6 weeks) and lower viral titres at late gestation (40 weeks) irrespective of the viral isolate used (MR766 or Nica-2/16), corresponding to higher levels of Axl, TIM1 and Tyro-3 being expressed at week 22.6.

Figure: ZIKV isolates from Africa and Asia (SZ01) might induce a strong antiviral response via different pathways

In conclusion, it might therefore be the case that isolates from the Asian lineage -in particular H/PF/2013-  including those from the Americas replicate efficiently in neural tissue due to failure to induce or counteract antiviral signalling whereas (primary) isolates from the African and/or Asian lineage with the exception those related to the 2013 outbreak in French Polynesia might induce antiviral signalling and thus prevent not only persistent infection of neural and non-neural tissue but also induce apoptosis of foetal neural (precursor) cells, leading potentially to early miscarriage rather than persistent infection. In this context, the ZIKV MR766 isolate might be an exception in so far as antiviral signalling is inhibited by downregulating the expression of genes encoding for proteins involved in antiviral signalling such as MyD88 and IFNAR-1. The inability of African isolates to counteract antiviral signalling might explain why the outbreaks in Africa have been sporadic and low in terms of infected individuals as well as the absence of ZIKV associated cases of neurological infection which is a feature of the outbreak in French Polynesia and in particular the current outbreak in the Americas. Future work however is needed to establish if individual genes are contributing to these differences or if the combination of viral proteins is required; here recently developed reverse genetic systems will be of use.

ZIKV and vertical transmission: role of the vaginal mucosa

Although the primary mode of ZIKV transmission is via infected mosquitoes, especially Aedes Agypti, a substantial number of infections are transmitted by sexual contact, with both female to male transmission and male to female transmission as well as male to male transmission. Indeed, following the infection with ZIKV, viral particles can be detected in the male semen for as long as 62 days after the onset of symptoms. ZIKV therefore joins a list of other viruses that can be transmitted via semen for a considerable amount of time following the onset of symptoms, with EBOV one of the most recent additions prior to the emergence of ZIKV.

For obvious reasons, male to female transmission poses an fictional risk factor for women who plan to become pregnant since infection of vaginal tissues with ZIKV might also lead to the infection of the placenta and thus the embryo during the first trimester of the pregnancy. It is therefore of utmost importance to investigate if ZIKV not only infects but also replicates in cells of the vaginal cells (in a similar way, the infection of testicular cells is of importance). IFNAR-1 -/- male mice infected with ZIKV H/PF/2013 s.c. leads to high viral tires in the brain, spleen and testes and mating of these mice with female wt mice leads to offspring with intrauterine growth restriction (IUGR) similar to microcephaly in human foetuses, indicating that male to female transmission of ZIKV can infect placental tissue and subsequently infect embryonal cells. Intravaginal (ivag) infection of wt C57BL/6 with ZIKV FSS1305 does not decrease survival nor weight loss of infected mice, despite viral replication in vaginal tissue from day 1-4 p.i. with viral RNA being detectable by qRT-PCR as early as 6 hrs p.i., indicating that ZIKV can replicate in the vaginal mucosa of wt mice asymptomatic. In contrast, infection of wt mice with ZIKV FSS1305 i.p. only results in low viral titres in the spleen at 24 hrs p.i. and undetectable ZIKV RNA at 72 hrs p.i., whilst no viral RNA can be detected in vaginal tissue, suggesting that systemic infection does not result in ZIKV infection of the vaginal mucosa.

Similar to NSC, and astrocytes, ZIKV infection of the vaginal mucosa induces the Type-I Interferon response since female Ifnar-1 -/- mice infected ivag with ZIKV FSS1305 exhibit high viral titres as early as 48 hrs p.i., exceeding those observed in wt mice. The notion that ZIKV FSS1305 induces the Type-I Interferon response is supported by that in MAVS -/- TLR-7 -/- mice ivag infection induces viral titres that are a magnitude higher when compared to wt mice, suggesting that ZIKV primarily induces a pathway that is dependent on IRF-3 and IRF-7 which is also supported by results that indicate that in IRF-3 -/- IRF-7-/-/ mice, viral titres are also significantly increased, reaching peak titres at 4-5 days p.i.. As outlined above however, it is not clear if ZIKV FSS1305 induces the expression of IRF-7 similar to ZIKV ArB41644 or not. Consistently, mice that have been infected ivag do not exhibit any signs of illness, which might be important since in women which are infected during sexual intercourse with her male partner might exhibit an asymptomatic infection that -in the case of a pregnancy might lead to the infection of the placenta and subsequent infection of the embryo.

Accordingly, ivag infection of pregnant wt mice with ZIKV FSS1305 at embryonic day E4.5 (corresponding to the late blastocyst stage of embryonal development) or E8.5 (late gastrulation/beginning of organogenesis) results in productive ZIKV replication of the vaginal mucosa with the offspring of mice infected at E4.5 exhibiting a significant growth defect despite the absence of ZIKV RNA in the placenta. This result indicates that rather infecting placental tissue directly ZIKV might transmitted to the embryo via Hofbauer cells or other maternal cells. In contrast to wt mice, the placenta of Ifnar-1 -/- mice infected with ZIKV FSS1305 at E8.5 (or 10 days p.i.) exhibit high levels of ZIKV RNA that exceed those detected in the placenta of infected IRF-3 -/- IRF-7-/-/ mice, indicating that vaginal infection of mice with ZIKV can indeed infect the placenta although in wt mice viral replication in the placenta might be inhibited or extremely low.

Following the infection of pregnant wt mice with ZIKV FSS1305 at either E4.5 or E8.5, the foetal brain does contain viral particles despite the absence of viral RNA (due to limitations of the qRT-PCR assay used) as evidenced by positive staining for the viral NS1 protein in neural and glial cells of the cerebellum and cortex despite normal foetal size. These results suggest that the current assays used for detecting ZIKV associated cases of microcephaly might not be sensitive enough to detect viral RNA in cases of foetal deaths associated with neurological symptoms.

In summary, the male to female transmission of ZIKV allows the replication of ZIKV in vaginal tissue and potentially infection of embryos and thus abnormal neuronal development. Strain specific differences might however account for the absence of microcephaly in previous outbreaks.

Further reading

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