Virology tidbits

Wednesday, 13 April 2016

Zika Virus and induction of apoptosis: combination of IFN-β and downregulation of gene expression?

As described in a previous post, Zika virus (ZIKV) infection of primary human skin fibroblasts induces the upregulation of TLR3 mRNA as well as inducing the expression of MDA-5 and RIG-1, components of the antiviral response induced not only ZIKV but by RNA viruses in general. As a result of the activation of RIG-1 and MDA-5, the expression of both Interferon-α (IFN-α) and Interferon-β (IFN-β) is increased. In mouse models such as A129, AG129 or Ifnar1 -/- mice that are deficient for either the receptor for IFN-α or both for IFN-α and IFN-β, higher viral loads are established in a number of tissues compared to wt mice, suggesting that the induction of IFN-α and IFN-β by ZIKV limits viral replication. Interestingly, in human neural progenitor cells (hNPC) infected with ZIKV the expression of IFNAR-1 (the human equivalent of Ifnar-1) is downregulated whereas the expression of the IFN-γ receptor (IFNGR-1) is upregulated. The induction of the type I IFN response can however also contribute to the severity of viral infections as shown for SARS-CoV in mice. In infected mice, the delay of the induction of type I IFN by SARS-CoV contributes to the accumulation of pathogenic inflammatory monocyte derived macrophages, resulting in elevated levels cytokines and chemokines as well as vascular leakage and the impairment of virus specific T cell responses, thus contributing to decreased survival of infected mice compared to Ifnar-1 -/- mice. Furthermore, blocking the IFNAR-1 receptor and deleting IFN-β in mice promotes the clearance of lymphocytic choriomengitis virus (LCMV) in mice whereas the activation of IFN-α inhibits early dissemination of LCMV. In contrast to SARS-CoV and LCMV however, Ifnar1 -/-, A129 and AG129 mice show a decreased survival rate, indicating that IFN-α is necessary for viral clearance and survival of infected mice.

The infection of hNPC with ZIKV MR766 induces the downregulation of a number of genes promoting cell survival including those encoding for anti-apoptotic proteins, cell cycle regulators, factors involved in the DNA damage response pathway and autophagy as well inducing apoptosis that is preceded by an arrest in G2/M phase of the cell cycle.

In addition, human neurospheres and brain organoids infected with ZIKV MR766 exhibit extensive caspase-3 dependent cell death as early as 3 days p.i. which is in agreement with previous results that showed that 56 hrs p.i. the expression of Caspase-3 is upregulated, suggesting that in human neuronal cells ZIKV induces Caspase-3 dependent apoptosis. None of these experiments however determined the contributing factors, i.e. the pathway of apoptosis induction.

Figure: Changes of gene expression in hNPC infected with ZIKV compared to Mock infected
cells; RECQL4, IFNAR-1, IRF-7, IFIT-2

The infection of human lung epithelial A549 cells with ZIKV PF/25013/18 results in effective viral replication as measured by immunofluorescence for the viral E protein and dsRNA, flow cytometry for E protein positive cells, and determination of viral titres that showed maximum titres within 48 hrs p.i. concomitant with the induction of PARP cleavage and decreased cell viability. RT-PCR analysis of ZIKV infected cells revealed that ZIKV infection induces a 200-fold increase of both IFIT-1 (ISG56) and IFIT-2 (ISG54), both of which are regulated by IRF-3 and IRF-7 as well as STAT-1/-2 (in addition to A549 cells, ZIKV also upregulates IFIT-2 and IRF-7 expression in hNPC). Since IFIT-2 induces the depolarization of mitochondria and thus contributes to the induction of Caspase-3 and -9 dependent apoptosis, it might be possible that the formation of the complex consisting of IFIT-1 and IFIT-2 is induced by ZIKV and induces apoptosis in a Bak and Bax dependent manner. Unfortunately, the current data do not contain experiments using either Bak -/- Bax -/- cells nor cells that are deficient for either IFIT-1/-2 or cells treated with siRNA targeting Bak, Bax, IFIT-1 or IFIT-2. Downstream of IFIT-1 and IFIT2, the infection of A549 cells with ZIKV triggers the production of mitochondrial reactive oxygen species (ROS) as detected by MitoSOX without increasing cytoplasmic ROS levels similar to cells infected with DENV by increasing the expression of mitochondrial SOD2 although in the case of DENV infected cells cytoplasmic ROS levels are increased as the result of DENV induced activation of the ER stress response. If the downregulation of RecQL4 expression in ZIKV infected cells contributes to the activation of SOD2 is a contributing factor is not known, but possible. Alternatively, ZIKV proteins localizing to the ER might induce the ER stress response and thus induce the expression of mitochondrial ROS independent of IFIT-1/-2.

Also it remains to be seen if the expression of IFN-β in ZIKV infected cells can be induced via the relocalisation of STING to perinuclear punctae.

Figure: Induction of STING signalling by delocalisation of STING to perinuclear punctae

The infection of primary human skin fibroblasts with ZIKV induces the expression of pro-inflammatory cytokines which can also be detected in patients infected with ZIKV. Accordingly, the infection of A549 cells induces the expression of IL-1β, IL-6 and MCP-1 at 24-48 hrs p.i.. IFN-β expression and secretion can be detected at early times post infection (12 hrs p.i., increasing between 18-24 hrs p.i.), suggesting that the induction of IFN-β increases the expression of both IFIT-1 and IFIT-2 contributing to the induction of apoptosis. Interestingly, pretreatment of A549 cells with IFN-β not only reduces viral titres but also reduces caspase-3 activity suggesting that other factors than the IFIT-1/IFIT-2 complex trigger apoptosis. Since IFN-β not only triggers an antiviral response and apoptosis but also autophagy, pretreatment of A549 cells might induce the formation of autophagosomes that not only promotes the degradation of viral particles upon entry but also promotes the degradation of viral RNA in a process called RNautophagy. Further studies are however needed to verify and explore this pathway. Since the infection of mouse embryonic fibroblasts (MEF) with West Nile Virus (WNV) or Japanese Encephalitis Virus (JEV) activates the inflammasome, pretreatment of A549 cells might prime cells to MEFV dependent inactivation of the inflammasome and thus apoptosis following inflammasome activation by ZIKV.

Figure: Induction of SOD2: dependent on downregulation of RECQL4 and/or induction of
ER stress response

In conclusion, the infection of A549 cells with ZIKV results in productive infection and triggers an antiviral response that includes the expression of chemokines, IFN-β, and IFIT-1 as well as IFIT-2, similar to observations from ZIKV patients or ZIKV infected hNPC. Similar to hNPC, neurospheres, and brain organoids, ZIKV triggers caspase dependent apoptosis probably in a IFIT-1/-2 independent manner, although further experiments are warranted. Pretreatment and treatment up to 2 hrs p.i. of A549 cells with IFN-β results in decreased viral titres and decreased caspase-3 activation suggesting that IFN-β has a pro-survival rather than a pro-apoptotic role in ZIKV infection, a notion that is supported by observations from existing mouse models. The increase of mitochondrial ROS by ZIKV might induce DNA damage that due to the potential inhibition of the DNA damage response by ZIKV might induce cell cycle arrest and subsequent apoptosis as well MAVS/RIG1 mediated induction of IFN expression and/or MAPK/NFκ-B mediated increase of chemokines.

Monday, 11 April 2016

ZIKV pathogenesis: development of animal models

Zika Virus (ZIKV) is a emerging positive strand RNA virus that belongs to the genus of Flaviviridae. Human infections are mostly asymptomatic although a causative link to microcephaly and Guillan-Barre Syndrome (GBS) have been proposed.

So far however no reliable animal model to study ZIKV associated disease has been identified but in order to test a potential vaccine and order to gain a better understanding of ZIKV pathogenesis animal models are being developed. Similar to DENV, these models may involve immunocompetent and humanized mice as well nonhuman primates.

Here recent advances to establish a mouse model are presented.

ZIKV in mice

In the original mouse model, ZIKV was first isolated from Swiss albino mice intracerebrallly injected with the original ZIKV 766 strain that was isolated from the sentinel rhesus monkey as well as virus suspensions obtained from infected Aedes Africanus mosquitoes. Following the 16 passages of ZIKV strain 766 in mice, 100% mortality can be observed and subsequent infections of mice with (mouse adapted) ZIKV 766 lead to a mean survival time of 10.6 days with an incubation period of 6 days.

Both the infection of mice with ZIKV 758 isolated from rhesus monkeys and the E/1 strain isolated from infected A. Africanus resulted in 100% mortality at passage 16 and 15 indicating that the intracerebral injection of ZIKV suspensions in and subsequent adaption of rhesus and mosquitoe derived ZIKV to mice results in sickness (indicated by roughness of the fur and inactivity), paralysis (motor weakness and paralysis of limbs) and death within 24 to 48 hrs p.i. .

Figure: ZIKV in Swiss albino mice: number of healthy, sick and paralysed mice

Figure: ZIKV in Swiss albino mice: number of dead mice

Subsequent intracerebral (i.c.) infection of newborn and 5 week old mice with ZIKV MP1751 strain showed that ZIKV replicates in both neurons and astroglial cells as well as extensive cell death in the hippocampus and cortex of infected mice, suggesting that apoptosis or necrosis of brain cells might be the cause of paralysis. Recently published data obtained from human cortical neuron progenitor cells infected with ZIKV 766 indicate that infected neuronal cell indeed undergo apoptosis probably by ZIKV induced downregulation of anti-apoptotic genes, thus explaining the loss of neuronal cells in ZIKV (intercelebrally; i.c.) infected mice. The initial experiments also revealed that mice infected intraperitoneally (i.p.) at 7 days of age exhibited a higher LD₅₀ value than those 14 days or older at time of infection although mice older than 6 weeks are slightly less susceptible than younger mice to i.c. infection.

Figure: LD50 values are influenced by age of mice at time of infection

Interestingly, recent data indicate that the infection of 7 days old mice intraperitoneally infected with ZIKV Dakar 41519 reduces viability by 33% compared to mice infected subcutaneously (s.c.), suggesting that the route of infection as well as the age at time of infection indeed effects the survival rate.

Initially, ZIKV was not detected in any other organs following infection i.c., but in more recent studies in A129 and AG129 mice infected either i.p. or intradermal (i.d.) with ZIKV FSS13025 (Asian lineage) showed high viral titres in the kidney, spleen, testes, and brain of infected mice suggesting that the route of infection might not only determine the survival rate but also sites or viral replication. Comparing these results with those obtained originally using Swiss albino mice suggest that ZIKV infection in mice induces the antiviral Interferon response since A129 mice are deficient for both Interferon-α and –β (IFN-α /-β) whereas AG129 are deficient not only for IFN-α /-β but also for IFN-γ. The notion that ZIKV induces the interferon response is supported by recently published studies in primary skin fibroblasts and human placenta cells as well as in triple knockout mice.

ZIKV: induction of the Interferon response

Infection of primary human skin fibroblasts with low passage ZIKV strain derived from a viremic patient in Tahiti/French Polynesia, PF-25013-18, results in the activation of the innate immune response namely the induction of Pattern Recognition Receptors (PRRs) by the viral RNA. As indicated by analysing the expression of genes using qRT-PCR, the expression of TLR3, RIG-1/DDX58 and MDA5/IFIH1 as well as CXCL10, Interleukin-1β, AIM2 is upregulated whereas the expression of others such as TLR7, TLR8, or TNF is downregulated as soon as 6 hrs p.i.. Activation of TLR3, RIG1 and MDA5 expression increases also the expression of Interferon stimulated genes (ISG) including OAS2, ISG15 and MX1, suggesting that ZIKV RNA activates PRRs dependent downstream signaling pathways by binding to TLR3, MDA5 and RIG-1 which subsequently stimulate the expression of ISG via upregulation of IRF7 expression. Since IRF7 is a transcription factor for type I interferons, the infection of primary skin cells with ZIKV induces an antiviral response that decreases viral titres.

Figure: Genes that are unregulated in PHT cells

Figure: Induction of the IFN response by ZIKV RNA

Since human placental trophoblasts (PHT) are resistant to infection with both ZIKV FSS13025, a strain isolated in Cambodia and the original ZIKV MR766 strain, ZIKV might induce an antiviral response similar to those observed in primary human skin cells. Indeed, ZIKV infected PHT cells exhibit a high level of type III Interferon, in particular Interferon-λ1 (IFNλ1) which is constitutively expressed by PHT cells. Similar to primary skin fibroblast cells the expression of OAS2, TLR3 and MX2 is unregulated when compared to human choriocarcinoma JEG-3 that support ZIKV replication, suggesting that ZIKV induces identical antiviral signaling pathways in both PHT and skin cells. Furthermore, conditioned media from infected PHT cells inhibits the replication of ZIKV in JEG-3 cells by inducing the expression of ISG, suggesting that PHT inhibit ZIKV replication by inducing the expression in a autocrine and paracrine manner although further work is needed verify the hypothesis.

The finding that ZIKV replication in PHT might be inhibited by IFNλ1 causes however some problems in the utilization of mice as a model to investigate the potential relationship between ZIKV and microcephaly in neonates and/or other embryonal development defects since not only is there a difference in the morphology of the placenta but also in the expression of IFNλ subtypes; mice only express IFNλ2 and IFNλ3 whereas PHT only low levels of IFNλ2 and no IFNλ3.

The importance of the role of the interferon response in controlling the replication of ZIKV and preventing the onset of neurological symptoms resembling those observed in infected humans is further strengthened by studies using mice deficient for components of the interferon and comparing them to fully immunocompetent mice. These experiments differ from those conducted in Swiss albino mice described above in so far, as this model allows not only the use of wt ZIKV strains or ZIKV strains that exhibit only low passage number in Vero or C6/36 mosquitoe cells but also prevents the need of infection via intracelebral injection, thus avoiding the need for the virus to cross the blood-brain barrier.

Similar to Swiss albino mice injected i.c. with ZIKV 766, the injection of immunocompetent C57BL/6 5 to 6 week old mice with ZIKV MR766 or H/PF/2013 either s.c. or intravenous (i.v.) does not induce the development of any neurological symptoms up to 10 days p.i. nor weight loss. As mentioned above, the infection of immunocompetent mice 7 days of age with ZIKV Dakar however decreases survival by 33% at day 30 p.i. , suggesting that either age or route of infection might play a role in the severity of the disease or alternatively that ZIKV strains of the African lineage are more virulent than those of the Asian lineage.

In order to determine if components of the interferon pathway prevent the onset of symptoms and/or death of infected mice after the infection with ZIKV, 4-6 week old mice deficient for the transcription factors IRF3, IRF5 and IRF7 as well as deficient for the Ubiquitin-like protein ISG15 were infected with ZIKV MR766 or ZIKV H/PF/2013 i.v. or s.c. and monitored for signs of infection for 10 days p.i. . In contrast to the single knockout mice, triple knockout (TKO) IRF3 -/- IRF5 -/- IRF7 -/- mice exhibited symptoms such as paralysis as early as 3 days p.i. regardless of the infection route. In addition, up to 100% of infected TKO mice were dead 10 days p.i. whereas single knockouts survived. In a similar way, single Ifnar-1 (Interferon-α receptor) knockout mice infected with ZIKV Dakar 41519, ZIKV MR766 or ZIKV H/PF/2013 exhibited 100% mortality 10 days p.i. whereas infected single MAVS -/- knockout mice did exhibit any mortality. Blocking the the IFNAR-1 receptor and thus IFN- α/β using a monoclonal antibody one day prior ZIKV infection renders wt mice susceptible to ZIKV which is similar to results described for West Nile Virus (WNV), although blockage of IFNAR-1 does not increase mortality but induces higher viral titres. Interestingly older Ifnar1 -/- mice exhibit a higher survivial rate with 40-80% of infected mice despite weight loss; whether viral loads are lower as well has not been investigated.

Similar to ZIKV infected A129 and AG129 mice, in Ifnar1 -/- mice the tissues with the highest viral load are spleen, testes, kidneys, spinal cord, brain and liver thus confirming previous reports that ZIKV is a neurotropic virus. In addition, the brain and testes of surviving Ifnar1 -/- exhibit high viral RNA levels up to 28 days p.i., a finding that might explain the occurrence of neurological symptoms as well as horizontal transmission of ZIKV to previously uninfected women. From the present epidemiological data however it is not clear if ZIKV induces encephalitis at a similar rate than those observed in patients infected with DENV or WNV.

Figure: Survival rates of immunocompetent and immunodeficient mice

Taken together with the gene expression data obtained from JEG-3 and PHT cells, these results suggest that the interferon response does protect not only placenta derived cells but also mice from ZIKV induced death similar to WNV. Further data however are needed if ZIKV neurovirulence in the embryo or foetus dependent on the resistance of ZIKV to interferon induced signalling or due to the induction of cell cycle arrest and subsequent apoptosis or neural progenitor cells.

ZIKV in rhesus macaque monkeys

In general monkeys infected with ZIKV do not exhibit any or only mild clinical signs of infections although virus can be detected as early as 3 days p.i. as suggested by historic data.

In order to assess the the infectivity of the original ZIKV MR766 isolate from Africa, experiments are currently conducted at the University of Wisconsin-Madison that determine the load of viral RNA in the blood, urine, saliva and cerebrospinal fluid (CSF)

of rhesus macaque monkeys infected with different doses of ZIKV. In both experiments, 2 out of 3 animals exhibit maximum load viral RNA in plasma at 3-5 days p.i. and in the urine at 7-9 days p.i. .

Figure: Load of viral RNA in rhesus macaque infected with ZIKV

In addition, similar studies are currently performed to determine if the infection of monkeys with ZIKV induces the formation of malformations in the brain during the foetal development, but as the time of writing these studies have not concluded.

In conclusion, the infection of immunocompetent mice with ZIKV is not lethal nor do those mice present themselves with clinical symptoms such as paralysis. Mice deficient for interferon dependent antiviral signaling however succumb to infection 10 days p.i., indicating that ZIKV is not resistant to interferon signaling per se. In addition, in primary human placental trophoblasts ZIKV replication is inhibited by IFNλ1, whereas previous studies suggest that human NPC and human stem cells are supporting ZIKV replication. This suggests a model where ZIKV needs to breach the placental-foetal barrier in order to infect embryonal cells without infecting placental cells, suggesting that infected maternal cells need to cross the barrier.

The activation of the interferon response by ZIKV RNA however might not only induce antiviral signaling but also induce the up- and downregulation of gene expression via inducing STAT1 and 2 mediated signaling. In this model, STAT-1/-2 regulates the expression of genes encoding for proteins regulating diverse processes as DNA repair, cell cycle control and autophagy, thus contributing to cell death observed in infected hNPC as discussed before.

Figure: ZIKV RNA induces not only antiviral signalling but also regulates gene expression