Virology tidbits

Saturday, 6 February 2016

Zika Virus, Microcephaly and Brazil

As discussed before, Zika Virus (ZIKV) is an emerging arbovirus, spread by Aedes Agypti and Aedes albopictus, which was first isolated in 1947 in Uganda from a Macaca monkey with the first human case being detected in Nigeria (1954). In subsequent decades sporadic cases linked to ZIKV have been reported in Africa and Asia, with a first epidemic reported in 2008 (Yap/Federated States of Micronesia) and a larger one in French Polynesia and Oceania 2013-2014 with the first cases in the Americas were identified in Natal/Brazil in March 2015 in samples from patients displaying dengue-like symptoms.

Table1: Outbreaks of ZIKV 1952-2014

Although most patients experience only a fever or a rash –or even are asymptomatic- following ZIKV infection, during the outbreak in French Polynesia an increase in patients suffering from an otherwise rare neurological disorder, Guillan-Barre Syndrome (GBS), has been reported and associated with a previous ZIKV infection, suggesting that ZIKV might be a neurotropic virus (similar to DENGV or CHIKV), a view supported by studies that suggest that ZIKV can infect and replicate in neuronal cells. If however the infection of ZIKV with neuronal cells is causing GBS has not been established and it might be possible that the immune response –rather than the infection per se- causes the neurological abnormalities reported during the 2013-2014 outbreak.

ZIKV in Brazil

The transmission of ZIKV from the Pacific Islands to Brazil appears to have happened either during the 2014 FIFA World Cup and/or an international canoe-racing event attended exclusively by competitors from the Pacific. Although the first ZIKV were isolated from patients in the city of Natal, patients with symptoms suggesting ZIKV infection were reported in the city of San Salvador from February 15 onwards, and ever since a total of 28 Brazilian states have reported cases of Zika (as of February 06 2016). Following the detection of ZIKV in Brazil, confirmed cases have been reported in throughout Central and South America (with the exception being Chile) as well as the Caribbean.

Travellers to these countries also lead to the appearance of confirmed cases in Europe, the Near East, Australia, and the US. With the exception of the US, so far however no local transmission has been reported in these countries. In the US, the only cases of transmission occurred horizontally by sexual intercourse and not via mosquitoes. Given the ubiquitous presence of the vector, it is expected that ZIKV being locally transmitted not only in Central and South America (with the exception of Chile) as well as the Caribbean but also in the southern part of the US with expected number of both symptomatic and asymptomatic cases of 3-4 million for 2016 (based on mathematical models).

Following the reports of ZIKV infection in Brazil, the national Ministry of Health (MOH) reported an increase in cases of microcephaly, a normal rare congenital disorder characterised according to Estudio Colaborativo Latino Americano de Malformaciones Congenitas (ECLAMC) initially as a small head defined by a head circumference more than three standard deviations bellow the average in appropriated charts for sex and age, i.e. a head circumference below 33 cm (later changed by the Brazilian MOH to 32 cm). As of January 30 2016, the Brazilian MOH reported 3670 cases being investigated for microcephaly and 404 cases being confirmed either by clinical signs (congenital infection,intracranial calcification, dilatation of cerebral ventricles or changes in the posterior fossa and other clinical signs) or a linkage to ZIKV infection (maternal and/or fatal).

Figure: Reported cases of microcephaly and confirmed cases
in Brazil May 2015- January 2016

In contrast, the MOH of Colombia (another hotspot of confirmed cases of ZIKV) has not reported any increase in microcephaly cases related to ZIKV despite 2100 pregnant women being infected with ZIKV, raising the possibility that ZIKV is not the causative agent of microcephaly in neonates.

During the epidemic in French Polynesia, an increase in autoimmune diseases (leucopenia and thrombocytopenic purpura) and neurological diseases (GBS and meningoencephalitis) as well as a small increase in microcephaly has been observed, but –as in Brazil- co-infections with both CHIKV and DENGV are possible, suggesting that ZIKV per se is not causing microcephaly.

Several factors may account for the discrepancy between Brazil and Colombia regarding a link between ZIKV and the increase in reported microcephaly cases. First, in Brazil the local SINASC birth database records only 1% of birth defects in live births whereas the expected percentage is 3%, i.e. birth defects in Brazil are underreported and with focusing now on a potential link with ZIKV more cases of microcephaly are reported, fostered not only increased attention of the media but also due to mandatory reporting. Second, cases of microcephaly always have been higher in the northeastern region of Brazil. Third, assuming that the number of pregnant women infected with ZIKV is similar to those infected in the 2007 Yap/Micronesia outbreak, the expected number of microcephaly cases would have been around 360. The key problem might however the reporting system itself, since some states report all cases of microcephaly regardless if any link to ZIKV (either previous infection of the mother or infection of the fetus in utero) has been shown into the registry that originally was set up to report only those cases that may be linked to viral infection either of the mother or the fetus/neonate. Consequently, confirmed cases include also cases without ZIKV.

In the absence of any ZIKV vaccine and clear evidence that ZIKV is causing microcephaly however precautions should be taken. Mass extermination of mosquitoes in Brazil and other countries will not only combat ZIKV but also other arboviruses such as Yellow Fever Virus, CHIKV, and DENGV. The number of diagnostic laboratories needs to be increased and being equipped to detect and distinguish emerging viruses and the public health system needs to be improved. Funds need to be make available to care for children born with microcephaly. Given that ZIKV has been shown to be transmitted by sexual intercourse and blood transfusion, issues like access to birth control need to be addressed in particular if ZIKV can be linked to an increase in microcephaly.

In order to understand ZIKV, we need an animal model – not an easy task since ZIKV only replicates in mice if injected intracelebral. As discussed in a previous post, differences between strains circulating in Asia and in Africa as well as those circulating in monkeys need to be addressed as well. Finally the role of transmission of ZIKV via saliva, urine and sexual intercourse needs to be determined as well, although they might only play a minor role compared to the transmission of ZIKV by mosquitoes. Finally so far the focus is on pregnant woman, ignoring the prevalence of ZIKV among cohabiting couples. Future epidemiology however will give us hopefully a full picture of the presence of ZIKV among different socioeconomic groups.

Sunday, 31 January 2016

Zika Virus (ZIKV): similarities to other arboviruses

Zika Virus (ZIKV) is an arbovirus belonging to the Flaviviridae transmitted primarily by mosquitoes (including Aedes Agypti and Aedes albopictus). Although first identified in a rhesus monkey from the forests in Uganda in the year 1947 -with an estimated first emergence probably in 1920- the first human case was only reported in 1952 in Nigeria. ZIKV has been shown to be distributed Northern Africa as well as in Southeast Asia and the Pacific; however only a few human cases in Africa and Asia were identified until 2007, when a ZIKV outbreak was reported in Yap/Micronesia followed by an outbreak in French Polynesia and New Caledonia 2013 and currently in Central and South America as well as the Caribbean.

Most epidemiological studies that are based on the seroprevalence of neutralizing antibodies, suggest that up to 73% of the population (6.1-73%) have been exposed to ZIKV.

Table 1: Outbreaks of ZIKV in Africa, Asia and the Pacific prior 2016 and seroprevalence of
ZIKV

Being a Flavivirus, ZIKV -like the related West Nile Virus (WNV), Japanese Encephalitis Virus (JEV), Chikungunya (CHIKV) and Dengue Virus (DENGV)- has a single strand, positive sense RNA genome, encoding for a polyprotein that ultimately is processed into three structural proteins, the Capsid (C), precursor of Membrane protein (prM), and Envelope (E) protein as well as seven non-structural proteins (NS1-5).

Figure: ZIKV structure

Figure: ZIKV polyprotein

Table 2: ZIKV proteins

Similar to CHIKV, the infection with ZIKV is a relative mild disease, characterised symptoms ranging from mild fever, headaches, conjunctivitis, maculopapular rashes, vertigo, or myalgia with low mortality and often is asymptomatic.

Since the clinical presentation is similar to infections with other arboviruses, in particular CHIKV and DENGV, diagnostics is difficult and mostly done by using RT-PCR of samples that are CHIKV and DENGV negative. Although serological tests (ELISA, Immunofluorescence) have been used in the past, they are less reliable due to cross-reactivity with other flaviviruses such as DENGV or Yellow Fever Virus. Currently no commercial kit is available to test for ZIKV in patients.

Being transmitted by infected mosquitoes, the initial target cells are in the skin compartment, both skin fibroblasts, epidermal keratinocytes and dendritic cells are highly permissible for ZIKV which enters the host cell a number cellular receptors, in particular DC-SIGN, AXL, Tyro-3, and (albeit to a lesser extent) TIM-1. Primary skin fibroblasts infected with ZIKV support viral replication whereas in infected primary epidermal keratinocytes, similar to keratinocytes infected with DENV, exhibit large cytoplasmic vacuolation and pyknotic nuclei can be observed, suggesting that ZIKV induces apoptosis in these cells despite supporting viral replication.

Similar to JEV and CHIKV infected cell, ZIKV induces the formation of autophagosome-like structure that form the scaffold for the assembly of viral replication centres. If the expression of the viral preE2/E1 and Capsid proteins also induces ER stress (similar to CHIKV) and if the ER stress induced by ZIKV contributes to the induction of autophagy and/or caspase dependent apoptosis has not been demonstrated yet, but seems to be very likely.

Figure: CHIKV and the ER stress response

Figure: JEV and the ER stress response

Figure: JEV and the induction of autophagosome formation via non-structural and structural
proteins located at the ER

Likewise it has not been demonstrated if the formation of autophagosomes by ZIKV in primary skin fibroblasts is -as in the case of CHIKV- dependent on LC3-C and NDP52. Given that only the Asian lineage, in contrast to the African lineage, of ZIKV has been able to cause prolonged epidemics in the human populations (in 2007 and currently in the Americas) it has been speculated that the viral NS1 gene adaptions increases viral fitness in humans. Therefore, it might be necessary to investigate the ability of different isolates from different lineages to induce the formation of replication centres in primary human cells. If the decrease in autophagic flux or viral induced ER stress in infected primary dermal and skin cells or in keratinocytes induces apoptosis has not been demonstrated.

Figure: ZIKV induction of the antiviral response

Antiviral response induced by ZIKV : role of the antiviral interferon response

In primary skin cells, following viral entry and release of the viral genome, the viral RNA induces the transcription of Interferon stimulated genes (ISG) such as OAS2, MX1, and ISG15, as well as the transcription of RIG-1, MDA-5, and Toll-like receptor (TLR-3) in a Interferon-β (IFN-β).As a result, viral replication is inhibited.

Since the infection of primary human dermal fibroblasts and primary human foreskin fibroblast cells (HFF) with ZIKV also induces the formation of LC3-II positive membrane vesicles , it might be possible that TLR-3 mediated NF-κB signaling induces autophagy in a DRAM-1 dependent manner.

If these vesicles however represent viral replication centres or are involved in antiviral signaling by degrading viral components (or are even involved in the presentation of viral antigens via the MHC- Class I and II complexes) is not clear.

Figure: TLR-3 and autophagy; formation of autophagosomes via DRAM-1

The intracelebral inoculation of mice with ZIKV results in productive viral replication in both neuronal and astroglial cells, with autophagosome-like structures present that resemble viral replication centres. A subset of infected cells however has been shown to undergo necrosis, suggesting that the infection of neuronal cells with ZIKV -similar to human medullablastoma TE-671 cells, astrocytes and neuronal cells infected with JEV- induces both the formation of autophagosome-like structures as well as cell death. If however ZIKV induced cell death is dependent on the induction of caspase-3 and -9 in addition to ROS dependent activation of apoptosis signaling kinase (ASK)-1 and p38 MAPK signaling pathways has not been demonstrated. Despite the close proximity of ZIKV replication centres, an involvement of the ER stress response in the formation of ZIKV RC has not been demonstrated as well.

ZIKV and the nucleolus

As discussed in a previous post, a number of viral proteins localises to the nucleolus, thus (potentially) inducing the formation of autophagosomes and/or inducing via activation of p53. In the case of the small isoform of West Nile Virus (WNV) Capsid protein for instance, p53 dependent apoptosis is induced by sequestration of Hdm2 to the nucleolus whilst the large isoform inhibits apoptosis and activates mTOR-dependent p70S6K, thus inducing translation of viral genes. If either isoform, affects also the formation of LC3-II positive vesicles has not been demonstrated; it should be noted however that the NS4A and NS4B proteins from all WNV isolates (except NY99) induce autophagy as a result of ER stress. Since the NS4B protein derived from the WNV Kunjin subtype also localises to the nucleolus, an involvement of the nucleolus in the formation of WNV RC cannot be ruled out.

Table3: Flavivirus proteins that localise to the nucleolus

It might therefore possible that the nucleolar localisation of ZIKV protein(s) might induce a cell cycle delay, promote apoptosis or induce autophagy.

Figure: ZIKV might promote autophagy or induce apoptosis by sequestering p53

ZIKV, Microcephaly, and Guillan-Barre Syndrome

In the case of viral induced microcephaly, in utero infection with Human Cytomegalovirus (HCMV) has been associated with cases of microcephaly in newborn infants, but if ZIKV is a causative agent of microcephaly as well or if other factors -such as co-infection of pregnant women with other neurotrophic viruses such as DENGV or CHIKV, and/or parasites such as malaria as well as environmental factors despite the isolation of viral RNA from the placenta and amniotic fluid also play a role is not clear.

During the current outbreak of ZIKV in the Americas as well as during the outbreak of ZIKV in French Polynesia, an increase in patients exhibiting Guillan-Barre Syndrome (GBS), a rare neurological disorder caused by an autoimmune response, has been reported. Although so far no direct link between ZIKV and GBS has been reported, it might be possible that the decrease in autophagic flux in ZIKV infected neuronal cells induces apoptosis or if the observed increase is related to an autoimmune response as a result of the immune response (as in cases of rheumatoid arthritis in CHIKV positive patients) remains to be seen.

In summary, based on results published on other members of the Flaviviridae, ZIKV might exhibit similar features regarding the interaction with host cell, including the formation of replication centres by inducing the ER stress response, the localisation of viral proteins to the nucleolus and -probably most importantly-the induction of antiviral Interferon signaling via the induction of TLR-3 mediated NF-κB signaling (that might also induce the formation of autophagosomes, supporting viral replication and/or degrading viral proteins and RNA) and the induction of Interferon stimulated genes (ISG), thus inhibiting viral replication.