Once the attached viral particles are internalized into the cell, the viral genome should be released

inside
the cytoplasm to start translation and replication.
o The mechanism of penetration of the flavivirus genome into the cytoplasm is initiated by the
fusion of the viral envelope with the membranes of the cellular endosomes from the host cell, a
process triggered by acidic pH inside cellular endosomes
o This mechanism of penetration is consistent with, as mentioned before, an early observation
showing that ZIKV particles were sensible to acidic pH, and were inactivated by treatment with
acidic pH lower than 6.2
o Along this line, the sensitivity of ZIKV particles to acidic pH is consistent with the observation
performed with other flaviviruses indicating that, in the absence of target membranes, the
exposure of flavivirus virions to acidic pH induces rearrangements of the E glycoprotein, which
result in a loss of infectivity
The viral RNA acts as mRNA inside the cytoplasm of the infected cell, and negative-strand viral RNA is
synthesized and directs positive-strand RNA synthesis in association with a virus- induced network of
membranes derived from the endoplasmic reticulum, ER
Although flavivirus replication is thought to occur in the cellular cytoplasm, it should be noted that one
study reported that ZIKV antigens could be found in infected cell nuclei
De novo synthesized positive strand-RNA has to be packaged in progeny virions that bud into the ER to
form enveloped immature virions
These virions traffic through the Golgi complex and, then, the prM is cleaved in the trans- Golgi network
for particle maturation prior to release from the infected cell

Knowledge regarding the cellular response to ZIKV infection is still scarce.
However, it has been experimentally probed that replication of ZIKV provokes an innate antiviral response,
inducing the transcription of TLR3, RIG-I, and MDA5, as well as that of several interferon stimulated
genes, such as OAS2, ISG15, and MX1, characterized by a strongly enhanced beta interferon gene
expression (Hamel et al., 2015).
In addition, ZIKV infection is sensitive to interferon (IFN) signaling, as pretreatment of primary skin
fibroblasts with IFN-alpha, beta, and gamma reduces ZIKV infection (Hamel et al., 2015).
ZIKV infection also upregulates the autophagic pathway in infected skin fibroblasts (Hamel et al., 2015),
which is consistent with the observations for other related flaviviruses
Moreover, the autophagic marker LC3 colocalizes with viral proteins within ZIKV-infected cells, and the
infection can be reduced by treatment with the autophagy inhibitor 3- methyladenine, whereas upregulation
of autophagy using Torin 1 increases ZIKV replication

Zika appears to use the C-type lectin receptor DC-SIGN and members of the TIM and TAM families of
phosphatidylserine receptors on host cell surface to gain access to the cytoplasm via recep- tor-mediated
endocytosis.
dermal fibroblast, keratinocytes, and immature dendritic cells can all be readily infected and support viral
replication. Skin den- dritic cells
In infected primary human fibroblasts, the virus triggers an innate immune response, including expression
of interferons, and both type I and type 2 interferons inhibit viral replication.
At the molecular level TLR3, which recognizes double-stranded RNA appears to be particularly central to
the innate immune response to Zika infection.
This is reminiscent of other flaviviruses that have been studied.
At the cellular level the virus induces autophagosome formation to promote replication and may trigger
apoptosis to foster viral dissemination.

While Guillain-Barre syndrome, a weakness or paralysis caused by immune attack on the peripheral
nervous system
microcephaly impacts the developing central nervous system and is likely due to teratogenic effects of the
virus, rather than the immune response,

After mosquito inoculation of a human host, cellular entry likely resembles that of other flaviviruses,
whereby the virus enters skin cells through cellular receptors, enabling migration to the lymph nodes
and bloodstream.
Few studies have investigated the pathogenesis of Zika virus infection.
 One study showed that human skin fibroblasts, keratinocytes, and immature dendritic cells
allow entry of Zika virus.[19]
Several entry and adhesion factors (e.g., AXL receptor tyrosine kinase) facilitate infection, and
cellular autophagy, needed for flaviviral replication, enhances Zika virus replication in skin
fibroblasts.[19]
After cellular entry, flaviviruses typically replicate within endoplasmic reticulum-derived vesicles.
However, Zika virus antigens were found exclusively in the nuclei of infected cells; this finding
suggests a location for replication that differs from that of other flaviviruses and merits further
investigation.

Aspects of Zika virus pathogenesis remain unclear. Zika virus's association with neurologic sequelae,
including potential neuropathophysiologic mechanisms, is being actively investigated.

Structure of ZIKV

Similar to other flaviviruses, ZIKV is an enveloped, icosahedral virus with a non-segmented, single-
stranded, positive-sense RNA genome, closely related to the Spondweni virus.

The genome is 10,794 bases long, and has two non-coding flanking regions, the 5 NCR and the 3 NCR.

It comprises a single long open reading frame: 5-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3.

The NS1, NS3, and NS5 are large, highly-conserved proteins, while the NS2A, NS2B, NS4A, and NS4B
proteins are smaller, hydrophobic proteins.

The ORF codes for a polyprotein that subsequently cleaves into the capsid (C), precursor membrane (prM),
envelope (E), and non-structural proteins (NS).

The 5 end has a methylated nucleotide cap.

The non-polyadenylated 3 terminus has a looped structure.

The secondary structure leads to the formation of a sub-genomic flavivirus RNA (sfRNA), which is essential
for pathogenicity.

The E protein composes the majority of the virion surface and is involved with aspects of replication, such
as host cell binding and membrane fusion.

Cryo-electron microscopic studies showed ZIKV to be similar to other flaviviruses.

The envelope (E) protein is very similar to the West Nile virus, Japanese encephalitis virus, and the dengue
virus [13].

The virion is about 40 nm in diameter, having 5-10 nm surface projections arranged in an icosahedral
symmetry.

The ZIKV contains a nucleocapsid (approx. 25-30 nm in diameter), surrounded by a lipid bilayer, containing
the prM and E envelope proteins [13].

Replication of ZIKV

Using the PF-13 ZIKV strain isolated during an outbreak in French Polynesia, Hamel et al. extensively studied
the interaction of ZIKV in humans[14].

They observed that fibroblast, keratinocytes, and immature dendritic cells are all susceptible to ZIKV
infection, and support viral replication.

Once the virus is deposited on the epidermis and dermis of the host, there is a rapid increase in RNA copy
numbers and ZIKV particles, which indicate active viral replication.

replicate initially in the dendritic cells at the site of infection where it then spreads to other areas of the
body via the bloodstream and lymphatic system. Viral replication typically occurs in the cytoplasm of
infected cells, but there is a study that shows ZIKV antigens can also be found in the nuclei of
infected host cells
 The ZIKV infection of the epidermal keratinocytes results in cell apoptosis, indicated by the appearance of
cytoplasmic vacuolation and the presence of pyknotic nuclei in the stratum granulosum.

Inducing apoptotic cell death prevents the antiviral immune response, and increases the dissemination
of the virions.

The E protein is responsible for the fusion and entry of the virus through the host membrane.

However, the exact mode of entry is not yet understood.

Post dissemination, the virus continues to spread to the lymph nodes and blood stream.

Although flaviviruses are generally known to replicate in the cytoplasm, ZIKA antigens have also been
detected in the nuclei of infected cells [15].

Zika virus life cycle

The life cycle of ZIKV is similar to other known flaviviruses.
Briefly, the E proteins are involved in the attachment of the virus to receptors on the host membrane.
Subsequently, the virus gets internalised via endocytosis.
The viral RNA is released into the cytoplasm following fusion of the viral and host membranes.
The ssRNA is then translated, and the resulting polyprotein further gets cleaved into various structural and
non-structural proteins (C, prM, E and NS proteins) [16].
Replication takes place on the ER surface.
ZIKA antigens have also been detected in the nuclei of infected cells
Transcription and replication of the dsDNA result in the formation of new viral mRNA and ssRNA,
respectively.
Following viral assembly at the ER, the virion budding utilises the host ESCRT (endosomal sorting
complexes required for transport) machinery to transport to the Golgi apparatus.
The prM protein is cleaved, and the virion is rendered mature.
Finally, the mature virus exits the cell via exocytosis [16].

Host Immune Response [14]

Following viral infection the hosts immune system recognises viral proteins and elicits an antiviral response.

Previous studies on flaviviruses showed that both TLR3 and TLR7 (Toll-like receptors) are able to recognise
molecules on these pathogens.

Following recognition, the TLRs induce the host defense, initiating various signalling pathways, which lead
to the production of type I Interferons (IFNs), inflammatory cytokines and chemokines.

However, an in vitro study by Hamel et al on human fibroblast cell lines showed that ZIKV infection only
enhanced TLR3, but not TLR7.

The increased levels of TLR3 were accompanied with the production of IFN- and IFN- in the infected
cells, a corresponding increase in the transcriptional levels of interferon regulatory factor 7 (IRF7), and the
upregulation of expression of several IFN-stimulated genes.

Simultaneously, there is an increase in the expression of two chemokines (CXCL10 and CXCL11), which are
known to play crucial roles in adaptive as well as innate immunity.

Upregulation of CCL5, another chemokine known for its antiviral activity, was also observed.

As an antiviral host defence mechanism, the researchers found that both types I and II interferons are
capable of inhibiting virus replication.

The Hamel et al study also showed that ZIKV infection triggered autophagy in the infected fibroblasts.

Generally, autophagy-mediated degradation of viral proteins prevents the replication of the virus; however,
some viruses, including ZIKV, utilize certain components of the autophagy cycle, and promote viral
replication and dissemination.

Electron microscopic studies also revealed membrane vesicles (70 100 nm) in close association with the
ER; this indicates that ZIKV replication occurs intimately with the host cell membrane.
 Reproductive Cycle of a Zika virus in a Host Cell

The reproductive cycle of ZIKV follows that of other known flaviviruses.

First, the virion attaches to the host cell membrane receptors via the envelope protein which
induces virion endocytosis.

Next, the virus membrane fuses with the endosomal membrane and the ssRNA genome of the
virus is released into the cytoplasm of the host cell.

It is then translated into a polyprotein that is subsequently cleaved to form all structural and non-
structural proteins.

Replication then takes place at intracellular compartments known as cytoplasmic viral factories
in the endoplasmic reticulum resulting in a dsRNA genome.

The dsRNA genome is then transcribed resulting in additional ssRNA genomes.

Assembly then occurs within the endoplasmic retiiculum and the new virions are transported to
the Golgi apparatus and then excreted into the intracellular space where the new virions can
infect new host cells. [6]