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Zika Virus Infection, Basic and Clinical Aspects: A Review Article.

Background: Zika virus infection has recently attracted the attention of medical community. While clinical manifestations of the infection in adult cases are not severe and disease is not associated with high mortality rates, Zika virus infection can have an impact on fetal development and lead to severe neurodevelopmental abnormalities. Methods: To gain insight into different aspects of Zika virus infection, a comprehensive literature review was performed. With regard to epidemiology and geographical distribution of Zika virus infection, relevant information was extracted from CDC and WHO websites. Results: In this review, we discuss different basic and clinical aspects of Zika virus infection including virology, epidemiology and pathogenesis of disease. Laboratory methods required for the diagnosis of disease together with ethical issues associated with Zika virus infection will also be discussed in detail. Conclusion: Herein, we have tried to provide a multi-faceted view of Zika virus infection, with greater emphasis on disease status in Eastern Mediterranean Region.


Introduction
Flaviviruses are responsible for several important human diseases including Dengue, West Nile and Yellow fevers, Japanese encephalitis, Tick-borne encephalitis, and Zika fever. The major route of transmission for most Flaviviruses is through arthropod vectors and central nervous system inju-ry and hemorrhagic fevers represent major clinical outcomes. In recent years, Zika virus infections have attracted the attention of international medical community, chiefly because of their role in causing microcephaly and other neurodevelopmental abnormalities which occur as a consequence of maternal infections (1). Zika virus was first isolated in 1947 in the Zika forest in Uganda from a rhesus macaque (2). Serological studies performed on human serum samples in Uganda later showed the presence of neutralizing antibodies against the virus, providing the first evidence that the virus can infect humans (3). Later studies revealed the presence of infection in other regions of Africa as well as Asian countries (4). Until 2007 Zika virus infections were mostly considered an infection of limited geographical distribution; however, later outbreaks of infection in Pacific islands and South America, associated with reports that the virus might cause nervous system abnormalities in newborns attracted the attention of a wide range of people in the scientific community. The situation reached its peak in 2015 when a striking increase occurred in reports of Zika virus infections in Brazil (5). The outbreak then spread to other countries in South, Central, and North America. The infections were associated with a significant increase in the number of microcephaly and Guillain-Barré syndrome cases in the infected regions, and this led to the declaration of a Public Health Emergency of International Concern (PHEIC) by the WHO in early 2016 (6)(7)(8). Since then, a strong international effort has been started to investigate epidemiological, molecular, pathogenic and clinical aspects of Zika virus infections. In this review, we have discussed the current knowledge about various aspects of Zika virus infection and recent developments including antiviral treatments for this infection. The first four sections of this review cover virology, epidemiology, pathogenesis, and laboratory diagnosis of Zika virus infection. While Zika virus infection is a global concern, in the epidemiology section we have put some more emphasis on findings from Eastern Mediterranean Region (EMRO) countries. Considering the ethical considerations relat-ed to feto-maternal and neurodevelopmental dimensions of the Zika virus infection, the final section of this review provides a detailed account of ethical aspects of Zika virus infection (Fig. 1).

Virology of the Zika virus
Zika virus is a new emerging mosquito-borne virus belonging to the Flaviviridae family of viruses (9). This family is comprised of 4 genera: Flavivirus, Hepacivirus, Pegivirus, and Pestivirus (10). Zika virus belongs to the Flavivirus genus, which antigenically and phylogenetically is related to the Spondweni virus (9,11). Many important human pathogens are included in this genus, for instance, Dengue, West Nile, Yellow fever, tickborne encephalitis, Japanese encephalitis, Murray Valley encephalitis and St. Louis encephalitis viruses. These viruses are associated with a range of infections from asymptomatic or self-limiting febrile infections to some fatal diseases such as hemorrhage, shock, meningitis, and encephalitis (12). In Flaviviridae family, all members have enveloped viruses with a single-stranded RNA genome of positive polarity (10) which contained one open reading frame (ORF) with two flanking noncoding regions (at 5' and 3' end) (13). The genomes are 5' capped without a 3' poly (A) tail. A polyprotein is coded by the ORF then processed into three structural proteins (the envelope (E), the capsid (C) and the precursor of the membrane (prM)) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (1,12). The ssRNA is held within an icosahedral capsid shaped from 12-kDa protein blocks; the nucleocapsid is surrounded by a hostderived membrane contained two viral glycoproteins (14). Similar replication strategies are employed by the members of Flaviviridae family despite significant differences in tissue tropism, transmission, and pathogenesis (10).
However, considering rapid geographic expansion of Zika virus, its severe neurological complications and devastating effects on the fetus, investigations are underway to find safe and potent drugs and therapies. These approaches range from repurposing earlier approved drugs to the screening and testing of in silico designed drugs (16). The antiviral candidates comprise agents targeting cellular components in addition to viral ones (15). All of the steps of Zika virus life cycle in the host cell from entry to release can be targeted by antiviral agents. In order to block the viral entry into the cell, various strategies have been used. On one hand, blocking the entry by receptor binding agents such as nanchangmycin (previously used as antibacterial and insecticidal agent), ZINC33683341 and ZINC49605556 (both in silico designed) has been proposed. The other approaches include the inhibition of endosomal fusion using compounds that reduce the acidity of endolysosomal vesicles like chloroquine (an anti-malaria drug) and disrupting the electrostatic interaction between cell and virus membrane using squalamine (a cationic chemical) (16). RdRP activity of NS5 is an outstanding target for antiviral drugs. Therefore, several nucleoside analogues are investigated for their ability to inhibit Zika virus replication. Despite demonstrated efficacy of these drugs in cell cultures and animal models, some of them were unsuccessful in the clinical trials. Sofosbuvir (a nucleotide analog inhibitor) is a RdRP inhibitor approved by FDA for chronic HCV treatment. It also inhibits Zika virus replication (15)(16)(17). Zika virus N2B-NS3 protease and NS3 helicase, which play essential roles in virus replication, constitute potential tar-gets for antiviral drugs (15). As the Zika virus helicase is similar to those of the other members of Flaviviridae, antiviral agents targeted their helicase could also be used for the Zika virus (18). Likewise, berberine previously used against dengue virus showed a high binding affinity to the Zika virus NS3 protease (19) as well. Despite the many studies done on Zika antiviral treatments, so far no FDA approved category A drug has been found safe to use in mothers and fetuses (16). In early 2015, several cases presenting a "denguelike syndrome" investigated in Brazil (a non-Dengue virus and non-Chikungunya virus infection) and Zika virus was detected by reverse transcription polymerase chain reaction (RT-PCR) assay and confirmed by DNA sequencing (25,26). The Brazil Zika virus strain shares a common ancestor with the Zika virus strain that circulated in French Polynesia (27). On Jul 2015, in the State of Bahía, Brazil, an increase in the number of Guillain-Barré cases in which half of them had reported symptoms consistent with Zika virus infection (28). In Jan 2016, an unusually significant increase of GBS was reported in El Salvador. An Emergency Committee was convened by the Director-General of WHO, under the International Health Regulations (2005) on 1 Feb 2016, and finally announced "the recent cluster of microcephaly and other neurologic disorders reported in Brazil to be a PHEIC (28).

Situation and risk analysis in Eastern Mediterranean Region, regional strategic plan
Although no countries of the Eastern Mediterranean Region of WHO have reported importation of Zika virus disease or autochthonous transmission, however, of the 22 countries in the Region, the following 8 have reported dengue outbreaks in recent years and/or have the presence of com-petent Aedes mosquitoes: Djibouti, Egypt, Oman, Pakistan, Saudi Arabia, Somalia, Sudan and Yemen. Some of these countries are particularly vulnerable to emerging of Zika virus, because of the fragility of health systems, weakness of disease surveillance systems, the inadequacy of response capacities, and a suboptimal level of public health preparedness (29-31). Pakistan is the most-at-risk country in the Region, primarily due to a large number of travelers to and from the Americas, dense urban populations, a documented large epidemic of dengue, and the most favorable climatic conditions for the reproduction of Aedes mosquitoes (29). In Iran, national committee of Aedes-borne diseases recommended enhancement of surveillance after 2016 and updated national guidelines of surveillance and clinical management of suspected cases, especially for travelers returning from at-risk countries. As of Sep 2018, all of the samples from suspected cases of Zika virus sent to the National Laboratory of Aedesborne diseases were shown to be negative. Prevention of Aedes-borne diseases needs comprehensive national strategic action plan with "one health" approach and close collaboration between community, academic, and public health authorities. Global map of Zika virus infection demonstrated in (Fig. 2) and areas potentially at risk of Zika in (Table 1).

Pathogenesis of Zika virus infection
Studies on the pathogenesis of Zika virus have shown similarities with the pathogenesis of other Flavivirus infections. Following transmission by the mosquito bite, Zika virus can infect several different cell types including skin keratinocytes, dermal fibroblasts and dendritic cells (DCs) (9). In vitro studies on fibroblasts exposed to Zika virus have shown high infection rates in these cells 24 to 48 h after infection. Flow cytometric analyses of DCs exposed to the virus have also shown that up to 60% of DCs express viral antigens around 24 h after infection (9). Different cell surface receptors have been proposed to mediate Zika infection of permissive cells; including DC-SIGN, AXL and Tyro3 molecules (9) (Fig.  3). Following cell entry, Zika virus induces strong interferon responses in infected cells. Studies on Zika virus-infected human primary fibroblasts have shown strong upregulation of interferonbeta transcripts 24 to 48 h after infection (9). A milder response has been reported for interferonalpha. Upregulation of intracellular "pattern recognition receptors" (PRRs) involved in sensing non-self-nucleic acids seems to be a crucial part of the initial immune response to the virus (9). Studies on Zika virus-infected fibroblasts have revealed strong induction of RIG-I and MDA-5 transcripts. Both of these molecules are capable of initiating a signaling process following the detection of intracytoplasmic viral RNA molecules. Innate immune responses are followed by adaptive immune events including the activation of T cells; Zika virus-infected DCs migrate to regional lymph nodes where they stimulate T cell proliferation, differentiation and cytokine production (32). Productive infection of dermal fibroblasts and dendritic cells together with insufficient control of infection by innate and adaptive immune mechanisms usually leads to Zika virus viremia, which underlies the non-specific clinical symptoms which might last for a few days. In a pregnant woman, maternal viremia might lead to fetal viremia. It is not well known how the virus is transmitted to developing nervous system following the establishment of fetal viremia. While in circulation, Zika virus can infect fetal monocytes and these monocytes can carry the infection to the developing nervous system. Studies on fetal brain tissue derived from Zika virus-infected mothers who have undergone abortion have shown viral particles in neural cells, indicating that the virus can proliferate inside neural cells in a developing brain (5). While inside the CNS, Zika virus can exert direct neurovirulence, similar to other Flaviviruses. There is also evidence that Zika virus might cause indirect neurovirulence through the activation of immune mechanisms including microglial activation and macrophage infiltration (5). Indeed, in adults Zika virus might be an etiological factor for "Acute Disseminated Encephalomyelitis" (ADEM) (33), an immunemediated disease of the CNS, which occurs subsequent to varieties of viral and nonviral infections (34). Whether an ADEM-like process might affect developing brain remains an open question.

Laboratory diagnosis of Zika virus infection
Depending on the purpose of the investigation, laboratory diagnosis of Zika virus can be conducted by virus isolation, antigen detection, viral RNA detection with molecular assays and anti-Zika virus antibodies detection with serological assays (35).

Virus Isolation
The first isolation of Zika virus was performed by intracerebral mouse inoculation considered as the reference assay for arboviruses isolation (25,36).

Antigen Detection
Antigen detection is a valuable assay to approved Zika virus infection in autopsy tissues. Using immunohistochemistry (IHC) technique, Zika virus antigen has been detected in brain and placental tissues from congenitally infected newborns with microcephaly and miscarriages (41,42). Recently, new assays for detection of Zika virus antigen including NS3 protein identification by flow cytometry in whole blood (43), aptamer-based ELISA assay for targeting of Zika virus NS1 protein (44), NS1 protein based competitive ELISA (45), NS1 protein-based rapid tests (46) have been developed.

Serological Assays
Despite the fact that the molecular diagnosis possesses high sensitivity and specificity, short period of viremia can negatively affect the Zika virus RNA detection (65). Therefore, detection of anti-Zika virus antibodies by serological assays could be an advantageous option for a wider diagnostic window.
Detection of anti-Zika virus antibodies can be performed by different serological tests including complement fixation, haemagglutination inhibition, immunofluorescence (IF) assay, ELISA and neutralization tests (13,35 Because of cross-reactivity with other Flaviviruses such as dengue and yellow fever viruses, and the possibility of nonspecific reactivity, results of IgM detection assays should be interpreted with caution and positive or equivocal results must be confirmed by plaque-reduction neutralization testing (PRNT) (66).

Ethical aspects and challenges of Zika virus infection
Addressing ethical questions and moral conflicts of Zika virus infection may be essential to develop a comprehensive approach to control this public health crisis (70). Therefore, the health authorities commitment to allocating a fair portion of health resources for responding to this situation is morally required (71). The government should priorities paying the noticeable amount of money for preventive measures in order to control the Zika virus spread (72). This priority setting is usually the most difficult step while encountering such situations, especially is the absence of enough scientific evidence to measure the real burden of disease and also the presence of social and media pressure, amplified by 2016 Olympic games in Brazil (73).
National and international health authorities should observe the principle of veracity, by clear and transparent informing system for raising the public awareness regarding the real situation (74). At the international level, the "Cosmopolitan Solidarity" for encountering such universal pattern of disease spread seems to be inevitable (75). Like Ebola outbreak, responsiveness to actions required by responsible international bodies especially WHO is another responsibility of states to form a global health governance network (76). In mosquito control, governments should try to keep the moral obligation of observing biosafety and protecting the ecosystems in the mind while trying to restrict the insect spread and reproduction (77). During policy-making for disease control, deliberative ethical evaluation is required in order to protect of population basic civil and human rights such as people's free mobilization or reproductive rights in case that border or birth control are among the solutions (78,79). Similarly, confidentiality of patients' health information and protecting people against stigmatization must be observed when policies for reporting the probable cases to health authorities are discussed (80). The fear raised from high probability of congenital problems, mainly microcephaly, of neonates delivered by mothers with Zika virus infection in their first trimester of pregnancy (81), puts abortion at the center of moral concerns (82). The position of various schools of ethics regarding abortion has a range from strict prohibition to complete moral justification based on the moral status they consider for the fetus in each development stage. Accordingly, abortion laws are still different in various countries according to the social, political, religious and cultural context (83,84). In Iran despite of general prohibition of abortion by Criminal Law, the "Therapeutic Abortion Act" of 2005 allowed performing abortion in presence of a "definite diagnosis of retardation or malformation of the fetus that is unbearable for the mother…" only before 4 months (19th wk) after conception and any attempt for abortion after this gestational age is illegal and even criminal (85).

Conclusion
Together with Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), Zika virus infection qualifies as a "newly emerging" infectious disease, with the potential to cause serious public health issues. Unlike the other "newly emerging" infections which can lead to severe morbidity and mortality in infected adults or pediatric hosts, Zika infection does not pose a significant threat to infected adults and its risk is more due to the potential to cause fetal abnormalities, provided that the infection occurs during pregnancy. Indeed, among the four recent PHEIC declarations by WHO (i.e. 2009 Swine flu declaration, 2014 Polio and Ebola declarations and 2016 Zika virus declaration), Zika is unique in the sense that it is a member of the TORCH group of infections; i.e. the group of pathogens with the ability to lead to congenital infections/anomalies. This might influence both disease prevention and management strategies as well as raising ethical and sociological issues. Thus, increased awareness of the medical community together with improvements in vector control and disease surveillance systems are of utmost importance for controlling any potential Zika virus-related threats in different countries.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.