INTRODUCTION:

Zika virus is an arbovirus of the genus Flavivirus, in the family Flaviviridae, which was first identified in 1947, in the Zika Forest in Uganda during a monitoring program on wild yellow fever. It is related to other flaviviruses, including the viruses that cause dengue, yellow fever and West Nile fever^[1]. Zika virus has been isolated from Ae. Africanus, Ae. Apicoargenteus, Ae. Luteocephalus, Ae. Aegypti, Ae. Vitattus and Ae.

furcifer mosquitoes and the most common clinical manifestations in patients with Zika infections included high fever, malaise, stomach ache, diarrhea, conjunctivitis, dizziness, and anorexia other less frequent manifestations included myalgia, headache, retro-orbital pain, edema, and vomit^[2].

Eradication of Zika virus poses substantial challenges because of its sylvatic transmission cycle between aedes mosquitoes and non-human primates. Although Zika virus is known to have circulated in parts of Africa and the Asia-Pacific region, a series of epidemics during the past decade have transported this virus eastward across islands in the Paciﬁc Ocean^[3].

In 1954, the first 3 cases of human infection were reported in Nigeria. Serosurveillance studies in humans suggest that Zika virus is widespread throughout Africa, Asia, and Oceania^[4]. In 2007 the first big Zika outbreak was reported, in the Yap Island in Micronesia. Other Pacific islands- Fiji, Vanuatu have had periodic outbreaks since then. The current outbreak has now spread to other countries in South America, Central America, and the Caribbeans. WHO has reported 23 countries and territories in Americas from where local transmission of Zika virus has been reported.^[5]

However ZIKAV transmission was only confined in the north-eastern states of Brazil in May 2015. This event was followed by rapid spread throughout the country, and subsequently to most of the countries in the Americas. In 2016, it was reported that an outbreak of zika virus was occurring in Americas, the pacific and the Caribbean^[6].

ZIKA Virus:

The Aedes mosquito that deposits the virus in the epidermis and dermis of the bitten host during a blood meal transmits ZIKV. Indeed, both skin fibroblasts and epidermal keratinocytes were found to be highly permissive to infection with ZIKV Infection of skin fibroblasts rapidly resulted in the presence of high levels of RNA copy numbers and a gradual increase in the production or ZIKV particles over time, indicating active viral replication in the infected cells. Virus-GAG interactions is due to an electrostatic interaction between the positive amino acids of E glycoprotein and the negative charge of sulfate groups on the polysaccharide, making this interaction for concentrating the viral particles around the cell before involving the receptors for entry. Despite several researchers trying to elucidate the receptors that mediate the flavivirus entry, so for this process is not well understood. A large number of molecules have been described as flavivirus candidate receptors in different cell types, but their precise role in virus endocytosis remains obscure. Currently, the best-characterized protein families that bind to and enhance flavivirus infection are C-type (calcium-dependent) lectin receptors and the recently identified phosphatidylserine receptors, T-cell immunoglobulin and mucin domain (TIM) and TYRO3, AXL and MER (TAM), and αvß3 integrin. In the case of ZIKV, it was reported that entry is mediated by DC-SIGN, AXL, Tyro3 and, although to a lesser extent, by TIM-1.

Zika virus (ZIKV) was discovered in the Zika Forest of Uganda during research supported by the Rockefeller Foundation to study the enzootic or sylvatic cycle of yellow fever virus and to identify additional arboviruses. Sentinel Rhesus monkeys were placed into the canopy in April, 1947 and one developed fever and viremia, leading to ZIKV isolation by inoculation of its blood intracranially into infant mice. The following January, ZIKV was isolated from the canopy-dwelling mosquito Aedes africanus collected at the same site. These cases were accompanied by fever, headache, diffuse joint pain, and in one case, slight jaundice. Two years later, experimental infection of a yellow fever-vaccinated human volunteer with a 6th mouse brain-passaged ZIKV strain from the 1954 Nigerian human isolates resulted in a relatively mild febrile illness (slight headache, malaise, fever) associated with natural infection. Attempts to demonstrate transmission by Aedes (Stegomyia) aegypti mosquitoes fed on the infected volunteer did not succeed. The first direct detection of ZIKV in Asia as well as the first evidence of transmission by an urban vector occurred when the virus was isolated from A. aegypti mosquitoes collected in Malaysia in 1966. Eleven years later, the first human infections in Asia were diagnosed from central Java in Indonesia by seroconversion in seven patients presented with fever, malaise, stomach ache, anorexia and dizziness. In addition to direct evidence of the range of ZIKV circulation from these isolations and patient diagnoses, human serosurveys have suggested a broad range in both Africa and Asia.^[7]

Zika was later identified from multiple countries in West and Central Africa, where it was also believed to persist in forest cycles between arboreal mosquito vectors and simian hosts. Molecular analyses of ZIKV lineages suggested two introductions from Uganda into West Africa, the first beginning around 1935 and the second around 1940.

Evidence of Zika in Pakistan, Malaysia, and Indonesia indicated that Zika became established in Asia, perhaps around 1960. This Asian lineage was believed to be the source of an introduction of Zika to Yap Island, Micronesia, which resulted in 2007 in the first epidemic attributable to this virus. Although the 49 clinical cases on Yap had relatively mild symptoms (rash, fever, arthritis, conjunctivitis), household surveys validated that more than 73% of the inhabitants of the island had been infected and that this pathogen could easily be transported by infective travelers to other islands in Oceania, or even to the Americas. During the health crisis, no hospitalizations or deaths were recorded, but it was the first time that Zika fever was found to be hemorrhagic. Additionally, this was the first time that ZIKV was discovered outside of the typical geographical range – Africa and Asia. It was hypothesized that ZIKV could spread to other Pacific Islands ^[17]. A larger outbreak of Zika in French Polynesia during 2013, also derived from the Asian lineage, followed the epidemic in Yap. An epidemic in northeastern Brazil in early 2015 is suspected to have begun with the introduction of Zika by a traveler from French Polynesia. More than twenty countries in the tropical Americas, as well as the United States, the Commonwealth of Puerto Rico, and the US Virgin Islands, currently recognize active ZIKV transmission.^[8]

Structure of ZIKA Virus:

The structure of zika virus bears similarities with the other viruses of the Family of Flaviviridae and the Genus of Flavivirus, especially Dengue Virus (DENV). Flaviviruses are classified in Taxonomy under the group ‘ssRNA positive-strand viruses, no DNA stage where RNA strand directly provides the template for viral protein synthesis without any intermediate DNA step. ZV contains a positive sense RNA genome at the core covered by a capsid followed by a lipid bilayer and an icosahedral outermost shell.

Fig.1 Structure of Zika Virus

Envelope (E) Protein:

These proteins are attached to the underlying lipid membrane by their respective transmembrane regions. E protein consists of an E protein complex of three domains named DI, DII and DIII present on the surface and a fourth stem-transmembrane domain acting as an anchor. The DI, II and III domains are arranged such that DI is in the center, joining the DII on one side and DIII on the other, creating a monomer. One monomer joins another monomer in antiparallel fashion making a dimer.

Fusions Loop of DII Domain:

The DII domain has a ‘fusion loop’ (FL) at its tip where it joins the DIII of other monomer completing the dimer structure. There is a small gap in this area consisting of all hydrophobic residues 98-109. The fusion loop is a highly conserved sequence of residues in Flavivirus Family that plays a key role in the fusion of the host cell membrane and viral membrane.

Membrane Protein:

Each E protein has M protein hidden under it consisting of the M loop and stem-transmembrane domains (M-TM). M loop is at the N terminus of protein while the M-TM domains are responsible for anchoring the M protein in the lipid membrane.

Non Structural Protein:

The nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5, are seven in number. The evidence suggests that NS1 glycoprotein plays an anchoring role in fetal pathologies especially the microcephaly. NS1 helps in immune evasion as it is synthesized in the host cell and secreted out on the surface of its cell membrane.^[9]

Fig.2 Zika Virus Transmission

Methods of ZIKA Virus Transmission:

Human to human transmission of zika virus predominantly occur from the bite of an infected mosquito of the Aedes species. Zika virus RNA has been isolated in blood, semen, urine, saliva, amniotic fluid, breast milk and cerebrospinal fluid. There has been one reported case of laboratory-acquired zika virus disease in the US.Cross-section of Zika virus, showing the viral envelope composed of envelope proteins (red) and membrane proteins (purple) embedded in the lipid membrane (white): The capsid proteins (orange) are shown interacting with the RNA genome (yellow) at the center of the virus.

Sexual Transmission:

Zika virus is the only arbovirus isolated from human semen which supports the hypothesis that zika virus can be transmitted by sexual intercourse. The first case of sexually transmitted zika virus was reported in 2011, between an infected male who was exposed to the virus while traveling to a zika virus-infected area and later transmitting the virus to his female partner. Another case reported transmission between a male traveler infected with zika virus who transmitted the virus through anal intercourse with his male partner. Although both men had exhibited Zika symptoms, the male traveler had a confirmed infection of zika virus and dengue, while his partner appeared to have only contracted zika virus. Semen samples collected 17 and 24 days post-onset of the illness showed that only the traveler had detectable levels of zika virus present in semen while his partner did not. Other studies have detected zika virus in semen supporting the notion of sexual transmission. The virus has also been detected in breast milk, although breast feeding-associated transmission has not yet been reported.^[10].

Blood Transfusion:

The potential for Zika virus transfusion transmitted infection was suspected in French Polynesia after viral RNA was detected in 2·8% of asymptomatic blood donors in 2014, and further confirmed in Puerto Rico in 2016 with 1·1% of blood donors identified as viraemic. In 2017, Zika virus RNA-positive asymptomatic blood donors were detected in Florida and Texas. Transfusion transmitted infection was confirmed in Brazil in 2016. As with sexual transmission, transfusion -transmitted infection is difficult to prove in endemic areas. With the poor availability of molecular biology based assays and the challenges associated with interpretation of serology data, the number of documented cases of transfusion transmitted infection is probably underestimated. Zika virus is a new challenge for the blood supply. WHO, the US Food and Drug Administration (FDA), and the American Association for Blood Banks have issued recommendations to prevent transfusion-transmitted infections. As most infections are asymptomatic, the most effective mitigation strategies to prevent transfusion transmitted infection are nucleic acid testing of blood donations or pathogen inactivation. A commercially available licensed pathogen inactivation system has been shown to inactivate a wide range of pathogens, including Zika virus, after photochemical treatment of plasma and platelets. Robust inactivation of Zika virus has also been shown with a pathogen inactivation system under development for the chemical treatment of red blood cells.56 The FDA has also issued guidance for donors of other substances of human origin, even though Zika virus transmission has not been reported through organ transplantation.^[11]

Mosquito Transmission:

A sylvatic cycle of ZIKV transmission between non-human primates and arboreal zoophilic Aedes spp. mosquitoes also exists in African and Asian forests, in which humans may be incidentally infected. In Africa, the virus has been isolated or detected by PCR in Ae. Africanus, Ae. Furcifer, Ae. Vittatus, Ae. Taylori, Ae. Luteocephalus, Ae. Albopictus and other Aedes species mosquitoes, while in Asia it has been detected in Ae. Aegypti, which is considered the main ZIKV epidemic vector outside Africa. Aedes hensilli, the most abundant mosquito specie in Yap Island and the competent one for ZIKV transmission, was conceivably the main vector for ZIKV transmission during the 2007 outbreak, even though the virus could not be detected in any mosquito samples. Aedes aegypti and Ae. Polynesiens is, the most common mosquito species in French Polynesia, were probably involved in local ZIKV transmission, while Ae. Aegypti and possibly also Ae. Albopictus are considered the vectors responsible for transmission in Brazil and in other countries in South and Central America, although reports on ZIKV isolation from field mosquitoes are lack. Both Ae. Aegypti and Ae. Albopictus are competent vectors for ZIKV, but transmission from Ae. Albopictus is less efficient than from Ae. aegypti due to a longer extrinsic incubation period. ^[12]

Mother to Fetus Transmission:

Substantial evidence now indicates that Zika virus can be transmitted from the mother to the fetus during pregnancy. Zika virus RNA has been identified in the amniotic fluid of mothers whose fetuses had cerebral abnormalities detected by ultrasonography, and viral antigen and RNA have been identified in the brain tissue and placentas of children who were born with microcephaly and died soon after birth. Among the few reports of teratogenic effects of flaviviruses, investigators described the brain and eyes as the main targets. No presence of virus and no pathological changes were detected in any other fetal organs apart from the brain, which suggests a strong neurotropism of the virus. The frequency of and risk factors for transmission are unknown.

Breast Milk Transmission:

Transmission through breast milk has not been documented, although the breast milk of a woman who became symptomatic with Zika virus infection on the day of delivery contained infective Zika viral particles in high titer.^[13]

Signs and Symptoms:

The common symptoms of ZIKV are quite broad in nature and are characteristic of many flaviviruses, such as Dengue. These symptoms can persist for 3 - 12 days and include arthralgia/ arthritis, edema, fever (mild-low grade), headaches, retro-orbital pain, conjunctivitis, maculopapular rash on face and limbs, myalgia, vomiting and other digestive disorders. Less commonly reported symptoms of flaviviruses are lymphadenopathy, prostatitis, anorexia, vertigo and hematospermia. Before the outbreak in French Polynesia (2013), there was neither severe presentations of these symptoms nor hospitalization due to ZIKV infection reported. Currently, there is an association between Zika fever and severe neurological complications in adults and congenital malformations in fetuses that has been the cause of some deaths.^[14]

Treatment:

No specific anti-viral drug is available to combat ZIKV infection and to protect from its dreadful pandemic threats. Designing of effective/ specific antiviral drugs and therapeutics for safeguarding the health of humans against ZIKV is under high progress . Some relief to the patient can be provided by timely symptomatic treatment in accordance with the clinical signs. Supportive therapy includes.

Treatment:

1) Sufficient rest .

2) Administration of fluids for preventing dehydration .

3) Analgesics for pain relief.

4) Antipyretics (acetaminophen or dipyrone) for reducing fever .

5) Checking pruritic rashes by the use of anti-histamines.

However, the use of aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) is contra-indicated to avoid any complications such as hemorrhages as noticed in the case of other flaviviruses (DENV and Chikungunya) and also need not be used during pregnancy.^[15]

Diagnosis:

The diagnosis of ZIKV infection is quite difficult because of the overlap of symptoms with other arboviral diseases and because of the broad cross-reactivity among flaviviruses of the antibodies induced by infection. Thus, laboratory diagnosis of acute ZIKV infection relies on the use of molecular tests for the direct detection of viral nucleic acids in blood and other biological specimens .

Zika virus can be tested in following samples; blood, saliva and urine. Mostly blood tests are performed. Following tests can be used for detection :-

a) PCR Test:

It is useful in the first 3-5 days after the onset of symptoms. It helps in the direct detection of Zika virus RNA or specific viral antigens in clinical specimens.

b) Serology Test:

It detects the presence of antibodies but are useful only after five days^.

ZIKV can be isolated from cell culture but the protocol is reserved to specialized laboratories. Zika fever diagnosis relies in routine on the detection of ZIKV RNA by molecular tools. Detection of ZIKV RNA is possible on blood and saliva collected at the acute phase of the disease . The use of saliva sample is of particular interest when blood samples are difficult to collect . Detection of ZIKV RNA after the first week after symptoms onset can be performed in urines.

These two institutions would be the apex laboratories to support the outbreak investigation and for confirmation of laboratory diagnosis. Ten additional laboratories would be strengthened by ICMR to expand the scope of laboratory diagnosis. RT- PCR test would remain the standard test. As of now there is no commercially available test for Zika virus disease. Serological tests are not recommended by them for the detection.

Prevention:

No vaccines for protection against Zika are currently available, so protecting against mosquito bites is the primary method of prevention. Given the possibility that the virus may be sexually transmitted, couples should take precautions against direct transmission of the virus from their partners . While several candidate vaccines are in development, FDA approved vaccines are not likely to be available for several years.

Steps for preventing mosquito bites:

· Use long-sleeved shirts and long trousers/pants.

· Stay in enclosed places with air conditioning or places with windows and doors that have screens to prevent the entry of mosquitos.

· Sleep under mosquito nets.

· Use registered insect repellents. When these are used as instructed, they are safe and effective, even during pregnancy or breastfeeding.^[16]

Vaccine:

Despite a limited number of available full-length Zika virus sequences, the molecular data are sufficient to reveal patterns of viral evolution and movement . The current data suggest that any vaccine product developed against any strain of Zika virus should be protective against all strains. The very nature of the close relatedness among the flaviviruses is responsible for the challenges in developing diagnostic algorithms for distinguishing among these viruses . World Health Organization experts have suggested that the priority should be to develop inactivated vaccines and other non-live vaccines, which will be safe to use in pregnant women and those of childbearing age . Bharat Biotech International (India) reported in early February 2016, that it was working on vaccines for Zika using two approaches: "recombinant", involving genetic engineering, and "inactivated", where the virus is incapable of reproducing itself but can still trigger an immune response with animal trials of the inactivated version to commence in late February.

Plasmid DNAVaccine:

The plasmid DNA vaccine containing the genes of the prM and E (prM-E) protein from the Brazilian zika virus strain. This vaccine has been tested on Balb/c mice inoculated intramuscularly with 50 ug of DNA. A single dose of this vaccine induces complete protection at 4 or 8 weeks post-vaccination after the injection of 105 particle /ml of wild zika virus .They demonstrated that the protection come only from the production of IgG-specific antibodies , without cellular immunity involved initially, although it is likely that memory T-lymphocytes will be essential for long-term protection.^[17]

m-RNA Vaccine:

The mRNA vaccine candidate composed of prM-Env from the French Polynesian 2013 ZIKV strain encapsulated in lipid nanoparticles (LNPs), which facilitate biodelivery and presentation. The nucleoside modification (methylpseudouridine) was designed to minimize innate immune system recognition. Inoculation of C57BL/6 and BALB/c mice with this vaccine induced a strong neutralizing antibody response in both mouse strains, and a specific CD4+ T cell response was also detected in the BALB/c mice. This vaccine was also tested in rhesus macaques, which were vaccinated and challenged 5 weeks later with Puerto Rico ZIKV strain (PRVABC59). Where as the placebo-immunized monkeys developed viremia, them RNA-LNP-vaccinated monkeys were completely protected.

Purified Inactivated Particle Vaccine:

The immunogenecity and protective efficacy of a vaccine formed by purified and inactivated virus particle derived from the Puerto Rico PRVAB59 strain of zika virus were evaluated . The administration of 1 ug of this aluminium -adjuvanted vaccine was tested both intramucularly and subcutaneously on Balb/c mice. Protective raised titres to prevent post-vaccination exogenous infection (wild- type zika virus ) were only obtained with intramuscular administration.

This initial study was completed with the testing of these vaccines in non-human primates, as the replication of zika virus in mice depends on the strain and their genetic characterstics and results connot be extraplotted to humans. A total of 16 chimpanzees were immunised subcutaneously with 5 ug of the vaccine consisting of purified and inactivated virus particles adjuvanted with aluminium at weeks 0 and 4. All the animal developed antibodies specific to E protein .After inoculation of the wild-type strain, all chimpanzees showed complete protection against the virus, and it could not be detected in any biologocal fluids .

Live Attenuated Vaccne:

The design of live attenuated ZIKV vaccines has followed a similar strategy to that deployed for the DENV live attenuated tetravalent chimeric vaccine (Dengvaxia), which is licensed on a country by country basis. Dengvaxia is a mixture of DENV serotypes 1–4 coupled to NS genes from Yellow fever strain 17D; the Yellow fever vaccine based on these genes has one of the best safety, efficacy, and durability profiles among anti-viral vaccines. In general,the development of live attenuated viruses requires a balance between immunogenicity and safety.^[18]

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Received on 04.12.2018 Accepted on 24.01.2019

Asian J. Pharm. Res. 2019; 9(1): 35-41.

DOI: 10.5958/2231-5691.2019.00007.8