Juan-Carlos Saiz; Ángela Vázquez-Calvo; Ana B. Blázquez; Teresa Merino-Ramos; Estela Escribano-Romero; Miguel A. Martín-Acebes

“ Recently, and albeit an estimated 80% of ZIKV infected people are asymptomatic3, during the ongoing outbreak in Brazil, ZIKV RNA has been identified in brain, placenta, and amniotic fluid specimens, and its presence has been associated to microcephaly in infants and miscarries during pregnancy ( [70] ; [71] ) .
One important aspect that still has to be clarified is whether ZIKV infection in humans drives to viral titers enough to initiate a new cycle when an infected person is bitten by a naïve mosquito. ”

“ Clinical Manifestations and Pathogeny Humans Zika virus infection has been reported to be symptomatic only in around 18% of the cases ( [110] ) , in which it causes a mild, self-limiting disease with an incubation period of up to 10 days, often mistaken with other arboviral infections like dengue or chikungunya. Clinical manifestations in symptomatic cases resemble that of an influenza-like syndrome ( [68] ) , being the most common symptoms fever, rash, arthralgia, and conjunctivitis ”

“ Abstract Since the beginning of this century, humanity has been facing a new emerging, or re-emerging, virus threat almost every year: West Nile, Influenza A, avian flu, dengue, Chikungunya, SARS, MERS, Ebola, and now Zika, the latest newcomer. Zika virus (ZIKV) , a flavivirus transmitted by Aedes mosquitoes, was identified in 1947 in a sentinel monkey in Uganda, and later on in humans in Nigeria. ”

“ The arbovirus life cycle imposes several barriers to non-synonymous mutations in some important genes as a consequence of the intrinsic constraints associated with dual replication in mammalian and invertebrate hosts, thus driving to a more slowly fixation of mutations of these viruses when compared with RNA viruses transmitted by other routes. ”

“ 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 (Figure 2) . Electron microscopy studies of virus infected cells showed that ZIKV virions are found in short chains within tubular elements of the ER, which appeared to be in continuity with distended cisternae ( [4] ) . ”

“ However, since the end of 2015, an increase in the number of GBS associated cases and an astonishing number of microcephaly in fetus and new-borns in Brazil have been related to ZIKV infection, raising serious worldwide public health concerns. Clarifying such worrisome relationships is, thus, a current unavoidable goal. Here, we extensively review what is currently known about ZIKV, from molecular biology, transmission routes, ecology, and epidemiology, to clinical manifestations, pathogenesis, diagnosis, prophylaxis, and public health. ”

“ Human-to-human transmission of the Asian ZIKV strains along the Pacific Islands and South America has been associated with significant NS1 codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titers ( [53] ) . Furthermore, this report predicted the presence of several epitopes in the NS1 protein that are shared between ZIKV and DENV, pointing to a significant dependence of the recent human ZIKV spread on NS1 translational selection. ”

“ Therefore, methods of control for a species may not be accurate to control the other one. Furthermore, when the populations of A. aegypti is reduced, the opportunistic invasive A. albopictus may rapidly move into the area ( [63] ) .
Aedes aegypti and, to a lesser extent, A. albopticus are clearly involved in ZIKV transmission and spread, but other Aedes spp., such as A. polynesienis, are suspected to have also contributed to it, as it was the case during the 2013–2014 outbreak in the French Polynesia ( [64] ) . ”

“ In Africa, ZIKV natural transmission cycle involves primarily Cercopithecus aethiops and Erythrocebus patas monkeys ( [76] ) . In Asia (Borneo) , antibodies against ZIKV have been detected among semi-captive and wild orangutans ( [77] ) . However, this study reported a higher prevalence of anti-ZIKV antibodies in humans than in orangutans, suggesting a possible incidental infection of these animals through contact with mosquitoes infected by viremic people, or from recently established sylvatic cycles. ”

“ In August 2008, a scientist was bitten many times while studying mosquitoes in south-eastern Senegal. Six days after returning to his home in Colorado (U.S.) , he felt ill with symptoms of Zika fever and hematospermia. By then, he had had unprotected intercourse with his wife, who had not been outside the U.S. during the previous year, which subsequently developed symptoms of Zika fever. Virus infection was confirmed by serologic testing in both, but the presence of ZIKV in the semen of the patient was not investigated ( [83] ) . ”

“ In any case, it should be noted that all infected monkeys showed viremia during the 1st week after inoculation, as well as induction of specific antibodies about 14 days post-inoculation ( [3] ) . This induction of viremia in monkeys is supposed to play an essential role for the establishment of the enzootic transmission cycle between non-human primates and mosquitoes. ”

“ In resume, although ZIKV transmission by routes other than mosquito bites has been so far sporadic, further studies are mandatory to clearly establish their possible role on ZIKV epidemics.
Ecology Most arboviruses are perpetuated in transmission cycles independent of human hosts, but those with sylvatic cycles often infect people who accidentally intrude on their natural habitats. Nonetheless, in many instances, humans are dead-end hosts in complex transmission cycles that involve different wild and domestic vertebrate hosts, as in the case of JEV ( [77] ) or WNV ( [23] ) . ”

“ Transmission Cycle Mosquitoes The arthropod vectors of the ZIKV natural transmission cycle are mosquitoes of the genus Aedes ( [54] ) . As mentioned early, the virus was first isolated from A. africanus ( [2] ) and, since then, ZIKV has been isolated from A. aegypti ( [47] ) , the main vector of the virus, and also from A. albopictus ( [55] ) , confirming that both species are competent vectors.
Aedes aegypti is currently distributed in Asia and Oceania, the Americas, and in a few regions of Africa and Europe (Madeira and the north-eastern Black Sea coast) 2 ”

“ Table 1 "Zika vector mosquito species." As in other arboviral infections, local overwintering could be an important aspect for maintenance and spread of ZIKV. Detection of the virus in a pool of A. furcifer males in 2011 in Senegal, even though no infected A. furcifer females were collected, strongly suggested that ZIKV is vertically transmitted, at least in this species, and that this transmission route may be an important mechanism of local maintenance ( [65] ) . ”

“ As a result of these data, it has been suggested to collect both blood and saliva samples to increase the sensitivity of molecular detection of viral RNA for Zika fever diagnosis in acute phase. On the other hand, urine sample can be associated at the late stages of the disease. Thus, when detection of ZIKV is of particular importance, using a combination of samples (blood/saliva/urine) is recommended ( [86] ) .
Prophylaxis Currently there are no specific antiviral agents, vaccine, or prophylaxis for ZIKV. Treatment is generally directed to symptom relief with analgesics and anti-pyretics. ”

“ NS5 is the viral RNA dependent RNA polymerase that is in charge of genome replication, and that also displays a methyltransferase domain necessary for capping the 5′ end of the viral genomic RNA. Since flavivirus non-structural proteins constitute major targets for antiviral research ( [25] ; [26] ) , deciphering the specific role of the non-structural proteins in ZIKV infection could greatly contribute to the development of antiviral strategies against this pathogen.
Host Cell-Virus Interactions Zika virus can infect a broad range of cells from different tissues and species. ”

“ Although it is still unknown if ZIKV overwinters in geographical areas without reports of cases, it appears that, as in other flaviviral infections, such as that caused by WNV ( [23] ) , epidemics are more related to the specific mosquito species, its population density, competence, and behavior in a given area, as this can shape virus dynamics. ”

“ The NS1/NS2A is believed to be cleaved by an unknown cellular signalase ( [11] ) . Of key importance is the proteolytic cleavage of prM to give the pr peptide and M protein, which is produced by furin-like protease located in the trans-Golgi network during the egress of the particles and promote the maturation of the virions ( [6] ) .
The analysis of the polyprotein sequence predicts the presence of potential N-glycosylation sites in the ZIKV proteins prM, E, and NS1 ( [11] ; [14] ; [15] ) . ”

“ The sequence of the prototype strain of ZIKV MR766, which corresponds to a passaged virus derived from the initial ZIKV isolated by intracerebral inoculation of the serum of the febrile monkey (Rhesus 766) into mice in 1947 ( [3] ; [2] ) , revealed that the ZIKV genome was 10794 nucleotides in length ( [11] ) . The genome contains a single open reading frame (ORF) that encodes a polyprotein of about 3400 amino acids (Figure 1) that is expected to be cleaved into the mature viral proteins (see next section for polyprotein processing) . ”

“ The first evidence of the role of A. aegypti in the urban transmission cycle of ZIKV was suggested after its isolation from a pool of mosquitoes collected in 1966 in Malaysia, in what it was the first isolation from a mosquito other than A. africanus ( [47] ) .
Aedes albopictus, the so-called Asian tiger mosquito, is also widely distributed. ”

“ Remarkably, as aforementioned, during ZIKV outbreak reported in French Polynesia the incidence rate of GBS cases was approximately 20-fold higher than expected given the size of the French Polynesia population and its previously established incidence (1–2/100 000 population per year) ( [112] ) . Likewise, in Colombia, during the ongoing outbreak, 86 cases of GBS have been associated to ZIKV infection19. Based on the 600 000 expected Zika infections in the country, up to 1000 cases of GBS could be anticipated. ”

“ Both isolations were performed by intracerebral inoculation into Swiss albino mice of the samples containing the virus (serum from febrile monkey or mosquito homogenates) demonstrating that ZIKV was a filterable transmissible agent ( [2] ) . These early filtration studies indicated that the size of ZIKV was in the range of about 30–45 nm in diameter ( [3] ) . Further transmission electron microscopy analysis of ZIKV infected cells revealed that the virions were spherical particles with an overall diameter of 40–43 nm and a central electron dense core being 28–30 nm in diameter ( [4] ; [5] ) . ”

“ First ZIKV isolation from humans was reported in 1954 in Nigeria ( [68] ) , although antibodies to the virus had been previously found during early surveys on human sera in different regions of Africa ( [3] ; [69] ) . ”

“ In this sense, different Aedes species (A. aegypti, A. unilineatus, A. vittatus, and A. luteocephalus) were recently tested in their susceptibility to ZIKV oral infection, and, although all of them were susceptible, viral genome could be amplified from saliva only in the case of A. vittatus and A. luteocephalus mosquitoes ( [54] ) .
Humans and Non-human Primates During outbreaks, humans are the primary host for ZIKV ( [67] ) , and both urban ( [55] ) and sylvatic ( [15] ) viral transmission have been demonstrated. ”

“ Map showing worldwide autochthonous and imported ZIKV human cases since the last 9 months. See text for details. Asia In Asia, distinguishing ZIKV infection from other arboviral infections (dengue, yellow fever, and other tropical diseases) is also difficult, so that, early epidemiological data should be treated with caution. In the early 50’s, seroprevalence of ZIKV in humans was reported in several countries: India, Malaysia, Philippines, Vietnam, and Thailand with variable rates, 8–75% ( [102] ; [103] ; [104] ; [105] ; [47] ) . ”

“ The first laboratory confirmed case was notified in November 2013 in Germany ( [115] ) . Since then, from 2015, imported cases have been documented in Norway ( [116] ) , Italy ( [117] ) , and Germany ( [118] ) . However, after the recent explosive expansion of the virus in the Americas, many European countries have reported imported cases of ZIKV infection in travelers returning home (Figure 5) , including Austria, Denmark, Finland, France, Germany, Ireland, Italy, Portugal, the Netherlands, Spain, Slovenia, Sweden, Switzerland, and UK18. ”

“ Epidemiology Africa Since the first isolation of ZIKV in 1954 from an inhabitant of Nigeria ( [68] ) , many serological and entomological studies have reported the circulation of the virus across a widespread area of Africa, including Kenya ( [92] ) , Nigeria ( [93] ; [94] ; [95] ) , Sierra Leone ( [96] ) , Gabon ( [97] ; [55] ) , Uganda ( [75] ) , Central African Republic ( [98] ) , Senegal ( [66] ; [65] ; [99] ) , and Ivory Coast ( [100] ) , with prevalence ranging 1.3–52%. ”

“ Because of its nucleotide sequence specificity, NAT cannot be used to screen a wide range of pathogens with one run, being necessary the use of multiple assays if several pathogens are co-circulating in the same area, but multiple testing is expensive and time-consuming ( [143] ) , and it does not detect all infected blood donations, especially when nucleic acid loads are low and when sera are tested in large pools ( [86] ) . ”