key: cord-290385-0smnl70i
authors: Chan, Jasper F.W.; Choi, Garnet K.Y.; Yip, Cyril C.Y.; Cheng, Vincent C.C.; Yuen, Kwok-Yung
title: Zika fever and congenital Zika syndrome: An unexpected emerging arboviral disease
date: 2016-03-03
journal: J Infect
DOI: 10.1016/j.jinf.2016.02.011
sha: 
doc_id: 290385
cord_uid: 0smnl70i

Unlike its mosquito-borne relatives, such as dengue, West Nile, and Japanese encephalitis viruses, which can cause severe human diseases, Zika virus (ZIKV) has emerged from obscurity by its association with a suspected “congenital Zika syndrome”, while causing asymptomatic or mild exanthematous febrile infections which are dengue- or rubella-like in infected individuals. Despite having been discovered in Uganda for almost 60 years, <20 human cases were reported before 2007. The massive epidemics in the Pacific islands associated with the ZIKV Asian lineage in 2007 and 2013 were followed by explosive outbreaks in Latin America in 2015. Although increased mosquito breeding associated with the El Niño effect superimposed on global warming is suspected, genetic changes in its RNA virus genome may have led to better adaptation to mosquitoes, other animal reservoirs, and human. We reviewed the epidemiology, clinical manifestation, virology, pathogenesis, laboratory diagnosis, management, and prevention of this emerging infection. Laboratory diagnosis can be confounded by cross-reactivity with other circulating flaviviruses. Besides mosquito bite and transplacental transmission, the risk of other potential routes of transmission by transfusion, transplantation, sexual activity, breastfeeding, respiratory droplet, and animal bite is discussed. Epidemic control requires adequate clearance of mosquito breeding grounds, personal protection against mosquito bite, and hopefully a safe and effective vaccine.

Globalisation and urbanisation with increasingly frequent and large-scale movements of humans, animals, and commodities by aviation and water transport has led to the spread of previously geographically-restricted microbes and vectors to distant and isolated places. 1 Recent examples of emerging viruses that have spilled over to other continents from their original localities via exportation of travelrelated cases include coronaviruses (severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus), influenza viruses, and Ebola virus. 2e11 Moreover, global warming and climate changes have redefined the geographical distributions of important vectors of arthropod-borne viruses (arboviruses), such as the Aedes mosquitoes, and facilitated the global spread of these viruses. Dengue virus (DENV), West Nile virus (WNV), and Chikungunya virus (CHIKV), have been introduced (WNV in 1999 and CHIKV in 2013) and/or spread rapidly in the western hemisphere in the past two decades. 12 Zika virus (ZIKV) is an arbovirus that was little known before it caused a large outbreak on Yap Island of the Federated States of Micronesia in 2007. 13 Even then, ZIKV was not considered as an important emerging pathogen because clinical disease was generally mild. The recent report of a possible association between ZIKV infection and an epidemic of microcephaly among neonates in Brazil has attracted global attention. 14 The rapid spread of ZIKV beyond Africa and Asia to the Americas and Europe, and the potentially novel "congenital Zika syndrome" outbreak have led the World Health Organisation (WHO) to declare the ZIKV epidemic as a global public health emergency on 1 February 2016. 15 It would therefore be important to review the current knowledge on the epidemiology, virology, clinical manifestations, and laboratory diagnosis of ZIKV infection, and most importantly, to formulate clinical management options with special reference to perinatal care and control measures based on comparisons made with other mosquito-borne arboviruses.

Important historical and epidemiological events ZIKV (strain MR 766) was first isolated from the blood of a febrile sentinel rhesus monkey (Macaca mulatta), Rhesus 766, during a study on yellow fever virus (YFV) in Zika Forest of Uganda in April 1947 (Table 1) . 16 In 1948, ZIKV was isolated from Aedes africanus mosquitoes caught in Zika Forest, suggesting that the virus might be mosquito-borne. In 1954, ZIKV was isolated from the serum of a 10-year-old Nigerian girl who had fever and headache, implying its role as a possible human pathogen. 17 Further virological and/or serological evidence of human ZIKV infection was reported in African (Uganda, Tanzania, Egypt, Central African Republic, Sierra Leone, and Gabon) and Asian (India, Malaysia, the Philippines, Thailand, Vietnam, and Indonesia) countries. 18e26 ZIKV infection remained relatively restricted geographically with less than 20 sporadic cases reported in these areas in the first 60 years after its discovery. 13, 27 In 2007, ZIKV emerged outside Africa and Asia for the first time and caused a major outbreak on Yap Island of the Federated State of Micronesia. 13 Over 70% of the Yap residents who were !3 years were infected within 4 months. 13 The attack rate of ZIKV infection in this outbreak was 14.6 per 1000 residents (range, 3.5e21.5 per 1000 residents). Subsequently, another major outbreak was reported in French Polynesia in October 2013. An estimated 30,000 humans (>11% of the French Polynesian population) were infected by ZIKV. ZIKV infection then spread from French Polynesia to other Pacific Islands including New Caledonia, Cook Islands, Vanuatu, and Solomon Islands. 27e30 The first cases of human ZIKV infection in the western hemisphere occurred on Easter Island, Chile, in February 2014, possibly originating from French Polynesia during the annual Tapati festival. 31 Phylogenetic analysis revealed that the NS5 gene sequence of the Chilean strains had !99.8% nucleotide and 100% amino acid identity to the French Polynesian strains. 31 The epidemic continued to expand rapidly and autochthonous human cases were reported in many Latin American countries in the ensuing 2 years. 32 Brazil stands out as the hardest hit Latin American country with an estimated 500,000e1,500,000 cases of ZIKV infection since March 2015. 33 Based on the close phylogenetic relationship between the South American strains and Asian and Oceanic strains of ZIKV, the virus might have been introduced into Brazil by Asian travellers during the World Cup or participants from the Oceanic countries of the Va'a World Sprint Championship canoe race in the summer of 2014. 30,34e36 The climate changes associated with El Niño in north and eastern South America in 2015 on the background trend of global warming might have facilitated the rapid spread of Aedes mosquitoes and ZIKV. 37 Currently, >30 countries in Africa, Asia, South America, Oceania, and Micronesia have reported autochthonous cases of human ZIKV infection. 38 Travel-related cases from endemic and epidemic regions were also reported in Europe, North America, Australia, and Japan. 39e50 More worryingly, the Brazil Health Ministry reported the detection of an unusual increase in the number of cases of neonates with microcephaly in northeastern Brazil in October 2015, coinciding with the expanding ZIKV infection epidemic. 14 Over 3000 suspected cases including some fatal cases were reported during the second half of 2015 alone. 14 This represented a >20-fold increase in the rate of microcephaly as compared to previous years. 32 On 24 November 2015, the French Polynesia health authorities also reported an unusual increase in the number of foetal and neonatal central nervous system malformations in 2014 and 2015. 51 

Like other flaviviruses, ZIKV is mainly transmitted by mosquitoes. In addition to the sylvatic (enzootic) transmission cycle between the haematophagous mosquito vectors and susceptible primary vertebrate hosts, the recent large-scale epidemics suggest that ZIKV is also adapting to an urban transmission cycle. 52, 53 Among the various mosquito species, Aedes (Stegomyia) mosquitoes appear to be the most important vector for ZIKV transmission, although some Anopheles, Culex, Eretmapodites, and Mansonia species have also been proposed as possible vectors (Table 2) . 25,52,54e69 The animal reservoirs of ZIKV are unclear. Non-human primates including M. mulatta, Cercopithecus aethiops, C. ascanius schmidti, C. mona denti, C. albigena johnstoni, Chlorocebus sabaeus, Colobus abyssinicus, Erythrocebus patas, and Pongo pygmaeus, and other mammals including zebras, elephants, and rodents, have been suggested as possible vertebrate hosts of ZIKV in Africa and Asia, based on virological and/or serological evidence of infection. 16,54,65,68,70e72 Ae. africanus is the first mosquito species from which ZIKV was isolated, and is likely an important vector in the sylvatic transmission cycle of ZIKV. 16, 54 Inoculation of unfiltered supernatant of ZIKV-infected Ae. africanus into mice and rhesus macaques led to clinical disease and/or neutralising antibody response. 16, 54 Ae. hensilli is the most commonly found mosquito species on Yap Island, but no virus isolate was made from field-collected mosquitoes to ascertain its role as a vector for ZIKV transmission during the outbreak. 67 Ae. aegypti and Ae. albopictus, which have much wider geographical distributions than other Aedes mosquitoes, are considered to be more important vectors in the urban transmission cycle of ZIKV. 73 These Aedes mosquitoes are highly susceptible to ZIKV infection in vitro with potential for further transmission after an extrinsic incubation period of 5e10 days. 58, 63 They bite both indoors and outdoors, and mostly during daytime. 74 Non-vector-borne transmission routes of ZIKV have been proposed (Table 3) . Like other arboviruses, blood transfusion-related transmission of ZIKV is possible, especially in endemic regions or where blood products obtained from infected travellers immediately returning from endemic regions are used. ZIKV RNA was detected in the blood of 2.8% of the donors in French Polynesia during the epidemic. 75 Sexual transmission of ZIKV appears highly probable, especially in patients presenting with haematospermia with infectious viral particles and RNA in semen. 76, 77 Notably, no other arboviruses have been associated with haematospermia or isolated from human semen. 76 This might further complicate the control of the ZIKV epidemic, since most infected patients are asymptomatic. Inadvertent sexual transmission of ZIKV to the female partner may then lead to virus transmission to the foetus, which may be potentially associated with severe congenital anomalies. Besides transplacental transmission, perinatal transmission of ZIKV may also occur during delivery, via breastfeeding, and/or close contact after birth via exchange of saliva and other bodily fluids. ZIKV RNA could be detected in breast milk and saliva of infected women, although replicative virus particles have not been demonstrated 78, 79 Perinatal transmission of other arboviruses, including DENV, CHIKV, WNV, and YFV, has also been reported. 80e90 Other suspected routes of transmission of ZIVK infection are those reported for other flaviviruses. These include mucocutaneous exposure to the virus in infected blood or via monkey bite, haemodialysis, or organ transplantation. 91e96 Particularly, as ZIKV may be shed in the urine of infected patients for more than 30 days, the risk to recipients of donated kidneys from donors at or returning from endemic areas has to be considered. 40, 41, 49, 50, 97 It is unknown whether ZIKV could be transmitted via respiratory droplets as viral RNA could occasionally be detected in nasopharyngeal swab and saliva samples. 40, 78, 79, 96 Virology and pathogenesis ZIKV is an enveloped, positive-sense, single-stranded RNA virus belonging to the genus Flavivirus in the family Flaviviridae. It is closely related to Spondweni virus and the 2 viruses represent the only members of their clade within the mosquito-borne cluster of flaviviruses ( Fig. 1) . 98, 99 Phylogenetic analysis suggests that ZIKV has likely emerged between 1892 and 1943 in Uganda. 66 The two major lineages of ZIKV are the African (subdivided into West and East African) and Asian lineages, which are responsible for causing the majority of infections in Africa and Asia (as well as the Pacific and Americas), respectively. 34, 69, 100 The single-stranded RNA genome of ZIKV has a size of 10,794 nucleotides encoding 3419 amino acids, with 2 flanking untranslated regions (5 0 and 3 0 UTRs) and a single long open reading frame encoding a polyprotein, which is cleaved into capsid (C), precursor of membrane (prM), envelope (E), and 7 non-structural (NS) proteins 66, 101 Reverse transcription-polymerase chain reaction (RT-PCR) using primers targeting the E or NS5 gene is a key laboratory diagnostic tool for ZIKV infection in the recent outbreaks. 13, 102, 103 The E protein is a major virion surface protein that is involved in receptor binding and membrane fusion. The domain III of E protein contains a panel of antigenic epitopes that are important targets of serological assays, neutralising antibodies, and vaccines. 104, 105 Loss of the N154 glycosylation site in the E protein may be associated with adaptation to mosquito vectors and thus facilitate transmission. 66 A single amino acid mutation in the E protein (E1-A226V) of CHIKV has been reported to be associated with increased fitness of the virus in Ae. albopictus and allows CHIKV to disseminate in regions lacking the typical Ae. aegypti vector. 106 The recent spread of the Asian lineage of ZIKV to Oceania and the Americas may be associated with significant NS1 codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titres. 107 Mutations in the E and NS1 genes should be detected in ZIKV strains causing the current epidemic.

When an infected Aedes mosquito bites an infected patient, it ingests a blood meal containing ZIKV. As in other flaviviruses, ZIKV likely replicates in the midgut epithelial cells and subsequently the salivary gland cells. After an extrinsic incubation period of 5e10 days, ZIKV can be found in the mosquito's saliva which can then infect human. 58, 63 Moreover, the virus can likely be vertically transmitted transovarially as other flaviviruses. When the mosquito's saliva containing ZIKV is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the subcutaneous layer, and the Langerhans cells. 108 The keratinocytes and fibroblasts contain AXL, Tyro3, and TIM-1, which can serve as attachment factors or receptors for ZIKV. The Langerhans cells contain DC-SIGN, which can also serve as a receptor for virus entry. 108 ZIKV infection of primary skin fibroblasts is associated with the upregulation with TLR3 mRNA expression, and enhanced transcription of RIG-I and MDA5, which are known innate immune responses to RNA virus infection. This is followed by enhanced expression of interferon-alpha and -beta, and their downstream pathways of immune activation. Both types I and II interferons can suppress the viral load of infected cells. Moreover, ZIKV is capable of increasing its replication by the induction of autophagy in host cells. Thus, autophagy inhibitors can decrease the viral load of infected cells. 109 Infected cells of human skin explant exhibits cytoplasmic vacuolation, pyknotic nuclei, and oedema in the stratum granulosum. 108 After replication in Endemic/epidemic areas: + Universal nucleic acid testing of blood donors. + Temporary discontinuation of blood donation (Importation of blood products from blood blank centres in non-endemic regions). Non-endemic/epidemic areas: + Pre-donation questionnaire to identify donors with recent travel history to endemic/epidemic areas. + Deferral of blood donors who have travelled to endemic areas within the preceding !14 days. + Self-reporting of symptoms after blood donation ( 

these local tissue cells and the regional lymph nodes, ZIKV may then disseminate from the lymphatics and bloodstream to reach other organs/tissues, including the central nervous system, the skeletal muscles, myocardium, and perhaps transplacentally to the foetus. ZIKV was highly neurotropic in infected suckling mice. The brains of infected suckling mice show neuronal degeneration, cellular infiltration, and softening in the brain with virus replication in astroglial cells and neurons on histopathological examination. 16, 54, 110 Moreover, evidence of inflammation in skeletal muscles and myocardium has also been demonstrated in infected suckling mice. 111 AXL and Tyro3 are members of the TAM family of receptor tyrosine kinases (RTKs). They are also present in neurons and under the influence of gonadotropin releasing hormone (GrH), which in turn may affect neuronal survival and migration. Furthermore, flaviviruses such as YFV may persist for up to 159 days after intracerebral inoculation in rhesus macaques. 112 The neurotropism and persistence of ZIKV may therefore partially explain microcephaly and predominantly neurological complications and foetal anomalies in this suspected entity of congenital ZIKV infection.

Most patients with ZIKV infection are asymptomatic. In the outbreak of ZIKV infection on Yap Island, only 18% of cases were estimated to be symptomatic. 13 The incubation period of ZIKV infection is unclear, but is estimated to be similar to other mosquito-borne flaviviruses (2e14 days). 76, 113 The clinical syndromes of symptomatic ZIKV infection can be broadly divided into Zika fever and congenital infection ("congenital Zika syndrome") ( Table 4 ).

Zika fever is an acute "dengue fever-like" illness characterized by low-grade fever (37.8e38.5 C), rash, retroorbital headache, bilateral non-purulent conjunctivitis, myalgia, and arthritis/arthralgia with periarticular oedema of the small joints of hands and feet. 13 The rash in Zika fever is typically described as a generalized, erythematous, maculopapular rash that spreads downward from the face to the limbs. 30 Less commonly, some patients may have more prominent systemic symptoms including high-grade fever, chills, rigours, sore throat, hypotension, and cervical, submandibular, axillary, and/or inguinal lymphadenopathies. 74 Digestive tract symptoms including nausea, vomiting, diarrhoea, constipation, abdominal pain, and aphthous ulcers may also be present. 13, 17, 19 Patients with genitourinary symptoms including haematuria, dysuria, perineal pain, and haematospermia often have detectable viral RNA or infectious virus particles in urine and/or semen. 76, 77 Haematological and biochemical laboratory parameters are usually normal. However, some patients may have transient and mild leucopenia, neutropenia, lymphopenia or activated lymphocytes, monocytosis, thrombocytopaenia, and elevated serum levels of lactate dehydrogenase, aspartate aminotransferase, g-glutamyl transferase, fibrinogen, ferritin, C-reactive protein, and erythrocyte sedimentation rate during the viraemic phase. 29 associated with restoration of normal number of peripheral immune cells and normal function of antigen-presenting cells. 45 Notably, the clinical manifestations of Zika fever are non-specific and may mimic those seen in infectious diseases caused by other arthropod-borne pathogens, especially DENV and CHIKV. Some suggest that Zika fever may be distinguished from dengue fever and Chikungunya fever by more prominent oedema of the extremities, less severe headache and malaise, and milder degree of thrombocytopaenia seen in the former. 13, 115 Moreover, haemorrhagic complications seen in dengue fever have not been reported in Zika fever, and arthralgia in Zika fever is less severe than that in Chikungunya fever. 13 However, none of these features are pathognomonic and laboratory confirmation is required to exclude co-infections with these arboviruses and other causes of acute febrile illness in returned travellers from endemic regions, such as malaria. Zika fever is usually self-limiting with most clinical manifestations resolving completely within 3e7 days. 13, 49, 116 No death, hospitalisation, or haemorrhagic complication was reported during the outbreak on Yap Island. 13 However, some patients may experience more protracted symptoms and other non-haemorrhagic complications. Zika fever-related rash usually resolve within the first week, but may last for up to 14 days and may be pruritic. 13 Other exanthematous diseases, such as DENV, CHIKV, rubella virus, measles virus, parvovirus B19, adenovirus, enterovirus, and rickettsial infection, should be excluded. The median duration of arthralgia is 3.5 days, but some patients may develop persistent or recurrent arthralgia for more than a month after symptom onset, mimicking the post-infectious chronic arthritis seen in Chikungunya fever and Lyme disease. 13, 76 Lymphadenopathies may be present for 2 weeks after symptom onset, and alternative diagnoses such as infectious mononucleosis-like syndrome, Streptococcus pyogenes infection, and toxoplasmosis should be considered in refractory cases. 45 A post-infection asthenia appears to be frequent and further investigations may be necessary to determine possible association between ZIKV infection and chronic fatigue syndrome. 13, 117, 118 Immune-thrombocytopenic purpura and cardiac complication have also been reported in a few cases. 114 Jaundice was observed in patients with virological and/or serological evidence of ZIKV infection in Eastern Nigeria in the 1950s who had co-infections (malaria and microfilaraemia) and a patient with sickle cell anaemia. 17, 119 A possible association between ZIKV infection and severe neurological complications has been proposed during the recent epidemics in Oceania and South America, during which the incidence of GuillaineBarré syndrome has increased by 8e20 times in French Polynesia. 115,120 74/ 8750 (0.8%) patients with suspected ZIKV infection in the French Polynesia outbreak developed neurological syndromes after presenting with a Zika fever-like illness. 120 Forty-two of these 74 (56.8%) patients were diagnosed with GuillaineBarré syndrome. 12, 51, 121 Similarly, Guil-laineBarré syndrome has been reported among patients with Zika fever-like illness in South America. 51, 121 Other neurological complications potentially linked to ZIKV infection include encephalitis, meningoencephalitis, myelitis, paraesthesia, vertigo, facial paralysis, and 48,76,114,115,120e122 Suspected fatalities due to ZIKV-related GuillaineBarré syndrome have been reported. 123 While the neurotropism of ZIKV may partially explain these neurological manifestations, more details and serial studies on their cerebrospinal fluid and magnetic resonance images by case-control studies are required to ascertain their association. Zika fever-related death appears to be extremely rare but a number of probable cases have been reported, especially among immunocompromised patients and neonates with suspected congenital ZIKV infection. 51,119,121 A small number of patients with coinfection with DENV or HIV did not appear to have more severe disease. 29, 124 Further studies should be conducted to identify patients who are at risk of severe disease or death.

Microcephaly (head circumference !2 standard deviations below the mean for sex and gestational age at birth) is the most prominent and commonly reported clinical feature of suspected congenital Zika syndrome. 14, 125 Besides microcephaly, neonates and foetuses with suspected congenital ZIKV infection also had other malformations (Table 4 ). General features included low birth-weight, redundant scalp skin, anasarca, polyhydramnios, and arthrogryposis. Neurological abnormalities included cerebral lesions, polymalformative syndromes, brainstem dysfunction, and absence of swallowing. 51 Ophthalmological defects included cataract, asymmetrical eye sizes, intraocular calcifications, macular atrophy (well-defined macular neuroretinal atrophy and/or macular pigment mottling and foveal reflex loss), optic nerve hypoplasia, iris coloboma, and lens subluxation. 122, 126, 127 Notably, other features characteristic of intrauterine infections, such as hepatosplenomegaly, rash, and chorioretinitis have not been reported. 14 Ultrasonographic examination revealed cerebral atrophy, intracranial calcifications especially over the white matter of frontal lobes, caudate, lentostriatal vessels, cerebellum, or around the lateral and fourth ventricles, dysgenesis of corpus callosum, vermia, and thalami, enlarged cisterna magna, asymmetrical cerebral hemispheres, severe unilateral ventriculomegaly, displacement of the midline, and thinning of the parenchyma on the dilated side, pons and brainstem. 14,51,121,128 ZIKV particles and RNA may be detected by electron microscopy and RT-PCR, respectively, in autopsied samples. 125 Two important questions concerning congenital ZIKV infection remain unanswered. The first question is whether ZIKV is indeed the cause of microcephaly and other congenital anomalies in these patients. Severe consequences have been reported for materno-foetal transmission of other arboviruses, such as dengue virus (preterm delivery, foetal death, low birth-weight, prematurity, acute foetal distress during labour), WNV (chorioretinitis and focal cerebral destruction), and CHIKV (encephalopathy and haemorrhagic fever). 80, 82, 86 Preliminary analysis in the current epidemic of microcephaly has not yet completely excluded other infectious or environmental aetiologies. 51 Moreover, there is some virological evidence to support the association between congenital ZIKV infection and these anomalies. ZIKV RNA has been detected by RT-PCR in the amniotic fluid of 2 pregnant women whose foetuses had ultrasonographic evidence of microcephaly, in the blood and foetal tissues of a neonate with microcephaly and other congenital anomalies who died within the first 5 min of birth, and in the neonatal brain tissues of a few cases of full-term miscarriages and neonates with microcephaly. 14, 51, 121, 125 However, there is still no large-scale prospective cohort or caseecontrol study to demonstrate a causal link between the presence of ZIKV in the foetus and the congenital anomalies after exclusion of other infectious and toxic causes. Some have suggested that the apparent microcephaly surge might be attributable to the intense search for cases due to the heightened awareness of a possible association with the ZIKV outbreak or the use of larvicide. 129 Furthermore, detailed investigations for exclusion of other pathogens associated with congenital malformations have only been reported in a small number of cases. 14, 125 Microcephaly is well reported in congenital cytomegalovirus, rubella virus, and varicella zoster virus infection. Chorioretinitis and intracranial calcifications are common in congenital cytomegalovirus infection and toxoplasmosis, but the latter is more commonly associated with hydrocephalus. Cataract and cardiac anomalies are characteristic of congenital rubella syndrome, although cataract can also be found in congenital herpes simplex virus infection. Thus, the diagnosis of congenital Zika syndrome would depend on the exclusion of these "TORCH" infections in future studies using clinical criteria, histopathological findings, and serological, molecular and conventional cell culture techniques. If ZIKV is eventually confirmed to be the cause of these congenital anomalies, the second key question would be whether congenital Zika syndrome actually comprises a wider spectrum of varying clinical severities than that seen in the reported cases. As with other congenital infections, it is possible that the reported cases of microcephaly represent only the tip the iceberg, focussing on the more severely affected patients, and that the timing of infection is likely to be important in determining the severity and outcome of the affected foetus. Early infection during the first or even second trimester may be associated with congenital anomalies or even intrauterine death. 14, 125, 130 Indeed, preliminary data suggested that the greatest risk of microcephaly or congenital anomalies in the affected neonates appears to be associated with ZIKV infection in the first trimester of pregnancy. 51 Of 35 mothers with infants born with microcephaly, 57% and 14% had a rash during the first and second trimester of pregnancy, respectively. 14 Besides neurological defects, cardiac and muscular abnormalities should also be excluded, as suckling mice infected with ZIKV developed evidence of central nervous system infection, myositis and myocarditis. 111 Some suspected cases of congenital Zika syndrome developed severe arthrogryposis. 14, 51, 121, 128 It is possible that intrauterine ZIKV infections that occur at a later stage of the pregnancy may present differently, either with less severe manifestations, such as mental retardation, sensorineural deafness, and/or ophthalmological lesions, or as full-term miscarriages. 14 Neonates with probable perinatal transmission of ZIKV infection appear to have mild disease and favourable outcome. 78 Further investigations should be conducted to better define the spectrum of manifestations in different gestational stages of congenital ZIKV infection.

Definitive diagnosis of ZIKV infection requires laboratory confirmation as there are no pathognomonic clinical, biochemical, or radiological features that reliably distinguish Zika fever from other arboviruses, and congenital ZIKV infection from other infective, toxic, or genetic causes of congenital anomalies. Successful isolation of ZIKV in viral culture, the gold-standard of laboratory diagnosis of viral infections, mainly depends on the timing of specimen collection and viral loads in the specimens. ZIKV has been isolated in Vero and Vero E6 cells inoculated with infected patients' serum, urine, and/or semen samples (Table 5) . 40, 75, 77 However, infectious virus particles were not recovered by culture in most specimens with low viral loads. A positive serum immunoglobulin (Ig) M or 4-fold rise in the titre of neutralising antibodies in paired serum samples collected approximately 2 weeks apart also establishes the diagnosis of ZIKV infection. IgM may be detected by enzyme-linked immunoassay on as early as day 3 of symptom onset and may last for over 2 months. 40, 49 IgM antibodies to DENV and WNV usually persist for 3 months and 5 months, respectively. 131e134 The major limitation of these serological tests is possible cross-reactivity with other flaviviruses. Neutralising antibodies detected by plaque-reduction neutralisation test may be more specific than IgM detection by ELISA for primary ZIKV infection, but may also have indeterminate results for secondary infection, including patients with previous vaccination against or exposed to other flaviviruses. 13, 40, 102, 120 This is especially problematic in areas where there is cocirculation of multiple flaviviruses with the same Aedes mosquito vectors. 13, 64, 102, 120 Patients with primary ZIKV infection and past DENV infection are more likely to have higher titre (usually !4-fold) of IgM and/or neutralising antibodies against ZIKV than against DENV or other flaviviruses. 13, 102 A positive serum DENV NS1 antigen test without serial increase in IgM or the combination of a positive IgM response to DENV and lack of an IgG seroconversion in the convalescent-phase serum sample should prompt the clinician to investigate for another flavivirus such as ZIKV. Moreover, co-infections with other mosquito-borne arboviruses, such as DENV, CHIKV, WNV, and Japanese encephalitis virus, are always possible and should be excluded by more extensive laboratory testing if clinically indicated.

Rapid and accurate diagnosis of ZIKV infection during the recent epidemics has mainly been achieved by the application of RT-PCR using primers that target the E or NS5 gene of ZIKV. 13, 102, 103, 135, 136 Alternatively, RT-PCR sequencing using universal primers that target the conserved regions in the genomes, such as the NS5 gene, of multiple flaviviruses, may allow simultaneous detection of >50 different flaviviruses. 137 Serum samples should be collected in the early phase of the disease, because viraemia is usually shortlived (usually 5 days, rarely up to 11 days) and may be low-level ( 10 2 copies/ml). 97, 102 Alternatively, urine and semen samples may have higher viral RNA loads Table 5 Advantages, limitations, and uses of different diagnostic tests and types of specimens for laboratory diagnosis of ZIKV infection. 14,40,41,49,50,75e79,96,97,102,121,125,127,137e139,153 

May be useful to exclude concomitant infections in patients with persistent or atypical rash.

May be useful to exclude concomitant infections in patients with persistent or atypical rash.

May be useful to exclude concomitant infections in patients with persistent or recurrent arthritis.

May be useful to exclude concomitant infections in patients with persistent or recurrent arthritis.

May be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia.

May be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia.

Other tissues Brain, liver, spleen, and pooled visceral (kidney, lung, and heart) tissues were positive in a fatal case (an adult male with co-morbidities and immunosuppressive treatment).

May be useful to exclude concomitant infections in patients with unusually severe or fatal infection.

Abbreviations: RT-PCR, reverse transcription-polymerase chain reaction; ZIKV, Zika virus.

(>10 6 copies/ml) than serum samples, and may be persistently positive for >30 days and !62 days after symptom onset, respectively. 97, 138 In a few cases, ZIKV RNA has also been detected in saliva and nasopharyngeal swab samples of patients whose serum samples tested negative for ZIKV. These samples should therefore also be collected in suspected cases of ZIKV infection. 40, 78, 79, 96 Collection of amniotic fluid should be considered in pregnant women with positive ZIKV test result or if the foetuses show ultrasonographic evidence suggestive of congenital ZIKV infection. 14, 102, 128 Cerebrospinal fluid, placental, and/or umbilical cord tissues from neonates with suspected congenital ZIKV infection should be sent for virological and/or histopathological examinations to establish the diagnosis. 14,121,125 ZIKV RNA may also be detected in organ tissues in the rare cases of suspected ZIKV-related deaths. 121 Future studies should aim to better stratify the clinical use of these tests and to develop point-of-care tests (eg: antigen tests) that can be widely used in less developed regions without the facilities and expertise for molecular or serological tests.

Treatment is usually not required for patients with asymptomatic or uncomplicated Zika fever. The mainstay of treatment is supportive as there are no specific anti-ZIKV antiviral agents. Acetaminophen may be used to relieve fever and arthralgia. Anti-histamines may help to control pruritus. Adequate rehydration for fluid loss through sweating, vomiting, and insensible losses should be encouraged. Aspirin should be avoided due to the risks of bleeding in those with thrombocytopaenia and developing Reye's syndrome in children less than 12 years of age. Nonsteroidal antiinflammatory drugs are also contraindicated in cases where DENV and CHIKV infections cannot be confidently excluded in order to avoid haemorrhagic complications. Potential neurological complications, especially GuillaineBarré syndrome, should be diagnosed promptly to allow early use of intravenous immunoglobulins and/or plasmapheresis. The risk of immune enhancement should also be considered if convalescent-phase plasma therapy with neutralising antibodies against ZIKV is used for treatment of severe cases. Virological testing and foetal ultrasound to exclude ZIKV infection and foetal microcephaly or intracranial calcifications should be offered to pregnant women who develop Zika fever-like symptoms during or within 2 weeks of travel to areas with ZIKV transmission. 139 Besides collecting the appropriate specimens for virological tests, serial foetal ultrasound examinations should be performed every 3e4 weeks to monitor foetal anatomy and growth in suspected cases of congenital ZIKV infection. Foetal ultrasound and/ or aminocentesis should also be offered to asymptomatic and seropositive pregnant women with history of travel to affected areas. After delivery, serum should be collected either from the umbilical cord or directly from the neonate within 2 days of birth for RT-PCR, IgM and/ or neutralising antibodies against ZIKV. 140 Comprehensive physical examination including measurement of the occipitofrontal circumference, length, and weight, evaluation for neurological abnormalities, dysmorphic features, hepatosplenomegaly, rash, ophthalmological lesions, and auditory defects, and laboratory testing for TORCH screening should be performed. 140 The affected child and the family should be managed and counselled by a multidisciplinary team consisting of paediatric neurologist, clinical geneticist or dysmorphologist, infectious disease specialist, medical social worker, and other relevant specialists. 140 Long-term follow-up to monitor physical, intellectual, and functional progress of the child should be offered. 140 

Both vector control and personal preventive measures are important for interrupting the transmission of ZIKV. Systematic mosquito surveillance and control programs should be established and coordinated by health authorities. 51 Mass sanitation campaigns to eliminate mosquito breeding sites in household and high-risk areas such as garbage collection points, construction sites, illegal dumping grounds, and invalid car fields should be organised. Mosquitoes should be removed with a radius of at least 400 m around areas with high population densities, such as schools, transport terminals, churches, and healthcare facilities. In areas where autochthonous or imported cases of ZIKV are detected, the use of adulticide through spraying to remove infected adult mosquitoes should be considered. Residents in or travellers to affected areas should stay indoor with air conditioning, window and door screens if possible, wear long sleeves and pants, use permethrintreated clothing and gear, and use insect repellents when outdoor. 140 Most Environmental Protection Agency (EPA)registered insect repellents, including N,N-diethyl-mtoluamide (DEET), should be safe for pregnant and lactating women (20% DEET), and children (10% DEET) aged >2 months. 141 Individuals returning from affected areas to non-affected regions should continue to use insect repellents for at least an additional 14 days to prevent local non-infected mosquitoes from the acquisition of virus from the asymptomatically infected returned travellers. This will serve to interrupt the mosquito-human-mosquito transmission chain. Hospitalised laboratory-confirmed cases should be managed in designated wards to avoid mosquito bites. The effects of other novel mosquito-control measures, such as the Wolbachia biological control approach, should be evaluated. 142 Other animals such as rodents should also be investigated as potential animal reservoirs and controlled as findings indicate.

Non-vector-borne transmission of ZIKV may be prevented by specific measures (Table 3) . Concerning blood transfusion, universal nucleic acid testing of blood donors is recommended. The use of universal primers that can simultaneously detect multiple arboviruses such as DENV and ZIKV should be considered. Temporary discontinuation of blood donation should be considered during an outbreak situation. In non-endemic areas, pre-donation questionnaire to identify donors with recent travel history to regions with reported cases of ZIKV infection and deferral of blood donation from these donors until at least 14 days after returning from affected regions should be implemented. Most transfusion-related transmissions of arboviruses are associated with asymptomatic infections, and symptomatic donors who were RT-PCR-positive for ZIKV usually developed symptoms between 3 and 10 days after blood donation. 143 Newer pathogen reduction technologies for blood products should be considered. 143 Similarly, donated organs, especially kidneys, from individuals with travel history to affected areas should be tested for ZIKV as the virus may persist in the genitourinary tract for an undetermined period. 40, 41, 49, 50, 97 Barrier methods should be used to prevent sexual transmission through infected semen. Male returned travellers should continue the use of condom with pregnant sex partner throughout the whole duration of pregnancy. Future studies should evaluate the duration of virus shedding in semen and the infectiousness of RNApositive semen samples, in order to determine how long barrier methods should be used by men returning to nonendemic regions. Some regional authorities have advised women to avoid pregnancy until the epidemic is over. 144 Pregnant women or those planning for pregnancy should defer travelling to regions with reported cases of ZIKV infection. If such travel was unavoidable, they should strictly comply with personal protective measures to avoid mosquito bites. Further studies are needed to determine the risk of ZIKV transmission by breast milk and saliva. Other less common transmission routes, including mucocutaneous exposure to infected bodily fluid during laboratory and patient-care procedures, and bites by infected primates should be avoided with strict compliance to infection control measures. In the laboratory setting, ZIKV can be killed by potassium permanganate, ether, and heat (>60 C), but it is not effectively neutralised with low concentration (10%) of ethanol. 54 No ZIKV vaccine is available currently. Because the moratorium for pregnancy may be impractical for some people, a safe and effective ZIKV vaccine is urgently needed. Some realistic approaches include liveattenuated or killed vaccine from human cell lines (as in the case of YFV and Japanese encephalitis vaccines), attenuated chimeric vaccine (DENV vaccine using the YFV vaccine backbone, currently in Phase III clinical trial), DNA and recombinant protein vaccine. 145 Suitable animal models for evaluation of these potential vaccine candidates should be developed for ZIKV infection. The role of passive immunisation before and after exposure to ZIKV should also be assessed in future studies.

The ZIKV epidemic has emerged as an unexpected global health emergency as the rapidly expanding ZIKV epidemic may turn out to be a major cause of permanent and severe disability in a generation of newborns, which would constitute a huge socioeconomic burden to the affected countries. The future of the ZIKV epidemic is unpredictable, but the worldwide spread of DENV and CHIKV over the past two decades suggests that ZIKV has the potential to follow their paths. With more than half of the world's human population living in areas infested with Aedes mosquitoes, the ongoing adaptation of ZIKV to an urban cycle signify the virus' pandemic potential. 36 Research preparedness is urgently needed to improve mosquito-control measures, as well as to develop point-of-care laboratory diagnostics, antivirals, and vaccines which are suitable for use in pregnant women and foetuses.

Interspecies transmission and emergence of novel viruses: lessons from bats and birds

Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection

Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease

The emerging novel Middle East respiratory syndrome coronavirus: the "knowns" and "unknowns

Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic?

Two years after pandemic influenza A/2009/H1N1: what have we learned?

Avian influenza A H5N1 virus: a continuous threat to humans

From SARS coronavirus to novel animal and human coronaviruses

The emergence of influenza A H7N9 in human beings 16 years after influenza A H5N1: a tale of two cities

Emergence in China of human disease due to avian influenza A(H10N8) e cause for concern?

Ebola virus disease: a highly fatal infectious disease reemerging in West Africa

Zika virus in the Americas e yet another arbovirus threat

Zika virus outbreak on Yap Island, Federated States of Micronesia

Possible association between Zika virus infection and microcephaly e Brazil

WHO statement on the first meeting of the International Health Regulations

Zika virus. I. Isolations and serological specificity

Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria

Serological and entomological study on yellow fever in Sierra Leone

Zika virus, a cause of fever in Central Java

A serological survey of arboviruses in Gabon

Serological survey for the prevalence of certain arboviruses in the human population of the south-east area of Central African Republic

Serological survey for arbovirus antibodies in the human and simian populations of the South-East of Gabon

Surveillance of the circulation of arbovirus of medical interest in the region of eastern Senegal

A survey for arboviral antibodies in sera of humans and animals in Lombok, Republic of Indonesia

Potential of selected Senegalese Aedes spp. mosquitoes (Diptera: Culicidae) to transmit Zika virus

Epidemiological notes on some viruses isolated in Uganda; Yellow fever, Rift Valley fever, Bwamba fever

Notes on Zika virus e an emerging pathogen now present in the South Pacific

Concurrent outbreaks of dengue, chikungunya and Zika virus infections e an unprecedented epidemic wave of mosquito-borne viruses in the Pacific

Co-infection with Zika and dengue viruses in 2 patients

Zika virus transmission from French Polynesia to Brazil

A report on the outbreak of Zika virus on Easter Island

Epidemiological update e neurological syndrome, congenital anomalies, and Zika virus infection

Anticipating the international spread of Zika virus from Brazil

Zika virus genome from the Americas

Entry routes for Zika virus in Brazil after 2014 world cup: new possibilities

Zika virus in Brazil and the danger of infestation by Aedes (Stegomyia) mosquitoes. Rev Soc Bras Med Trop 2015. pii: S0037e86822015005003102

El Niño and climate change-contributing factors in the dispersal of Zika virus in the Americas?

Zika situation report e neurological syndrome and congenital anomalies

Zika virus infection acquired during brief travel to Indonesia

First case of Zika virus infection in a returning Canadian traveler

Two cases of Zika fever imported from French Polynesia to Japan

First case of laboratory-confirmed Zika virus infection imported into Europe

Zika virus infection after travel to Tahiti

Zika virus infection in a traveller returning to Europe from Brazil

Zika virus infections imported to Italy: clinical, immunological and virological findings, and public health implications

Cytokine kinetics of Zika virus-infected patients from acute to reconvalescent phase

Zika virus in an American recreational traveler

Acute Zika virus infection after travel to Malaysian Borneo

Zika fever imported from Thailand to Japan, and diagnosed by PCR in the urines

Zika virus infection in a traveller returning from the Maldives

Epidemiological alert e neurological syndrome, congenital malformations, and Zika virus infection. Implications for public health in the Americas

Zika virus emergence in mosquitoes in southeastern Senegal

Cross-species transmission and emergence of novel viruses from birds

Zika virus. II. Pathogenicity and physical properties

A simple technique for infection of mosquitoes with viruses; transmission of Zika virus

Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia

Investigation surrounding a fatal case of yellow fever in Cote d'Ivoire in 1999

Oral susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika virus

Twelve isolations of Zika virus from Aedes (Stegomyia) Africanus (Theobald) taken in and above a Uganda Forest

The occurrence of Chikungunya virus in Uganda. I. Isolation from mosquitoes

Critical review of the vector status of Aedes albopictus

Aedes albopictus, an arbovirus vector: from the darkness to the light

Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore

Zika virus in Gabon (Central Africa) e 2007: a new threat from Aedes albopictus

Yellow fever and Zika virus epizootics and enzootics in Uganda

Molecular evolution of Zika virus during its emergence in the 20(th) century

Aedes hensilli as a potential vector of Chikungunya and Zika viruses

Zika virus infections in Nigeria: virological and seroepidemiological investigations in Oyo State

Molecular characterization of three Zika flaviviruses obtained from sylvatic mosquitoes in the Central African Republic

A sero-epidemiological survey for certain arboviruses (Togaviridae) in Pakistan

Sylvatic transmission of arboviruses among Bornean orangutans

Health evaluation of free-ranging and semicaptive orangutans (Pongo pygmaeus pygmaeus) in Sabah, Malaysia

A new threat looming over the Mediterranean basin: emergence of viral diseases transmitted by Aedes albopictus mosquitoes

Dengue and dengue vectors in the WHO European region: past, present, and scenarios for the future

Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia

Probable non-vector-borne transmission of Zika virus

Potential sexual transmission of Zika virus

Evidence of perinatal transmission of Zika virus, French Polynesia

Detection of Zika virus in saliva

Dengue infection in pregnancy: prevalence, vertical transmission, and pregnancy outcome

Maternal dengue and pregnancy outcomes: a systematic review

Maternal and fetal consequences of dengue fever during pregnancy

Maternal and perinatal outcomes of dengue in PortSudan, Eastern Sudan

Breast milk as a possible route of vertical transmission of dengue virus?

Chikungunya virus infection during pregnancy

Multidisciplinary prospective study of mother-to-child chikungunya virus infections on the island of La Reunion

West Nile virus infection in pregnancy

Possible West Nile virus transmission to an infant through breastfeeding e Michigan

Perinatal transmission of yellow fever, Brazil

Case report: probable transmission of vaccine strain of yellow fever virus to an infant via breast milk

Transmission of dengue virus without a mosquito vector: nosocomial mucocutaneous transmission and other routes of transmission

Laboratory-acquired West Nile virus infections e United States

The dengue and dengue hemorrhagic fever epidemic in Puerto Rico

Transmission of West Nile virus from an organ donor to four transplant recipients

Possible dialysis-related West Nile virus transmission e Georgia

Zika virus infection in Australia following a monkey bite in Indonesia

Detection of Zika virus in urine

Phylogeny of the genus flavivirus

A multigene analysis of the phylogenetic relationships among the flaviviruses (Family: Flaviviridae) and the evolution of vector transmission

Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage

Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses

Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia

Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes

Molecular biology of flaviviruses

Moraes Figueiredo LT. Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization

A single mutation in chikungunya virus affects vector specificity and epidemic potential

Spread of the pandemic Zika virus lineage is associated with NS1 codon usage adaptation in humans

Biology of Zika virus infection in human skin cells

Autophagy and viral diseases transmitted by Aedes aegypti and Aedes albopictus

Zika virus infection of the central nervous system of mice

Zika virus: further isolations in the Zika area, and some studies on the strains isolated

Persistence of arboviruses and antiviral antibodies in vertebrate hosts: its occurrence and impacts

Incubation periods of mosquito-borne viral infections: a systematic review

Rapid risk assessment: Zika virus infection outbreak, French Polynesia

Current Zika virus epidemiology and recent epidemics

Zika virus outbreak

Zika virus infection in man

Zika virus infection, Cambodia

Fatal Zika virus infection in girl with sickle cell disease, Colombia. Emerg Infect Dis

Zika virus infection complicated by Guillain-Barre syndrome e case report

Rapid risk assessment e Zika virus epidemic in the Americas: potential association with microcephaly and Guillain-Barré syndrome

Zika virus in Brazil and macular atrophy in a child with microcephaly

British Broadcasting Corporation (BBC) News. Colombia links Zika to rare nerve disorder deaths

First detection of autochthonous Zika virus transmission in a HIV-infected patient in Rio de Janeiro, Brazil

Zika virus associated with microcephaly

Ophthalmological findings in infants with microcephaly and presumable intra-uterus Zika virus infection

Ocular findings in infants with microcephaly associated with presumed Zika virus congenital infection in Salvador, Brazil

Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?

Zika virus: Brazil's surge in small-headed babies questioned by report

Viral infections during pregnancy

Levels of IgM antibodies against dengue virus in Rio de Janeiro

Persistence of virus-reactive serum immunoglobulin m antibody in confirmed West Nile virus encephalitis cases

Development and persistence of West Nile virusspecific immunoglobulin M (IgM), IgA, and IgG in viremic blood donors

Virus and antibody dynamics in acute west nile virus infection

One-step RT-PCR for detection of Zika virus

A diagnostic polymerase chain reaction assay for Zika virus

Universal primers that amplify RNA from all three flavivirus subgroups

Detection of Zika virus in semen

Interim Guidelines for pregnant women during a Zika virus outbreak e United States

Interim Guidelines for the evaluation and testing of infants with possible congenital Zika virus infection e United States

Zika virus spreads to new areas e region of the Americas

Wolbachia and the biological control of mosquito-borne disease

Zika virus and the never-ending story of emerging pathogens and transfusion medicine

Jamaica advises women to avoid pregnancy as Zika virus approaches

A review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available

Arthropod-borne viral infections of man in Nigeria

Dengue and other arboviral diseases in South-East Asia

Zika virus, French polynesia, South pacific

Rapid spread of emerging Zika virus in the Pacific area

First report of autochthonous transmission of Zika virus in Brazil

Zika virus spreads across Americas as concerns mount over birth defects

Zika virus-related hypertensive iridocyclitis

Laboratory infection with Zika virus after vaccination against yellow fever

The study was partly supported by the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease of the Department of Health, The Food and Health Bureau, Hong Kong Special Administrative Region, China; and by the donations of Hui Hoy and Chow Sin Lan Charity Fund Limited and Mr. Larry Chi-Kin Yung.

The authors declare no conflict of interest.
