Recent progresses and remaining challenges for the detection of Zika virus

Zika virus (ZIKV) has emerged as a particularly notorious mosquito‐borne flavivirus, which can lead to a devastating congenital syndrome in the fetuses of pregnant mothers (e.g., microcephaly, spasticity, craniofacial disproportion, miscarriage, and ocular abnormalities) and cause the autoimmune disorder Guillain‐Barre' syndrome of adults. Due to its severity and rapid dispersal over several continents, ZIKV has been acknowledged to be a global health concern by the World Health Organization. Unfortunately, the ZIKV has recently resurged in India with the potential for devastating effects. Researchers from all around the world have worked tirelessly to develop effective detection strategies and vaccines for the prevention and control of ZIKV infection. In this review, we comprehensively summarize the most recent research into ZIKV, including the structural biology and evolution, historical overview, pathogenesis, symptoms, and transmission. We then focus on the detection strategies for ZIKV, including viral isolation, serological assays, molecular assays, sensing methods, reverse transcription loop mediated isothermal amplification, transcription‐mediated amplification technology, reverse transcription strand invasion based amplification, bioplasmonic paper‐based device, and reverse transcription isothermal recombinase polymerase amplification. To conclude, we examine the limitations of currently available strategies for the detection of ZIKV, and outline future opportunities and research challenges.

virions. While seven nonstructural proteins play a pivotal role in regulating viral replication and transcription together with the activation of host antiviral responses. 28,29 Based on previous reports, ZIKV evolved in Uganda probably between 1892 and 1943. 30 A nucleotide sequence-based study of the NS5 gene indicated that three lines of ZIKV exist: Asian (one strain analyzed), East African (one strain analyzed), and West African (three strains analyzed). Through the analysis of entire genomes (phylogenetic), there are two genetic lines of ZIKV originating from Africa and Asia. The Asian lineage was further classified into two factions: Micronesian and Malaysian strains. dermal fibroblasts, and epidermal keratinocytes are known to be accommodating to ZIKV infection. The TIM-1, DC-SIGN, AXL, and Tyro entry/adhesion factors permitted the entry of ZIKV. The replication of ZIKV could trigger an immune antiviral response and in infected cells produced type I interferon. 65 Similarly, T cells (e.g., Th1, Th2, Th9, and Th17) are activated during the acute phase of ZIKV infection. 66 Animal models can be used to help understand the pathogenesis of ZIKV infection. 67 For example, Li et al. have used a mouse model to demonstrate that 25-hydroxycholesterol (25HC) could protect hosts against ZIKV infection. The addition of 25HC inhibited ZIKV infection in vitro by blocking the entry of the virus. Moreover, treatment with 25HC could reduce viremia and could provide protection against ZIKV in rhesus macaques and mice. The results indicated that 25HC could be used as a potential natural antiviral agent to defeat ZIKV infection and prevent ZIKV-associated birth defect (such as microcephaly). 68 The primary neural progenitor cells for cortex development in the fetus are radial glial cells (RGs). Wu et al. found that when the abdominal cavities of pregnant mice were injected with a contemporary ZIKV strain, the RGs of the dorsal ventricular zone were infected, causing a remarkable reduction of these cortex founder cells in fetuses. Therefore, the study reinforced the conclusion of ZIKV vertical transmission and infection affecting fetal brain development. Moreover, the study also provided a promising animal model for the evaluation of therapeutic regimes and preventative measures. 69 Cugola et al. demonstrated that Brazilian ZIKV (ZIKV BR ) could infect fetuses and could lead to birth defects, including intrauterine growth restriction and microcephaly in mice. In addition, the virus could infect human cortical progenitor cells (hCNPCs) and even result in the increased cell death. It was verified that ZIKV BR could cross the placenta and lead to microcephaly by targeting cortical progenitor cells, and could induce cell death and impair neuronal development. 70 It has been confirmed that ZIKV infection can increase cell death by the dysregulation of cell-cycle progression and lead to the death of hCNPCs, causing cortical thinning and microcephaly. 71,72 Rana et al. demonstrated that a prototype strain of ZIKV, MR766, which could efficiently infect organoids and lead to a decrease in overall organoid size correlated with the kinetics of viral copy number.
The human embryonic stem cell derived organoid was successfully developed for recapitulating first trimester growth of fetal brain. Moreover, it was found that in neurosphere models that ZIKV could infect neural progenitor cells, and the ZIKV activated toll-like receptor 3 (TLR3) in cerebral organoids attenuating neurogenesis. 73 In another study, Kriegstein's group clarified the fundamental cellular and molecular mechanisms of neurological defects linked to ZIKV infection. Moreover, the expression of receptors during the entry of enveloped viruses such as ZIKV into various cell types of the developing brain has been investigated. 74 There is increasing evidence suggesting that microcephaly is connected with ZIKV particularly in the first trimester of pregnancy. [75][76][77][78][79] Nevertheless, the underlying mechanism still needs further detailed investigation.
Currently, numerous studies have evaluated the relationship between ZIKV and microcephaly through animal

| Vector transmission
It has been reported that the ZIKV incubates for a period of 4-5 days in the human host and can then infect other vectors during blood feeding. Then, the virus spends an extrinsic incubation period of 8-12 days in the vectors infected by the virus and is then transported in the saliva of vectors to infect other hosts. 34,114 Based on previous reports, the mosquitoes have been known as one of the most important carriers that spread and transport ZIKV.
However, Aedes are the most common mosquito species found as vectors of ZIKV transmission, including A. africanus, A. aegypti, A. furcifer, A. vitattus, A. apicoargenteus, A. vittatus, A. hensilii, and so forth. 19,114,124,166 For example, in the tropics and subtropics, A. aegypti is the main mosquito vector associated with ZIKV transmission. In addition, A. aegypti and the A. albopictus species are known to spread ZIKV within the Americas. 167 When Aedes mosquitoes bite people infected by ZIKV, they can be infected by the ZIKV from humans. Then, healthy individuals can be infected by infected mosquitoes during the process of blood feeding. 105,168 Since, ZIKV is associated with multiple mosquito species, the transmission dynamics of ZIKV still remain largely unknown. 169 In recent years, the vertical transmission of ZIKV between mosquitoes has been widely studied. To test whether ZIKV could be vertically transmitted, Thangamani et al. injected ZIKV into the female A. albopictus and A. aegypti. The results indicated that the vertical transmission of ZIKV in A. aegypti mosquitoes could offer a potential mechanism for the virus to survive under adverse conditions. 170  | 2045 also studied. [172][173][174] These studies suggested that vertical transmission of ZIKV in mosquitoes played potential roles in ZIKV spread and maintenance, and thus more mosquito control strategies should be proposed to regulate mosquitoes when a ZIKV epidemic occurs.

| Sexual transmission
Sexual transmission of ZIKV has been reported, and significant effort has been given to explore that transmission mode. 57,[175][176][177][178][179][180][181][182][183] The first report of the sexual transmission of ZIKV was in Colorado, USA (2008). 97 Similarly, in 2011, another report indicated sexual transmission of ZIKV. 163 While, another similar study indicated that a female diagnosed through the reverse transcriptase polymerase chain reaction (RT-PCR) was infected by ZIKV. The woman had sexual intercourse with an infected man who had recently returned from the Caribbean, and serologic testing confirmed that they were both infected by ZIKV. 184 In addition, viral particles were discovered in the semen of a man who had been infected by ZIKV. 185 Shortly afterward, the female-to-male sexual transmission of ZIKV was reported for the first time, which indicated that ZIKV was able to spread rapidly. 186 In addition to vaginal sex, anal sex has been confirmed to transmit ZIKV. Deckard et al. 187 reported a case of sexual transmission of ZIKV between a man (patient A) who recently traveled to an area of active ZIKV and his male partner who had not travelled (patient B), indicating that ZIKV could be transmitted through anal sex. The infectious viral load and ZIKV RNA detected in the semen of a male indicated the potential of sexual transmission. 176,188,189 To date, ZIKV is the first arbovirus to be detected in the semen of humans. 185 To study ZIKV sexual transmission and vaginal viral replication, Shresta et al. constructed a mouse model of ZIKV. Where, it was found that the infected mice were resistant to vaginal infection during the estrus-like phase. While, when the mice were infected during the diestruslike phase, they succumbed to infection or experienced transient illness. Based on these lethal and sublethal mouse models, it was shown that intravaginal deposition of ZIKV could lead to transgenital transmission, hormonal changes in the reproductive tract (FRT) of female mice, and the replication of ZIKV could persist in the FRT of female mice for several days. 190 Recently, some reports indicate that the RNA and infectious viral load of ZIKV can be detected in the urine and saliva of infected patients. Due to the related properties of behavior connected with sexual activity, it was quite difficult to distinguish between sexual transmission and urinary/salivary transmission. 97,[191][192][193]

| Maternal fetal transmission
Significant evidence exists to confirm that mothers infected by ZIKV can transmit the virus to their foetuses during pregnancy. 166,[194][195][196][197][198][199] In addition, it has been shown that ZIKV can cross the placenta and infect the nervous tissues of foetuses, the mechanism of which is supported by detecting ZIKV RNA and antigens in the placenta, amniotic fluid, and the brain tissues of babies. Moreover, ZIKV could damage the human placenta and display significant fetal neurotropism. 167,[200][201][202][203] To further study the mechanism of action, Miner et al. 204 found that ZIKV infection could lead to placental damage and fetal death during pregnancy using mouse models, which further supported the transmission by a trans-placental route. Babies may be born with congenital teratogenic disorders when mothers were infected by ZIKV during pregnancy. For example, in Brazil, infected pregnant women have reported that their babies are born with congenital teratogenic disorders, such as microcephaly. 41 Similarly, the ZIKV has been detected in the amniotic fluid of pregnant woman during the 17th week of pregnancy in Spain. Using an ultrasound scan, fetal malformations were observed, such as arthrogryposis and brain microcalcifications. 205 It has been reported that the RNA and infectious viral particles of ZIKV can be detected with high loads in the breast milk of infected mothers. 206,207 On this basis, the result indicated that the transmission of ZIKV can occur from a mother to her baby. For example, Besnard et al. evaluated two cases of mother-to-child ZIKV transmission and the RNA of ZIKV was found in the breast milk and serum of the two mothers. In addition, ZIKV RNA was also detected in the serum of their babies. 166 However, when the breast milk was inoculated with Vero cells, the replication of ZIKV could not be detected, which made the transmission by breast milk uncertain yet plausible.
Therefore, more evidence is required to support the ZIKV transmission through breast milk. 208

| Transmission through body fluids
It is known that the ZIKV can be transported by body fluids (e.g., blood, urine, and saliva), which has been confirmed through the detection of ZIKV RNA in urine, blood, saliva, and other body fluids using the RT-PCR. 19,[208][209][210][211][212][213][214] During a ZIKV outbreak in French Polynesia, there were about 3% asymptomatic blood donors detected with ZIKV RNA, and the potential risk of ZIKV transmission through blood transfusion received from infected patients has been reported by Musso et al. 215 In addition, there have been two cases of blood transfusionrelated ZIKV transmission in Brazil. 216 To solve this problem, strict guidelines were issued by the WHO for blood donation/transfusion in regions where ZIKV was endemic in February 2016. 180 In addition, the American Association of Blood Banks indicated that ZIKV represented a high risk and took preventative measures to control the ZIKV transmission through blood transfusion, such as pathogen inactivation, and nucleic acid test (NAT) of blood products. 19 There were two methods put forward to prevent ZIKV transmission through blood transfusion, one was the detection of ZIKV in donated blood by a simple yet precise test, another was avoiding blood donations from infected individuals in ZIKV endemic regions or those who have traveled to those regions. 217 To improve the safety of transfusion, rendering the pathogens in blood inactive through the pathogen reduction technologies is an extremely efficient approach. 218 However, detection strategies and the transmission of ZIKV from donated blood require more investigation. 2 ZIKV has been detected in saliva and urine using RT-PCR, and ZIKV is more frequently detected in saliva and urine than that in blood using the RT-PCR tests. Infected individuals display the highest concentrations of ZIKV in their saliva at disease onset. Therefore, the saliva can be used as a suitable sample for the routine detection of ZIKV RNA when blood collection is difficult, 193 particularly in neonates and children. In January 2016, Barzon et al. 192 demonstrated that ZIKV could be detected in the saliva of patients who had recently returned from the Dominican Republic with febrile illness. Urine samples have been used for the detection of ZIKV infection, particularly during the later phases of ZIKV infection. 191,193,219,220 Kutsuna et al. 221 reported that the ZIKV was detected in the urine of a Japanese traveler who had travelled to Bora Bora in January 2014. In addition, there is evidence indicating that ZIKV can be transported through urine. 213 For example, in June 2016, Bonaldo et al. reported that infectious ZIKV particles were isolated from the urine and saliva of patients with acute symptoms in Brazil. The urine and saliva samples were inoculated with Vero cells, and were detected through RT-PCR, NAT, and quantitative RT-PCR. However, the viral load found in urine samples was higher than that in saliva samples. 222 It was shown that urine samples were important for the diagnosis of ZIKV infections within 10 days after the onset of the disease. 19 ZIKV is a huge global threat for humans due to rapid worldwide dispersal. 116,223 Researchers from around the world have made significant effort to develop effective strategies for the detection of ZIKV infection (Figure3).
Unfortunately, recently, ZIKV has resurged in India with the potential for devastating effects. Therefore, novel, rapid, accurate, and sensitive strategies for the detection of ZIKV are urgently required to effectively prevent and control outbreaks of ZIKV. The discovery of ZIKV can be performed by detecting the viral components of ZIKV (e.g., viral proteins, RNA, or virus isolation), or measuring the antibody concentration against viral proteins of ZIKV (host immune response) through serological assays. 96 In recent years, there have been many strategies (Table 1) developed for the detection of ZIKV, including viral isolation, serological assays, molecular assays, and sensing methods. [224][225][226][227][228][229][230][231][232] Recently, there have been some novel detection strategies developed for the detection of ZIKV, such as reverse transcription loop mediated isothermal amplification (RT-LAMP), transcription-mediated amplification (TMA) technology, reverse transcription strand invasion based amplification (RT-SIBA), bioplasmonic ZHANG ET AL.
| 2047 paper-based device (BPD), reverse transcription isothermal recombinase polymerase amplification, near-infrared spectroscopy (NIRS), nanoparticle (NP)-enhanced viral lysate electrical sensing assay, and smartphone-based fluorescent lateral flow immunoassay. In addition, there are a great many kits developed for the detection of ZIKV, such as the kits based on nucleic assay and serological assays. The ZIKV diagnostic kits currently authorized by Emergency Use Authorization and commercially available kits for ZIKV detection have recently been evaluated. 233,234 While may reviews summarize the methods for the detection of ZIKV, they are not comprehensive, and just cover viral isolation, serological assays, and molecular assays. 19,225,226,234,235 Herein, we comprehensively summarize the strategies for detecting ZIKV (i.e., the viral isolation, serological assays, molecular assays, advanced sensing strategies, and other current state of the art methods).

| VIRAL ISOLATION
The viral isolation from cell cultures has been considered as the "gold standard" for the detection of viruses, and has been widely and universally performed since the early 1960s. Moreover, viral isolation has been the standard strategy that all other strategies are compared with. 310  Calf blood: 10 pM degeneration of cells, and changes of cells are collectively called the cytopathic effect (CPE). Unfortunately, viral isolation methods suffer from some disadvantages, such as requiring a long incubation period and the expertise of evaluating the CPE. In addition, when evaluating the CPE, it is worth noting that the viral load in the serum often presents at a low level with a fleeting viremic stage. Moreover, the viremic stage may cease after the onset of symptoms. 311 With technological advances, the development of monoclonal antibodies and introduction of molecular diagnostics have offered powerful strategies for the detection of viral infections. Currently, viral isolation in cell culture serves as an unpractical tool for most clinical situations, and as such is primarily used for research purposes. 226 112,114,224,225,236,248,[314][315][316][317][318][319][320][321][322][323][324] However, there are some performance limitations of serological assays. Because of the cross-reactivity between the antibodies of ZIKV and the antibodies against other homologous flaviviruses (e.g., denguevirus), low viral loads, and nonspecific binding, the serological assays of ZIKV are difficult. [325][326][327][328][329] Though they possess some disadvantages, serological assays still need to be further developed for overcoming the shortcomings of other methods.

| IgM antibody capture enzyme-linked immunosorbent assay
Due to its high specificity, sensitivity, and ability to directly detect complex biological samples, the ELISA technique has been viewed as an effective strategy for the detection of IgM in the serum of patients, and has been widely applied in multiple fields. [330][331][332][333][334][335][336][337][338][339][340][341][342] Over the past few decades, the outbreak of ZIKV represents a huge global threat for humans, especially for pregnant women. [343][344][345] The ELISA technique has been widely applied for the detection of ZIKV, because the IgM antibodies in the infections of ZIKV are generally generated during the later stages of infection and detected for up to 3 months. Moreover, after the development of IgM antibodies, the immunoglobulin G (IgG) antibodies are typically produced within a few days, and can be constantly detected for several months. 231,236,[346][347][348] As reported previously, the first step in MAC-ELISA ( Figure 4A) is adding the sample from patients into a well plate precoated with antibodies against human IgM. Following that, the virus-specific antigen is added into a well plate, if IgM is present in the patient sample, strong binding to antibodies in the well plate will occur. Otherwise, the sample from the patient will be washed away and there is no detectible signal when the secondary antibody (the antibody tagged with the enzyme like horseradish peroxidase) is added. After the secondary antibody is added, chromogenic substrates (e.g., 3,3′,5,5′-tetramethyl benzidine; 2,2′-azinobis(3ethylbenzothizoline-6-sulfonic-acid); and o-phenyl-enediamine) are employed for quantification. The samples from infected patients will lead to an optically detectible signal (e.g., fluorescence and absorbance), which can be measured and read using a spectrophotometer to determine the IgM concentration for the patient's samples. 235 At genetic properties of the ZIKV were associated with this epidemic. At that time, the immune responses due to the infection by ZIKV had been rarely described. 96 When the MAC-ELISA was employed for the assays of IgM with whole viral antigen (inactivated virus) and the monoclonal antibody (MAbs)-based capture ELISA was used for the detection of IgG, it was found that the IgM appeared as early as 3 days after the onset of symptoms but the IgG emerged after 10 days in an infected patient without a history of flavivirus infections. 350,351 As such, the detection of neutralizing antibodies against ZIKV as early as 5 days after the onset of symptoms is possible. 231 In 2016, the use of putative cross-reactive sera from infected patients with Euroimmun (Euroimmun, Lübeck, Germany) IgM antibodies against ZIKV and anti-ZIKV IgG in ELISA tests was reported by Huzly et al., which exhibited a high specificity and confirmed the applicability of Euroimmun ZIKV ELISA for specifically detecting the virus from individuals with suspected previous exposure to flavivirus or vaccine. Moreover, it demonstrated this ELISA strategy as an effective diagnostic tool that could be used for the counseling and screening of patients who had potentially been infected by ZIKV (e.g., the travelers and pregnant women commuting from the endemic regions of ZIKV). 236 356 PRNT possesses better specificity and sensitivity than MAC-ELISA. However, there are some disadvantages with PRNT. For example, the PRNT is laborintensive and expensive, and requires live virus. Moreover, antibodies against the conserved flavivirus domain can reduce detection specificity, especially during the acute stage and early convalescent period. 357 In a recent study, Collins et al. evaluated the late convalescent serum samples from the patients with previous exposure to ZIKV or DENV. It was found that most DENV infections could not induce lasting, high-level, and cross-neutralizing ZIKV antibodies. Moreover, ZIKV-specific antibody populations develop after ZIKV infection irrespective of prior DENV immunity. The development of ZIKV-specific antibody populations after the ZIKV infection was unrelated to prior DENV immunity. 358 Additionally, data from Puerto Rico demonstrated that PRNT provided an inaccurate confirmatory test, for populations with a high pre-exposure to DENV. 326,359 3.3 | Immunofluorescence assay IFA as one the most commonly used tests has been employed for the detection of ZIKV. [360][361][362][363][364][365][366] The first step of IFA is that the BHK21 cells are infected by various strains of flavivirus and fixed by ice-cold acetone. Then, the infected cells are incubated in the normal mouse serum or a 100-fold dilution of mAb 2A10G6, and the cells need to be washed three times with phosphate-buffered saline (PBS) after incubation for 60 min at 37°C. Subsequently, cells were treated at 37°C using a 200-fold dilution of FITC-conjugated anti-mouse IgG in 0.02% (w/v) Evans blue for 30 min. Finally, after being rinsed with PBS five times, the positive cells can be determined with a fluorescence microscope. 367 For example, Hamel et al. reported that IFA was used for fibroblasts infected by ZIKV ( Figure 4B). ZHANG ET AL.

| 2057
Moreover, it was found that the skin immune cells (e.g., immature dendritic cells, dermal fibroblasts, and epidermal keratinocytes) were all permissive to the infection of ZIKV. To conduct the laboratory confirmation of ZIKV infection in infected patients, Huzly et al. 112 used an indirect IFA to determine the ZIKV infection of infected patients as previously described. It was found that the anti-ZIKV IgM was determined by indirect IFA titers ranging from 1:1280 to 1:>20,480, and the anti-ZIKV IgG was determined by indirect IFA titers ranging from 1:320 to 1:>20,480. 236 Deng et al. studied the Asian lineage ZIKV imported to China, including its isolation, identification, and genomic characterization. Moreover, they further identified the newly isolated ZIKV by IFA by using the convalescent phase serum of a patient infected by imported ZIKV. It was found that the serum of the convalescent phase with 1:320 dilution could strongly react with the SZ01 virus which was newly isolated. 368 Recently, Li et al.
found that 25HC could protect host against ZIKV infection. It was found that the Vero cells could be protected from ZIKV with a 0.4 mM concentration of 25HC. 369 However, it was reported that the IFA also exhibited crossreactivity related to various flaviviruses. 96

| Multiplex microsphere immunoassays
Currently, serological assays of ZIKV mainly depend on IgM capture, which can cause some problems due to the cross-reactivity with other homologous flaviviruses. Nevertheless, the MIA could enhance the sensitivity and specificity of ZIKV detection by adding one or more antigens. Compared with other diagnostics tests, MIA shows advantages in that it captures the diagnostic ability of viral envelope proteins (EPs; which elicits robust but crossreactive antibodies against other flaviviruses) and the contradistinctive differential ability of viral nonstructural proteins (e.g., NS1 and NS5, which can increase the specificity (78-100%) of the assay). 224,255,356,[370][371][372] In 2017, Wong et al. reported the multiplex MIA for serologic diagnosis of ZIKV that combined viral structural with nonstructural proteins for the first time. The developed MIA platform could achieve rapid diagnosis (<4 h) with an improved diagnostic accuracy, and higher sensitivity than IgM-capture ELISA in detecting the ZIKV infection.
Moreover, only a small specimen volume (10 μl) was required for a single reaction. In addition, the obtained MIA platform could not only be used for the clinical diagnosis of ZIKV infection, but was also able to monitor immune responses in vaccine trials. 373 In a recent study, a novel multiplexed flavivirus IgM MIA (flaviMIA) was successfully developed and validated by Taylor et al., which could be used for differentiating ZIKV-specific IgM from the other flavivirus infections of humans. The developed flaviMIA could combine 12 inactivated flavivirus antigens (which were from yellow fever virus (YFV) and ZIKV) to form the unequal anti-flavivirus antibody coupled beads for interrogating the sera of infected patients (who were suspected to have a ZIKV infection after traveling to relevant countries). The obtained flaviMIA protocol displayed broad concurrent flavivirus serodiagnostic ability, and was easily operated. Moreover, the flaviMIA protocol was suitable for high throughput testing, and could achieve a rapid diagnosis and could generate repeatable results. 249

| Reporter virus neutralization test
Currently, the serologic assay of ZIKV infection mainly depends on the labor-intensive PRNT, which requires the long turnaround time (over 7 days) and results in a bottleneck for the ZIKV diagnosis of patients. To solve this problem, Shan et al. successfully developed a novel high-throughput assay (RVNT), which could be used for the diagnosis of ZIKV and DENV ( Figure 5). The newly developed RVNT was homogeneous, and neutralizing antibody could be quantified using the luciferase viruses in a 96-well format. It was found that RVNT had higher dynamic range and could keep the relative specificity as the current PRNT assay by using 91 human specimens. Moreover, the RVNT could improve assay throughput and shorten the turnaround time (<2 days). Furthermore, the study demonstrated that the RVNT could be employed as a first-line diagnostic test for vaccine clinical trials. 374 Shortly afterward, the same group evaluated the performance characteristics of the RVNT with 258 clinical serum specimens by employing the current PRNT as a reference strategy. The result demonstrated that the RVNT as a screening assay exhibited an excellent diagnostic specificity, sensitivity, and accuracy. Based on the confirmatory assay, the developed RVNT titers were verified to show 93.1% agreement with the current PRNT titers. Moreover, RVNT as an accurate and reliable strategy could be used for the detection of neutralizing antibodies in serum specimens of patients. In addition, RVNT could significantly shorten the turnaround time and greatly improve the assay throughput to detect many clinical specimens in a single run owing to its homogeneous 96-well format. As such RVNT has great potential in clinical diagnosis, serologic surveillance, and monitoring antibody response in vaccine trials. 250  for ZIKV infection using a virus-like particle-based method. The RVP assay was shown to work in a 96-well plate format, and its GFP readout could be quantitated using a simple fluorescence microscope. Moreover, the assay exhibited high specificity and reproducibility in the detection of ZIKV antibodies as PRNT. 375 Though RVNT exhibits many advantages compared with traditional PRNT, it still needs more well-characterized samples of infected patients to be tested in order further strengthen the validation of the novel RVNT. 322,376 F I G U R E 5 Experimental scheme of reporter virus-based infection assay to measure neutralization titers of specimens. Reprinted with permission from Ref. 246 Figure 6B). Using square wave voltammetry and electrochemical impedance spectroscopy (EIS), the developed biosensor exhibited high sensitivity and specificity for detecting the ZIKV antibodies in saliva and blood samples. The biosensor was used to quickly distinguish the specific antibodies of ZIKV from DENV. Moreover, because of the high sensitivity, the new biosensor could be applied to early infection detection when conventional ELISA approaches could not detect circulating antibodies whose levels were still very low. In addition, the biosensor showed the ability to detect the ZIKV antibodies in the saliva samples, which could solve the problem of collecting serum samples from volunteers or infected individuals. Furthermore, the saliva test could be used for rapid screening of people from sensitive areas during a ZIKV outbreak (e.g., the airports or border). 253 With the advances in NPs and digital health systems, portable and sensitive detection technologies have been quickly developed for timely managing emerging viral infections. For example, a nanomotor-based bead-motion cellphone (NBC) system was successfully developed by Draz et al. for the specific and sensitive immunological detection of ZIKV infection, which was rationally designed with nanotechnology, cellphone, and microfluidics. Interestingly, the platinum (Pt)-nanomotors could be accumulated on the surface of beads and would move in H 2 O 2 solution when the ZIKV was present in a test sample. The concentration of ZIKV was determined by correlation of changes in the beads motion. The obtained NBC system was able to detect the ZIKV in samples with a low concentration of 1 particle/μl. The newly developed NBC system displayed high specificity for detecting the ZIKV in the presence of closely related viruses and other neurotropic viruses (e.g., human cytomegalovirus and herpes simplex virus type 1). Furthermore, the developed NBC system allowed for simple and rapid testing of viral load, and could be potentially employed for immunoassays that need rapid and simple identification of viruses. 378 Recently, Sola et al.
developed a novel, straightforward, and robust strategy (Figure 7) to extend the range of immune-diagnostic probes by copresenting peptides which belong to the identical antigenic surface. The group focused on ZIKV NS1 protein antigen putative antigenic region, a diagnostic confidence was reached through the oriented and spatially controlled coimmobilization of the peptide sequences found adjacent within the protein fold, which cooperatively interacted to provide an enhanced immunoreactivity with respect to single linear epitopes. Based on the developed strategy, ZIKV infected individuals could be differentiated from healthy controls. The method could be included in high-throughput screening platforms of mixed and linear peptide libraries. Moreover, it may facilitate a fast identification of the conformational immunoreactive region. 379 F I G U R E 7 (A) The working principle for enhancing antibody serodiagnosis using a controlled peptide coimmobilization method. (B) Strategy to enhance immunoreactivity by spatially controlled copresentation of peptidic probes on microarray surfaces through "Click" reaction of yne-modified peptides on Copoly Azide. Reprinted with permission from Ref. 379

| MOLECULAR ASSAYS
As a consequence of the cross-reactivity with other homologous flaviviruses (e.g., DENV), the serological assays of ZIKV are extremely challenging (due to an uncertainty of the results). 225 Therefore, molecular assays have played an important role in confirming the detection of viruses. Molecular assays as an important tool for the detection of ZIKV can be performed using RT-PCR. Moreover, owing to its high sensitivity and high selectivity, the RT-PCR has been viewed as the gold standard for the molecular amplification and diagnostics of ZIKV. 235,300,381,382 To date, the ZIKV RNA has been found in various biofluids (e.g., semen, blood, urine, saliva, breast milk, and amniotic fluid) and could be detected by RT-PCR. 184,208,[383][384][385] It has been confirmed that ZIKV is more frequently detected in saliva and urine samples than in blood using the RT-PCR tests. Saliva samples are particularly interesting since they have increased the prospects of ZIKV detection at the acute phase of infection and provides appropriate results when blood is hard to be collected particularly in neonates and children. Nevertheless, viral load has been discovered to be higher in urine samples than that in saliva samples. Moreover, the detection of ZIKV in patients' urine more than 10 days after disease onset have indicated that the urine sample was of great importance for the diagnosis of ZIKV infections. 19,222 In this review, RT-PCR methods have been categorized into traditional RT-PCR, real-time RT-PCR, pan flavivirus RT-PCR, and nested RT-PCR. Among these assays, the pan flavivirus RT-PCR and nested RT-PCR are rarely used. In addition, several other molecular assays have been illustrated, such as real-time PCR based neutralization assay, localized surface plasmon resonance (LSPR), and DNA sequencing.

| Traditional RT-PCR
RT-PCR with high sensitivity and selectivity has been considered as the gold standard for the molecular amplification and assay of virus. In the early phase of the acute period of ZIKV infection, it is feasible to determine the ZIKV RNA in serum by RT-PCR. 225,355,[386][387][388] Based on previous reports, the RT-PCR has also been employed for detecting the ZIKV in multiple sample matrices, such as semen, plasma, saliva, serum, urine, and conjunctival fluid. 188,193,200,381,[389][390][391] For example, Bingham et al. compared the result of ZIKV RNA detected by RT-PCR in saliva, serum, and urine specimens from infected people with a history of traveling to Florida. It was found that the urine samples could be used as the most suitable specimen to identify acute infections of ZIKV. 392 Similarly, by studying small sample size or several cases using both the blood and urine samples, Lamb et al. found that the ZIKV RNA detected in urine possessed higher levels after the onset of ZIKV infection than that in blood. Based on the result, they recommend that serum and urine were collected for molecular assays and served as a standard for evaluating ZIKV infection of patients. Moreover, it was found that urine could serve as a good alternative sample when blood collection was difficult and ZIKV was hard to be detected in serum samples during early disease infection. 393,394 Interestingly, Tan et al. evaluated strategies to maximize the detection and quantitation of ZIKV RNA in urine samples, and determined that urine samples required special consideration because pre-analytical factors (e.g., the freeze-thaw, length of storage, and storage temperature) could adversely affect the stability of the urine samples. Moreover, it was found that urine samples stored at 4°C for more than 48 h could influence the detection of ZIKV RNA by RT-PCR, and urine samples frozen at −80°C could lead to the significant loss of detectable ZIKV RNA. Furthermore, it was found that the detection of ZIKV could be achieved by adding a nucleic acid stabilizer (e.g., Shield or ATL) when delays in testing were anticipated. 395 In another study, George et al. detected the envelope (E) protein and NS2B genes of ZIKV in serum and urine samples collected from infected patients (including newborns, pregnant women, travelers to ZIKV epidemic areas in New York, and suspected symptomatic GBS patients) using realtime RT-PCR. Given the short viremia time period of ZIKV infection, selecting appropriate samples for detection was a promising strategy, because the ZIKV RNA could not be detected in plasma or serum samples obtained 10 days after the onset of symptoms. However, the results indicated that urine samples were more dependable than serum samples due to their higher viral load. Moreover, compared with other serological assays, the test results of this strategy were more reliable. 396 To compare the detection of ZIKV RNA in multiple specimen types, Rossini et al. used the plasma, whole urine and blood as samples to detect ZIKV RNA obtained on the same day (3-28 days after the onset of symptoms) from 10 patients infected by ZIKV. The results indicated that ZIKV RNA was positive in urine samples at the 21st day after the onset of symptom while the ZIKV RNA could be detected up to 26 days after the onset of symptom. In addition, it was demonstrated that the simultaneous test of different specimen types could increase the probability of disease diagnosis. 397 ZIKV RNA has been detected in breast milk of mothers. However, it has not been reported that ZIKV can be transported by breast milk. Another study reported by Murray et al. indicated that ZIKV in whole blood samples could persist for 81 days, while ZIKV in serum samples could persist for 73 days. Therefore, ZIKV RNA could be detected using the whole blood samples collected from asymptomatic patients. 391,398 Soon afterward, Frankel et al. successfully developed a dual targeting RT-PCR assay for detecting the ZIKV RNA within urine, whole blood, plasma, and serum, which used an automated m2000 system to extract ZIKV RNA from samples. The assay was highly specific and sensitive with a limit of detection (LOD) of 120 copies/ml in whole blood, 40 copies/ml in urine and plasma, and 30 copies/ml in serum. 399 While, Hancock et al. 400 used electronic health records (RT-PCR) in a syndromic surveillance program to monitor asymptomatic pregnant women from American Samoa in order to establish a timeline to cease routine testing.

| Real-time RT-PCR
Compared with conventional RT-PCR, real-time RT-PCR has some advantages, including low false positives, rapidity, higher specificity and sensitivity of quantitative analysis. 401 In real-time RT-PCR, the RNA of a virus needs to be extracted from the infected samples, which is then added into a reaction mixture (made using the necessary factors, including the specific primers of ZIKV, reverse transcriptase, DNA polymerase and dNTPs, buffer solution, and the intercalating fluorescent dye used for quantification) for amplification. With the amplification of the target, the fluorescence in the samples enhances as the amount of product increases. Thus, the concentration of ZIKV in the samples can be determined by the fluorescence changes with high specificity and sensitivity. Owing to its high specificity, sensitivity, and ability for quantitative analysis, real-time RT-PCR has been viewed as the most powerful tool for molecular assays.
Moreover, real-time RT-PCR was recommended by the CDC for detecting and quantifying ZIKV RNA during the early stages of ZIKV infection ( Figure 8A). 12,224,226,323 According to reports, at the early onset of ZIKV infection, the ZIKV RNA could be detected by real-time RT-PCR. Furthermore, the real-time RT-PCR could easily detect the ZIKV RNA in urine samples with the advantage of collecting samples easily and noninvasively, which was extremely useful for evaluating travelers during an epidemic. 191,381 It was reported that ZIKV infection could be confirmed using the real-time RT-PCR. 402 By using the real-time RT-PCR to detect the ZIKV in plasma and urine samples, Pessôa et al. 403 confirmed that the viral loads were lower in urine than that in plasma samples. In another study, Corman et al. made a comparison between seven published real-time RT-PCR assays and two novel assays developed by themselves. By analyzing the sensitivity of each assay, they discovered that there were some detection methods unsuitable for the specific diagnosis of ZIKV because of the limited sensitivity and the potential incompatibility of some strains. 404  clinical specimens collected from the epididymis/testis and kidney compared to the ZIKV-E gene targeting real-time PCR. Furthermore, the ZIKV-5ʹ-UTR assay has no in vivo or in vitro cross-reactivity with YFV, DENV, Japanese encephalitis virus, hepatitis C virus, chikungunya virus (CHIKV), and West Nile virus. 407 Recently, Santiago et al. successfully developed a novel Trioplex real-time RT-PCR assay (Trioplex assay) for detecting ZIKV infections and differentiating ZIKV infections from CHIKV and DENV. The analytical performances of all Trioplex modalities were evaluated through the detection of these three viruses (i.e., ZIKV, CHIKV, and DENV) in whole blood and serum samples, and ZIKV in urine samples. The developed Trioplex assay had a LOD close to 10 3 GCE/ml for the three pathogens using different RNA extraction methods. Moreover, simultaneous detection of more than one sample from each patient could increase detection sensitivity by 6.4%. The results indicated that the developed Trioplex assay has a high sensitivity for confirming ZIKV infections. 409  Moreover, the one-step real-time PCR could be used to distinguish between ZIKV, CHIKV, and DENV, and is particularly suitable for whole blood samples. Moreover, there were no PCR inhibition and no cross-reactivity found for the clinical specimens. 270 Real-time RT-PCR has been widely used, and has exhibited numerous advantages such as high sensitivity and specificity, fewer false positives, and quantitative analysis. However, there are some limitations. The main limitation is that the real-time RT-PCR depends on a good laboratory infrastructure with skilled operators. Moreover, it is timeconsuming, and requires several hours to obtain a test results. Furthermore, the equipment for PCR is expensive, and requires thermocycling.
Droplet digital PCR (ddPCR) is a method for the quantitation of nucleic acid, where the PCR reaction mix is partitioned into approximately 20,000 nanodroplets. Then, when PCR is performed, an automated droplet flow cytometer is used for reading the droplets. The ddPCR method exhibits improved accuracy and higher sensitivity than that of realtime PCR. Moreover, ddPCR serves as an endpoint measurement and can achieve an absolute quantification of ZIKV RNA levels without the requirement of a calibration curve, which makes it a promising tool for the determination of brain viral load. 411 Utilizing the micro-ddPCR and real-time RT-PCR, Hui et al. established a new protocol for the ZIKV detection based on the NS5 gene amplification. Compared with real-time RT-PCR, the established ddPCR method achieved a linear range from 10 1 to 10 4 copy/μl, and a LOD as low as 1 copy/μl. Therefore, the proposed ddPCR method illustrated great potential for early diagnosis, laboratory evaluations, and ZIKV monitoring. 412

| Pan flavivirus RT-PCR and nested RT-PCR
The pan flavivirus RT-PCR may sometimes be employed using a degenerate primer to detect flavivirus. However, pan flavivirus RT-PCR can lead to a loss of sensitivity. Similarly, the nested RT-PCR is also rarely utilized. The nested RT-PCR needs two successive PCRs and two primer sets (an inner primer set and an outer primer set).
The primary primer or outer primer is targeted on a general area of the RNA. Subsequently, the nested primer or inner primer is targeted on a specific coding region. This method can reduce the amplification of an unforeseen binding site. 189,226 Even though the RT-PCR has been reported to have many advantages, the probes and primers having high specificity when applied in RT-PCR may cause significant errors. Therefore, the problem of missing unanticipated sites should be noted, which may lead to failure in amplification.

| Other molecular assays
According to previous reports, other molecular assays have been developed for detection of ZIKV. 323 For example, by combining real-time PCR with serum neutralization tests, Wilson et al. developed a new real-time PCR based neutralization assay. The real-time PCR was used for measuring the neutralization endpoint rather than by counting plaques to measure the neutralization endpoint, but the test took 72 h. The assay could be used to confirm test results (the serum samples were positive) obtained from an IgG/IgM ELISA. Moreover, the real-time PCR based neutralization assay had a high sensitivity (100%) for both DENV and ZIKV. 413 In another study, a one-step reverse transcription-insulated isothermal PCR (RT-iiPCR) reagent set ( Figure 8B) was developed for the detection of the ZIKV RNA in spiked samples. Comparison of the CDC and PAHO real-time RT-PCR assays for various spiked sample types including mosquitoes, the performance of ZIKV RT-iiPCR reagent set could be evaluated. Moreover, these assays displayed an exclusive specificity for the ZIKV (Asian and African lineages) with detection endpoints of 10 PFU/ml of infected tissue culture fluid, and the LOD ranged from 10 to 100 for in vitro transcribed RNA copies/μl. In addition, it was confirmed that there was 92% agreement between the real-time RT-PCR assays and the ZIKV RT-iiPCR. 408 Surface plasmon resonance based biosensors as label-free technology have been widely applied for both bioassays and analytical chemistry. 414 Figure 8C). It was found that the NPs-Qdot bimetallic hybrids (e.g., AuAgNP-Qdot646-MB) could detect trace amounts of ZIKV RNA over a sample with a range of 6-673 copies/ml. Moreover, compared to single metal NPs, bimetallic NP-Qdot-mediated fluorescence signals were stronger for detecting ZIKV RNA. In this case, due to the differences between NP-Qdots, it was very important to choose the most appropriate one for the detection of ZIKV RNA. 291 DNA sequencing has been widely applied and used to confirm ZIKV infection, and the relationship between ZIKV strains could be demonstrated by sequencing of the NS3, NS5, and E genes. 101,124,[418][419][420] To solve the problem with the lack of ZIKV genomic data, Metsky et al. used multiple sequencing strategies to produce 110 ZIKV genomes from mosquito and clinical samples collected from 10 countries and territories. Viral genetic diversity from the ZIKV outbreak in America was greatly expanded. Then by comparing the generated 110 ZIKV genomes with another 64 published genomes from the NCBI GenBank, it was found that the ZIKV outbreak started in Brazil. 421 Buechler et al. analyzed the ZIKV genome using deep sequencing. The results indicated that a comparison of the ZIKV genome and sequences from the data bank could be used to identify the nucleotide changes of ZIKV specific strains. 422 Recently, Gu et al. has studied the first whole-genome sequence of ZIKV strain AFMC-U using next generation sequencing, which was amplified from the urine specimens collected from a Korean traveler returning from the Philippines. The study indicated that urine samples were very important for the detection of ZIKV infection. 423

| ZIKV DETECTION BASED ON SENSING METHODS
Though serological and molecular assays play a key role on the detection of viruses, there are some disadvantages limiting their widespread applications (e.g., extensive sample preparation, false-positive and false-negative results, the requirement of expensive laboratory equipment, requiring skilled operators, and time consuming generation of results). 314,356,411,412 Fortunately, opportunities exist for the development of sensing technologies toward ZIKV detection. In particular, sensing strategies that have been developed for use in multiple fields, including biochemical analysis, medical diagnostics, environmental monitoring, food quality control, and so forth. [424][425][426][427][428][429][430][431][432][433][434][435][436] As such, numerous sensing strategies have been developed for the sensitive and selective detection of ZIKV infection, such as electrochemical, colorimetric, fluorescence, and surface-enhanced Raman spectroscopy (SERS) methods. 235,323,437

| Electrochemical sensing
In recent years, electrochemical sensing has been widely applied in biochemical analysis, food quality control, and environmental monitoring. [438][439][440][441][442][443][444][445] To develop an electrochemical sensing methodology for the detection of targeted disease, many advanced technologies have been utilized, such as nanostructured immobilizing materials, transduction techniques, molecular recognition, and novel sensing arrays. 446 proposed that a nano-enabled electrochemical immunosensing technology could be used for the detection of ZIKV infection in biofluid specimens collected from the infected patients, and particularly the pregnant women and newborn babies ( Figure 9A). The proposed miniaturized electrochemical nano-sensing systems could be used for point-of-care (POC) ZIKV detection, and could provide better disease management and treatment by promoting high-speed personalized health-care for patients. 230 The proposed electrochemical nano-sensing systems could be fabricated using nano-structured sensing materials, miniaturized sensing transducers, nano-/micro-electrodes and microelectronics for selectively monitoring, screening and detecting ZIKV infections. [459][460][461][462][463] Recently, by using an interdigitated micro-electrode by functionalization of a gold (IDE-Au) array, a novel electrochemical immunosensor for selectively and sensitively detecting the protein of ZIKV was developed. In the electrochemical immunosensor, the IDE-Au immunosensing chip was constructed by immobilizing the specific envelop protein antibody (Zev-Abs) of ZIKV onto the functionalized IDE-Au with an electrode width/gap of 10 µm. The electrical response of the IDE-Au immunosensing chip as a function of ZIKV-protein concentrations could be measured using EIS, and it was verified that the sensing chip could selectively detect ZIKV protein. Furthermore, the electrochemical immunosensor displayed a high sensitivity of 12 kΩM −1 with a low LOD of 10 pM, and the ZIKV-protein could be detected from 10 pM to 1 nM. In addition, by integrating with a miniaturized potentiostat (MP) which was interfaced with a smartphone (Figure 9B), the as-prepared IDE-Au immunosensing chip could be used to rapidly detect ZIKV infections during the early stages in a POC application. 273 In a very recent study, a novel electrochemical biosensor based on nanocarbon materials (graphene oxide) and imprinted polymers (SIPs) composites was successfully developed for the detection of ZIKV in both serum specimens and buffer ( Figure 9C). The electrochemical biosensor could be employed for the detection of ZIKV using variation of electrical signal with a change of ZIKV concentrations in both serum specimens and buffers. The electrochemical biosensor could detect ZIKV with a low concentration of 2 × 10 −4 PFU/ml (1 RNA copy/ml) in PBS. Moreover, the LOD for ZIKV was close to the LOD of the real-time RT-PCR approach. 464
| 2067 of ZIKV. The paper-based biosensor could be used to detect ZIKV RNA genome using a CRISPR/Cas9-based module. The biosensor displayed high selectivity because of the hybridization between the ZIKV RNA and the CRISPR/Cas9 system (which was specifically designed). Moreover, the study indicated that ZIKV could be detected at femtomolar (fM) concentrations. By using the sequences of DENV as a negative control, the selectivity of the prepared biosensor could be confirmed. In addition, the results indicated that the developed paper-based biosensor exhibited high specificity and selectivity and the capability of discriminating viral strains with single-base resolution by using CRISPR/Cas9. 277 Using the color change of leuco crystal violet dye, Song et al. developed a POC ZIKV sensing system ( Figure 10B) using the RT-LAMP with high sensitivity, which only used a chemically heated cup to adjust to the sensing temperature of the system without a requirement for electrical power.
Moreover, the sensor was considered as an instrument-free POC detection device, because the color variation could be examined by the naked eye without any instrumentation. In addition, it was shown that the sensor could detect ZIKV from saliva, blood, semen, and urine samples with a high sensitivity of 5 PFU in a short time (<40 min). 278 Shortly afterward, Müller et al. developed a high-throughput colorimetric assay for the detection of ZIKV infection. The colorimetric assay could be used to quantify ZIKV infections with a broad range of viral dilutions in both human and monkey cells. On this basis, the neutralizing antibody titers in ZIKV antisera could be defined or the inhibitory activities of antivirals that block ZIKV could be tested using the colorimetric assay. More importantly, the 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide (MTT)-based colorimetric assay could be evaluated by using the naked eye and analysed using computational tools (Graphpad Prism), achieving a good linearity over a broad range and did not require costly reagents or equipment. Therefore, the colorimetric assay as a cheap, simple, and fast strategy for the testing of antiviral compounds and the quantification of ZIKV neutralizing antibodies is a promising substitute for antibody-based detection, especially with large specimen numbers in resource-poor settings. 485 To meet the requirement of rapid and noninvasive ZIKV diagnostic screening assays for the prenatal care of women living in the areas where the ZIKV may outbreak, Calvert et al.
developed a novel RT-LAMP assay for the colorimetric detection of ZIKV RNA in serum and urine specimens with high specificity and sensitivity using a simple visual detection method. The results of the RT-LAMP assay indicated a LOD 10-fold higher than real-time RT-PCR. The developed RT-LAMP assay could detect ZIKV RNA collected from a panel of 178 diagnostic samples with a low LOD of 1.2 RNA copies/μl. Moreover, it was shown that the RT-LAMP assay exhibited a high specificity for the ZIKV RNA when the ZIKV was examined with diagnostic samples positive for the CHIKV and DENV. In addition, the developed assay could be potentially used as a fast, sensitive, specific, and reliable assay for detecting ZIKV in serum or urine specimens under the conditions of clinical/field setting with minimal technological expertise and equipment. 279 Smartphones have attracted widespread interest due to their potential application in pathogen detection and POC usage. 486 In a very recent study, Priye et al. reported that the smartphone CMOS sensor could be used for reliably and reproducibly quantifying the colorimetric sensing of nucleic acid specimens with no vision biases ( Figure 11). Using luminescence-based analysis, positive or negative discrimination was higher by about an order of magnitude compared with traditional RGB analysis. Moreover, the chromaticity part of the analysis could achieve a reliable multiplexed detection for different targets that were labeled using spectrally separated fluorophores. Using the end point RT-LAMP, a F I G U R E 9 (a) An interdigitated microelectrode is modified with different nanostructures for high loading of ZIKV-specific antibodies for the detection of ZIKV proteins at picomolar concentrations using an appropriate electrochemical transduction technique. Reprinted with permission from Ref. 230  chromaticity-luminance formulation could simultaneously detect ZIKV and CHIKV RNA. In addition, the chromaticity-luminance analysis was suitable for other types of multiplexed fluorescence detection using a smartphone camera. 487

| Fluorescence sensing
In recent years, fluorescence sensing has been employed for the detection of ZIKV infections. 488 For example, a new ultrasensitive electrogenerated chemiluminescence (ECL)-based immunoassay ( Figure 12A The smartphone app can wirelessly actuate the RGB LED excitation source and isothermal heater to achieve real-time monitoring and change of the heater temperature along with illumination of the samples using an appropriate excitation light source. The illuminated reagent can be captured through a smartphone camera with an interchangeable emission filter and the image is able to be subsequently analyzed. Reprinted with permission from Ref. 281  (QUASR) technique. A simple, portable, and cheap "LAMP box" using a smartphone (consumer class) could be used to conduct the reactions, which could be powered using a 5 V USB source (e.g., solar panel or USB power bank).
Compared with the detection using the naked eye or the traditional RGB intensity sensors, the use of a smartphone could increase fivefold the discrimination of positive or negative signals, because the smartphone exploited chromaticity to analyze fluorescence signals using an algorithm. The developed diagnostic device could directly detect ZIKV collected from saliva, blood, and urine specimens, which demonstrated that the device was suitable for clinical deployment. 281 Based on the multimode interference wave guide-based optofluidic-chip platform, Parks et al. developed a highly specific and sensitive multiplexed fluorescence assay for dual detection of ZIKV protein and nucleic acid. The device could be used to detect different types of molecular targets such as ZIKV protein and nucleic acid complexes with high specificity, sensitivity, and reliability. 489  | 2073 DNA and RNA of ZIKV (in heat-inactivated human blood serum and in buffer). In addition, it was shown that the modularity provided by DNA nanotechnology could enable multiplexing through combining orthogonal fluorescent labels for simultaneous detection of different sequences. 282 In another study, Xie et al. successfully fabricated a novel stable and water soluble 3D Cu-based zwitterionic metal-organic framework (MOF) for the simultaneous detection of the RNA sequences of ZIKV and DENV. The as-prepared MOFs could be used for forming the electrostatic, hydrogen bonding interactions and/or π stacking with the fluorophore-labeled DNA probes to construct two different sensing systems. The sensing systems displayed high sensitivity and selectivity, and could simultaneously detect the RNA sequences of ZIKV and DENV by using the single and synchronous fluorescence analysis. The single detection strategy could simultaneously detect the RNA sequences of ZIKV and DENV with a LOD of 192 and 332 pM, respectively. For the synchronous fluorescence detection strategy, the LODs of 121 and 184 pM were respectively obtained. Moreover, the two assays with high specificity had no interference with other mismatched RNA sequences, including single-base mismatched RNA sequences. In addition, two DNA probes could be used for synchronous ZIKV detection without cross-reaction. 284 In a recent study, Zhang et al. reported a novel Janus emulsion agglutination assay ( Figure 14) that could be used to detect interfacial protein-protein interactions.
Janus emulsion droplets could be functionalized using rcSso7d-ZNS1 (an antigen binding the hyperthermophilic rcSso7d protein) with thermal stability, which was used to detect ZIKV NS1 protein. In hydrocarbon and fluorocarbon phases, the emulsion droplets containing fluorescent dyes could be used to intensify the intrinsic optical signal, which could be determined through a simple optical fiber. In addition, two novel optical transduction strategies were successfully developed, which could be used for the analyte quantification. Through the incorporation of dyes in emulsion droplets and detection of the emitted light or backscatter, or the multiple emissions modulated by an inner filter effect (IFE), robust ratiometric signals were successfully used for the detection of agglutination. The proposed assay based on two optical schemes could be employed for the detection of ZIKV NS1 protein with a LOD of 100 nM. 285

| SERS sensing
Due to their merits of unique LSPR properties, excellent multiplexing ability, large dynamic range, and high sensitivity, SERS has been used in multiple fields, such as bioanalysis, disease diagnosis, immunoassays, environmental surveillance, food safety supervision, and biomedicine. 490-505 SERS has been widely investigated and applied for the detection of viruses. [506][507][508][509] Due to its high sensitivity in a multiplexed assay, SERS has recently been used to detect ZIKV infections. For example, Sánchez-Purrà et al. successfully developed the new SERS-based sandwich immunoassay (SERS-lateral flow assays [LFAs] platform; Figure 15A) for the multiplexed detection of ZIKV and DENV with lower LODs than those achieved using the colorimetric readout of LFAs. Interestingly, SERS encoded-Au nanostars could be conjugated to specific antibodies to distinguish the NS1 biomarkers between ZIKV and DENV using 4-mercaptobenzoic acid and 1,2-bis (4-pyridyl) ethylene (BPE) as Raman reporter molecules. By combining SERS with LFAs (the simplicity of a LFA was combined with the high sensitivity of SERS), the low sensitivity of LFAs for detecting low concentrations of biomarkers could be greatly improved, and the combined SERS-LFA platform exhibited high sensitivity and simplicity. Moreover, compared with the colorimetric LFAs, the SERS-LFA platform led to 7.2-fold LOD decrease for DENV NS1 and 15-fold for ZIKV NS1, respectively, resulting in LODs down to 7.67 ng/ml for DENV NS1 and 0.72 ng/ml for ZIKV NS1, respectively. In addition, the SERS-LFA platform could be connected wirelessly to the cloud or a PC, which could analyze and report the data of new disease cases in real-time during an outbreak. 286 In a recent study, to solve the problems of the high LOD and the cross-reactivity between DENV and ZIKV, Camacho et al. designed a novel immune-specific assay based on SERS nanoprobes ( Figure 15B). The gold shell-isolated nanoparticles (Au-SHINs) were fabricated using Au NPs (100 nm) and silica shell (4 nm) coated with Nile Blue (NB; Raman reporter). Following that, NB molecules were modified on the outer silica layer. After wrapping in a final silica shell, the SERS nanoprobes were functionalized with the monoclonal antibodies against ZIKV NS1. Owing to the specificity toward ZIKV antigens provided by the monoclonal anti-ZIKV NS1 antibodies functionalized SERS nanoprobes, the assay system possessed the capability of decreasing the cross-reactivity between ZIKV antigens and DENV antigens to avoid false positives. Moreover, it was found that the immune-specific assay based on the SERS nanoprobes could be used for detecting ZIKV antigens with very low concentration of 10 ng/ml, and no cross-reactivity with DENV NS1 antigens. 287

| Other sensing methods
There are some other sensing methods reported for the detection of ZIKV infection. 510 For example, using an immobilized monoclonal antibody with high specificity, Afsahi et al. developed a new portable and cost-effective graphene-enabled biosensor for the detection of ZIKV ( Figure 16A). By covalently linking the anti-ZIKV NS1 to the graphene, the biosensor could be used for the quantitative, real-time detection of ZIKV antigens through the incorporation of field effect biosensing technology. Moreover, the biosensor functionalized with anti-ZIKV NS1 could detect ZIKV antigens (ZIKV NS1) in buffer at concentrations as low as 0.45 nM. It was found that the biosensor could serve as a potential diagnostic tool for measuring ZIKV NS1 in simulated human serum. The selectivity of the biosensor was verified by using Japanese encephalitis NS1 (a homologous viral antigen with potential cross-reaction), no measurable cross-reactivity was observed. In addition, a graphene-enabled ZIKV biosensor with the merits of high speed, sensitivity, and selectivity was considered as an ideal candidate, which could be further developed as a medical diagnostic test. 511  with high sensitivity and specificity. The LAMP assay could directly detect ZIKV RNA in mosquitoes, cultured infected cells, and the body fluids of infected patient (e.g., semen, blood, urine, saliva, and plasma) and in plasma, semen, and serum specimens of infected patients without the requirement of ZIKV RNA isolation/reverse transcription. 295 By incorporating the optimized RT-LAMP and a LFA ( Figure 18A), a novel, user-friendly, simple strategy was developed by Lee's group for the highly sensitive detection of ZIKV. The method could be used to identify ZIKV RNA in the human whole blood and pure water. When the RT-LAMP reaction was optimized by adjusting the concentrations of dNTP and Mg 2+ ; this resulted in the complete elimination of nontarget   in serum, urine, and mosquito specimens without the requirement of RNA extraction from the urine and mosquitos samples. 518   indicating that the developed strategy may be used as a portable and powerful molecular diagnostic tool to detect the ZIKV. 300 6.4 | Bioplasmonic paper-based device BPD has been applied to the diagnosis of ZIKV through the detection of IgM and IgG antibodies to NS1 protein of ZIKV. 519  IgM and IgG antibodies to NS1 protein of ZIKV in human serum. Furthermore, the BPD also exhibited excellent stability at room temperature and elevated temperatures for 30 days, which was achieved using a MOF dependent biopreservation strategy. 520

| Reverse transcription isothermal recombinase polymerase amplification
Recombinase polymerase amplification (RPA) as a promising tool exhibits a great many merits, such as simple primer design, speed, power saving (runs at 37°C), robustness and no initial heating step for biological substances.  Figure 21). The developed RAMP could increase the sensitivity of the amplification process and displayed the merits of isothermal amplification (e.g., low power consumption and simple instrumentation). The RAMP was used to test specimens with similar targets like the various strains of ZIKV and the HPV, resulting in no false negatives or false positives. 304 Compared with the traditional multiplexing methods (e.g., isoPCR, mPCR and nmPCR), RAMP was faster and more inhibitor-tolerant, and took less time (<40 min), even when using lowabundance specimens. 523 Interestingly, Chan et al. described a repurposed 3D printer and RPA for the rapid and low-cost POC molecular diagnostics. In the device, a 3D printer was used to rapidly perform the nucleotide extraction with high throughput. The nucleotide extraction and isothermal amplification was performed within the same enclosure. With the amplification of RNA, the assay could produce a fluorescence signal that could be detected using a smartphone camera or portable detector. Moreover, the ability of this device was verified using ZIKV and foodborne pathogens. The device could extract and purify up to 12  However, it could not discriminate between the antibody classes, and could only be applied in selected laboratories with expensive equipment. 524 NIRS as a rapid, cost-effective and reagent-free tool has been used for the noninvasive detection of ZIKV in thoraces and heads of intact mosquitoes (A. aegypti mosquitoes). NIRS could be used to detect ZIKV using a beam of light targeted on the mosquitos resulting in a diagnostic spectrum. Surprisingly, a 94.2-99.3% accuracy relative to real-time RT-PCR was obtained.
Moreover, compared to real-time RT-PCR, the NIRS was 110 times cheaper and 18 times faster. 525 In another study, a novel NP-enhanced viral lysate electrical sensing assay was developed for the specific and sensitive detection of ZIKV on cellulose paper microchips using screen-printed electrodes ( Figure 22). ZIKV could be isolated from biological specimens using antibodies, and was able to be labeled by platinum NPs to enhance the electrical signal. Then, the captured ZIKV-platinum NP complexes were lysed employing a detergent to release the charged molecules associated with the intact virus and the platinum NPs on the The channel was integrated with the microsized pillars that increased the surface area allowing more aptamers to attach to incoming E protein molecules. Therefore, the overall sensitivity of the system was increased. The developed device relied on the construction of protein-mediated sandwich morphology which was obtained by using aptamers and gold nanoparticles (Au NPs) functionalized with aptamers. The colorimetric signal was produced upon the addition of silver reagents into the channel, which were selectively deposited on the surface of Au NP, forming a gray contrast in the testing zone. The obtained microfluidic channel approach resulted in a high specificity for the detection of ZIKV and CHIKV E proteins, with a detection limit of 10 pM in calf blood and 1 pM of viral EP targets in PBS. 309 While ZIKV has been known since 1947, it was not a huge global threat for human health until recently. It was reported that ZIKV could cause congenital abnormalities in the fetuses of infected mothers, such as microcephaly, fetal demise, and other brain damage in infants. Moreover, ZIKV can lead to GBS in adults. With the spread of ZIKV between different continents/countries over the last decade, it has resulted in a cause of concern for human health.
The rapid spread of ZIKV may be caused by multiple routes of transmission (including vector transmission, sexual transmission, maternal fetal transmission, and body fluid transmission) rather than just as a result of mosquito bites. Unfortunately, the ZIKV has recently resurged in India with the potential for devastating consequences.
Therefore, rapid detection strategies and efficient medicines for the appropriate prevention and efficient control of ZIKV need to be urgently developed. Furthermore, the exhaustive molecular mechanism of ZIKV and hostpathogen interactions still need to extensively evaluated to understand the enhanced pathogenicity of ZIKV infection, to develop novel effective detection strategies and drugs to prevent and control ZIKV infections.
Currently, numerous strategies have been developed for the detection of ZIKV. Among these strategies, the MAC-ELISA/PRNT and real-time RT-PCR are gold standards and have been recommended by the CDC for the detection of ZIKV during infection. However, serological assays and molecular assays both suffer from some limitations. For the serological assays, the cross-reactivity between ZIKV and other homologous flaviviruses (e.g., DENV) is a problem. For the molecular assays, the missing unanticipated sites may cause failed amplification. Thus, the serological assays and molecular assays need to be further improved to enhance the detection stability and accuracy. In addition, serological assays, and molecular assays both require a laboratory environment and expensive equipment that requires human expertise in order to perform these tests. Such equipment and expertise may be not available for all regions of the world. Therefore, affordable, rapid, reliable, and portable sensing methods and devices still need to be developed for the detection of ZIKV infection. To date, some sensing methods have been put forward for the detection of ZIKV. The performance of these sensing methods and devices should be further evaluated and verified before they are used for the clinical diagnosis of ZIKV. In addition, ZIKV pathogenesis and genetics need be better understood to facilitate the design of new diagnostic methods and effective drugs and vaccines to counter ZIKV. F I G U R E 24 (A) Polydimethylsiloxane-based microfluidic device for the multiplexed assay of viral E proteins (e.g., ZIKV and chikungunya) on a single platform using an aptamer-analyte interaction. (B) Illustration of the procedures established The surface was firstly conjugated with a linker molecule (i.e., PMPI) that attaches to the thiolated end of the aptamers (1); the control site was blocked by BSA. Specimens containing viral E proteins were introduced in to the channel where specific aptamers toward the proteins bind to the targets (2). AuNPs conjugated with protein-specific aptamers were subsequently added and to free epitopes on the previously captured E proteins for the formation of the sandwich morphology (3); if no arboviral E proteins are present, AuNPs are washed away. Finally, Ag reagents are introduced and deposited onto the bound AuNP surface, thus producing a colorimetric signal indicating existence of arboviral E proteins (4). Reprinted with permission from Ref. 309