Zika virus infection of cellular components of the blood-retinal barriers: implications for viral associated congenital ocular disease
© The Author(s). 2017
Received: 26 July 2016
Accepted: 25 February 2017
Published: 3 March 2017
Ocular abnormalities present in microcephalic infants with presumed Zika virus (ZIKV) congenital disease includes focal pigment mottling of the retina, chorioretinal atrophy, optic nerve abnormalities, and lens dislocation. Target cells in the ocular compartment for ZIKV infectivity are unknown. The cellular response of ocular cells to ZIKV infection has not been described. Mechanisms for viral dissemination in the ocular compartment of ZIKV-infected infants and adults have not been reported. Here, we identify target cells for ZIKV infectivity in both the inner and outer blood-retinal barriers (IBRB and OBRB), describe the cytokine expression profile in the IBRB after ZIKV exposure, and propose a mechanism for viral dissemination in the retina.
We expose primary cellular components of the IBRB including human retinal microvascular endothelial cells, retinal pericytes, and Müller cells as well as retinal pigmented epithelial cells of the OBRB to the PRVABC56 strain of ZIKV. Viral infectivity was analyzed by microscopy, immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR and qRT-PCR). Angiogenic and proinflammatory cytokines were measured by Luminex assays.
We find by immunofluorescent staining using the Flavivirus 4G2 monoclonal antibody that retinal endothelial cells and pericytes of the IBRB and retinal pigmented epithelial cells of the OBRB are fully permissive for ZIKV infection but not Müller cells when compared to mock-infected controls. We confirmed ZIKV infectivity in retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells by RT-PCR and qRT-PCR using ZIKV-specific oligonucleotide primers. Expression profiles by Luminex assays in retinal endothelial cells infected with ZIKV revealed a marginal increase in levels of beta-2 microglobulin (β2-m), granulocyte macrophage colony-stimulating factor (GMCSF), intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP1), and vascular cell adhesion molecule 1 (VCAM-1) and higher levels of regulated upon activation, normal T cell expressed and presumably secreted (RANTES) but lower levels of interleukin-4 (IL-4) compared to controls.
Retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells are fully permissive for ZIKV lytic replication and are primary target cells in the retinal barriers for infection. ZIKV infection of retinal endothelial cells and retinal pericytes induces significantly higher levels of RANTES that likely contributes to ocular inflammation.
KeywordsZika virus Blood-retinal barrier Retinal endothelial cells Retinal pigmented epithelial cells Retinal pericytes Cytokines Inflammation
Zika virus (ZIKV) is an arbovirus that belongs to the Flavivirus family which also includes West Nile virus, dengue, yellow fever, and Japanese encephalitis viruses and is transmitted to humans by Aedes species mosquitoes [1, 2]. ZIKV was first identified in a rhesus monkey in 1947 and first recognized in humans in 1952 [1, 3]. ZIKV has quickly spread to more than 70 countries in the Americas and the Caribbean infecting more than 2 million people [4, 5]. Currently, there is no treatment or vaccine for Zika virus. There is very limited information known about this emerging global health threat.
ZIKV infection has been associated with a sporadic increase in the incidence of microcephaly in infants [6–9]. Congenital ocular findings concomitant with microcephaly have also been associated with ZIKV infection during pregnancy [10–13]. A recent study showed ocular abnormalities present in 34.5% of microcephalic infants examined and involved the bilateral vision in 70% of them . The lesions included focal pigment mottling of the retina, chorioretinal atrophy, optic nerve abnormalities, bilateral iris coloboma (congenital fissure), and lens dislocation . These lesions are considered vision-threatening, and children should be screened as a process of differential diagnosis to rule out other causes such as West Nile virus infection, toxoplasmosis, cytomegalovirus, rubella, herpes simplex virus, and syphilis [13, 14]. Children born to mothers with little or no symptoms of ZIKV infection can still have microcephalic babies with severe ocular abnormalities . This finding would support the notion of ophthalmic screening for all babies born in epidemic regions. Risk factors for ocular involvement in infants with presumed ZIKV congenital infection include smaller cephalic diameters at birth and infants whose mother develop symptoms during the first trimester of pregnancy . Adults with acute ZIKV disease often experience hyperemic sclera, conjunctivitis, and retro-orbital pain, and uveitis has also been observed in a patient with ZIKV infection after an initial clinical presentation of conjunctival hyperemia [17–19]. The target cells for ZIKV-associated ocular disease are unknown. The cytokine dysregulation that contributes to ZIKV-induced ocular inflammation is yet to be identified. The route of viral dissemination in the ocular compartment has not been described. Here, we identify target cells in both the inner and outer blood-retinal barriers (IBRB and OBRB), describe the cytokine expression profile in retinal endothelial cells after ZIKV exposure, and propose a mechanism for viral dissemination in the retina.
Human primary retinal microvascular endothelial cells and retinal pericytes were obtained from Cell Systems Corporation (Kirkland, WA, USA) and were cultivated in Pericyte Media (PM) from ScienCell (Carlsbad, CA, USA). Primary human retinal pigmented epithelial cells and epithelial cell media (EpiCM) were obtained from ScienCell. The human Müller cell line MIO-M1 , derived from an adult retina, was kindly provided by Dr. John Penn (Vanderbilt University Medical Center Eye Institute). Acquisition of the MIO-M1 cell line was approved by the Internal Review Board and Ethics Committee of Vanderbilt University Medical Center in Nashville, Tennessee. Retinal pericytes and retinal endothelial cells were maintained at passage level 3 in PM media. The Müller cell line MIO-M1 was maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% Pen/Strep. All cells were trypsinized and plated in uncoated 100-cm2 dishes or uncoated 4.2-cm2 glass chamber slides at a density of and 2.5 × 105 cells per dish and well, respectively.
Viruses and virus cultivation
The Zika virus strain PRVABC59 provided by the Centers for Disease Control and Prevention (CDC) used in this study was originally isolated from a human serum specimen from Puerto Rico in December 2015, nucleotide (GenBank):KU501215 ZIKV strain PRVABC59, complete genome [21–23]. The virus was cultivated in Vero cells (Cercopithecus aethiops, African green monkey kidney cell line), and infectious supernatant was filtered using a 0.22-μm filter and the serum content adjusted to 15%. Viral titers were done by end-point dilution and infectivity measured by IFA staining with the 4G2 antibody (fluorescent focus assay (FFA) on Vero cells. Stock viral titer was adjusted to ~1 × 106 FFU/5 μl of infectious culture supernatant. Heat-killed ZIKV was prepared by heating the viral inoculum at 65 °C for 30 min in a water bath . The mild heat inactivation that we employ is unlikely to cause a global effect on thermolabile viral proteins. All experiments were carried out under biosafety level 2 containment as recommended. The use of ZIKV was approved by the Meharry Medical College Institutional Biosafety Committee.
ZIKV-infected Vero cells were used to validate the monoclonal antibody to the Flavivirus group antigen which binds to the fusion loop at the extremity of domain II of the E protein (D1-4G2-4-15, 4G2) (Millipore, Temecula, CA, USA) [25, 26]. ZIKV cytopathology in Vero cells included cell rounding and sloughing with a perinuclear staining profile using the 4G2 antibody by immunofluorescent staining (data not shown).
Immunofluorescent staining was performed as previously described . Briefly, chamber slide cultures containing ZIKV-infected or mock-infected retinal endothelial cells, retinal pericytes, Müller cells, or retinal pigmented epithelial cells were washed twice with PBS pH 7.4, air-dried, and fixed in absolute methanol for 10 min. Cells were air-dried for 15 min, hydrated in Tris-buffered saline (pH 7.4) for 5 min, and incubated separately for 1 h with monoclonal antibodies to von Willebrand factor (VWF) for retinal endothelial cells (Millipore, Temecula, CA, USA), or vimentin for retinal pigmented epithelial cells (Santa Cruz, CA, USA). All antibodies were diluted 1:50 in PBS pH 7.4. For ZIKV infection of retinal endothelial cells, retinal pericytes, Müller cells, and retinal pigmented epithelial cells, cells were incubated for 1 h with monoclonal antibodies to the 4G2 Flavivirus group antigen, at a 1:50 dilution in PBS pH 7.4. Donkey anti-mouse secondary antibodies conjugated to fluorescein isothiocyanate (FITC) were used to detect ZIKV-positive cells. Immunofluorescent staining was performed as previously described .
Total RNA was extracted from both ZIKV-infected retinal endothelial cells and retinal pigmented epithelial cells along with their respective mock-infected and heat-killed ZIKV control cells using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was DNase-treated prior to elution on the column according to the manufacturer’s recommendations. Messenger RNA in 0.5 μg of each sample was primed using oligo-dT and reverse-transcribed with a high-capacity complementary DNA (cDNA) reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Gene-specific primer pairs included ZIKV forward primer 5′TTYGAAGCCCTTGGATTCTT3′ and ZIKV reverse primer 5′CYCGGCCAATCAGTTCATC3′ and 50 ng of cDNA for RT-PCR amplification, using PuReTaq Ready-To-Go PCR beads (GE Healthcare, Buckinghamshire, UK). PCR was carried out in a MJ Mini thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) in a final volume of 25 μl. The cycling protocol used was 95 °C for 5 min, 55 °C for 30 s, and 72 °C for 1 min for 36 cycles, with a final extension at 72 °C for 10 min. PCR products were electrophoresed in 1.5% agarose and DNA bands visualized by ethidium bromide. Primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward primer 5′-TGATGACATCAAGAAGGTGGTGAA-3′ and reverse primer 5′-TCCTTGGAGGCCATGTGGGC CAT-3′ (256 bp) were amplified in mock and infected cells as a loading and quality control. Using ZIKV-infected cell total RNA, we amplified a 364-bp DNA fragment with the above primers, respectively, to positions 1538-1558 and 1902-1883 of the ZIKV genome sequence AY632535 .
Total RNA was extracted separately from ZIKV-infected retinal endothelial cells, retinal pericytes, and Müller cells, along with the respective mock-infected controls using a Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA) as previously described above. Messenger RNA in 0.5 μg of each sample was primed using oligo-dT and reverse-transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA). Real-time quantitative PCR was performed on iCycler using iQ Sybr Green Supermix (Bio-Rad). Samples were analyzed in triplicate and normalized to GAPDH RNA. Reaction mixture contained 250 nM of each primer and 200 to 400 ng of template cDNA in a final volume of 20 μl. The primers specific for ZIKV were as follows: forward 5′-AGGATCATAGGTGATGAAGAAAAGT-3′ and reverse 5′-CCTGACAACACTAAGATTGGTGC-3′ . RANTES primers used for qRT-PCR were as follows: forward 5′-GGCAGCCCTCGCTGTCATCCTCA-3′, reverse 5′-CTTGATGTGGGCACGGGGCAGTG-3′. GAPDH primers used for qRT-PCR were as follows: forward 5′-GAAGGTGAAGGTCGGAGT-3′ and reverse 5′-GAAGATGGTGATGGGATTTC-3′.
The inflammatory and angiogenic cytokine analysis was performed with 200 μl of supernatant from three pooled cultures of mock-infected, ZIKV-infected, and heat-killed ZIKV-exposed retinal endothelial cells for 96 h post-exposure using a Luminex instrument (Luminex Corporation, Austin, TX) and 100-plate viewer software. Luminex analysis on 47 different proinflammatory and angiogenic cytokines was performed on supernatants as previously described . Infections were performed in triplicate in chamber slides for 96 h. Replicate assays are inherent in the Luminex technology by counting 50 bead replicates per analyte and reporting the median. This is the equivalent of running 50 replicate assays per well. In addition, robotic pipetting was performed for all volume-critical steps, which minimizes well-to-well variability, and calibrators and controls were run in duplicate involving three levels of control per analyte in duplicate on every plate . Experiments presented in this study that involved ZIKV infections were performed in triplicate. Supernatants from mock-infected, ZIKV-infected, and heat-killed ZIKV-exposed retinal endothelial cells were separately taken from triplicate samples and pooled for Luminex analysis.
Experiments presented in this study were performed in triplicate (mock-infected, ZIKV-infected, and heat-killed ZIKV-exposed retinal endothelial cells, retinal pericytes, Müller cells, and retinal pigmented epithelial cells were used for RT-PCR and qRT-PCR amplification of ZIKV and RANTES cDNA). To compare the mean values between the two groups, the unpaired t test was used. Statistical significance was defined as P < 0.05. Data are presented as means ± SD. qRT-PCR experiments were replicated three times and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
Retinal endothelial and retinal pericytes are permissive for ZIKV infectivity but not retinal Müller cells
Retinal pigmented epithelial cells of the OBRB are permissive for ZIKV infectivity and exhibit low-level cytopathology
Dysregulation of angiogenic and proinflammatory cytokines in ZIKV-infected retinal endothelial cells
ZIKV blood-retinal barrier infection model
There is no information in the literature that defines target cell populations in the human eye related to ZIKV-associated ocular disease. This study provides information that is important for understanding ZIKV pathology in the ocular compartment and identifies important cell types in both the inner and outer blood-retinal barriers (IBRB and OBRB) that are permissive for ZIKV infection and dissemination in the eye. This in vitro study suggests that ZIKV traffics both retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells during infection but does not infect Müller cells. The highest levels of ZIKV transcription were observed in retinal pericytes compared in retinal pigmented epithelial cells and retinal endothelial cells. The model we proposed is hypothetical because primary cells in culture may not behave as cells in ocular tissue and will require validation in vivo. Cytokine and adhesion molecule profile analysis reveals a marginal increase in the levels of β2-m, GMCSF, and MCP1 and a moderate increase of ICAM-1, IL-6, and VCAM-1 expression; however, significantly higher levels of RANTES expression were observed in ZIKV-infected cells compared in controls (Fig. 6a). Recent studies show that patients with ZIKV infection have high levels of RANTES in their serum when compared with patients infected with dengue virus or Chikungunya virus . Upregulation of RANTES over time would lead to chronic inflammation and recruitment of inflammatory cells in the retinal microenvironment. The next steps for this study will be to directly examine eye washing or lacrimal fluid from patients with ZIKV-associated ocular hyperemia or ocular tissue from infants who have died of congenital ZIKV infection to determine viral dissemination patterns and cytokine expression profiles in vivo.
We have identified primary human retinal endothelial cells and retinal pericytes of the IBRB and human retinal pigmented epithelial cells of the OBRB as target cells for ZIKV infection in the eye. We have determined that ZIKV induces a moderate angiogenic and proinflammatory cytokine response with the exception of RANTES in infected retinal endothelial cells that likely play a major role in ocular inflammation in acute ZIKV ocular disease. The hypothetical model we proposed based on our findings suggests that ZIKV disseminates throughout the retinal bed via the retinal arteries and infects retinal capillary endothelial cells and retinal pericytes of the IBRB and traffics the choroid capillaries to infect retinal pigmented epithelial cells in the OBRB.
- 4G2 antibody:
Flavivirus group antigen monoclonal antibody
Blood brain barrier
Charge-coupled device camera
Centers for Disease Control and Prevention
Dulbecco’s Modified Eagle Medium
Epithelial cell medium
Glyceraldehyde 3-phosphate dehydrogenase
Granulocyte macrophage colony-stimulating factor
Inner blood-retinal barrier
Intercellular adhesion molecule 1
Monocyte chemotactic protein-1
Müller cell line from human retina
Multiplicity of infection
Outer blood-retinal barrier
Polymerase chain reaction
Penicillin streptomycin mixtures
Asian strain of Zika virus isolated in Puerto Rico in December 2015 from human serum
Quantitative reverse transcription polymerase chain reaction
Regulated upon activation normal T cell expressed and presumably secreted
Reverse transcription polymerase chain reaction
Vascular cell adhesion molecule 1
von Willebrand factor
We thank Jared Elzey for the critical reading of this manuscript. We acknowledge the Meharry Office of Scientific Editing and Publications (supported by NIH grant S21MD000104). We thank Drs. Waldemar Popik and Atanu Khatua for the technical assistance with qRT-PCR.
DJA was supported by the Meharry President, Dr. James E.K. Hildreth, Zika virus Research Startup Award. The research reported in this publication was also supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under the Award Number 3UH2TR000491-02S1 to DJA. TR was supported by the Vanderbilt Summer Science Academy grant from the National Heart Lung and Blood Institute under the Award Number R25 HL096223-06.
Availability of data and materials
The authors declare that they used the standard commercial software, database packages, and tools, for data analysis. In addition, the authors declare that they do not have a link to include for the data and materials and that all data and methods of the analysis are included in the manuscript and the raw data described will be available for the reviewers.
DJA conceived and designed the study. DJA and TR performed the experiments. DJA drafted the manuscript. Both authors have read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
This study was approved by the Meharry Medical College Institutional Biosafety Committee and is compliant with the standard IRB protocols.
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- Dick GW, Kitchen SF, Haddow AJ. Zika virus, isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952;5:509–20.View ArticleGoogle Scholar
- Dick GW. Zika virus (II): pathogenicity and physical properties. Trans R Soc Trop Med Hyg. 1952;5:521–34.View ArticleGoogle Scholar
- MacNamara FN. Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg. 1954;2:139–5.View ArticleGoogle Scholar
- Heukelbach J, Alencar CH, Kelvin AA, de Oliveira WK. Pamplona de Góes Cavalcanti L.J. Zika virus outbreak in Brazil. Infect Dev Ctries. 2016;2:116–20.Google Scholar
- World Health Organization. Epidemiological alert: neurological syndrome, congenital malformations, and Zika virus infection: implications for public health in the Americas. http: //www.paho.org/hq/index.php?option=com_docman&task=doc_view&Itemid=270&gid =32405&lang = en. Accessed January 27, 2015.
- Kleber de Oliveira W, Cortez-Escalante J, De Oliveira WT, do Carmo GM, Henriques CM, Coelho GE, et al. Increase in reported prevalence of microcephaly in infants born to women living in areas with confirmed Zika virus transmission during the first trimester of pregnancy-Brazil, 2015. Morb Mortal Wkly Rep. 2016;65:242–47.View ArticleGoogle Scholar
- Rasmussen SA, Jamieson DJ, Honein MA, Petersen LR. Zika virus and birth defects-reviewing the evidence for causality. N Engl J Med. 2016;374:1981–87.View ArticlePubMedGoogle Scholar
- Brasil Ministério da Saúde. Informe Epidemiológico N° 25-Semana Epidemiológica (SE) 18/2016 (01/05 a 07/05/2016)-Monitoramento dos casos de microcefalia no Brasil, 2016. http://portalsaude.saude.gov.br/images/pdf/2016/ maio/11/COES-Microcefalias-Informe Epidemiol-gico-25-SE-18-2016-09mai2016-12 h13.pdf. Accessed June 11, 2016.
- European Centre for Disease Prevention and Control. Microcephaly in Brazil potentially linked to the Zika virus epidemic. http://ecdc.europa.eu/en /publications/Publications/zika-microcephaly -Brazil-rapid-risk-assessment-Nov-2015.pdf. Accessed January 6, 2016.
- de Paula Freitas B, de Oliveira Dias JR, Prazeres J, Sacramento GA, Ko AI, Maia M, et al. Ocular findings in infants with microcephaly associated with presumed Zika virus congenital infection in Salvador, Brazil. JAMA Ophthalmol. 2016;5:529–35.View ArticleGoogle Scholar
- Ventura CV, Maia M, Ventura BV, Linden VV, Araújo EB, Ramos RC. Ophthalmological findings in infants with microcephaly and presumable intra-uterus Zika virus infection. Arq Bras Oftalmol. 2016;1:1–3.Google Scholar
- Ventura CV, Maia M, Bravo-Filho V, Góis AL, Belfort Jr R. Zika virus in Brazil and macular atrophy in a child with microcephaly. Lancet. 2016;10015:228.View ArticleGoogle Scholar
- Jampol LM, Goldstein DA. Zika virus infection and the eye. JAMA Ophthalmol. 2016;5:535–36.View ArticleGoogle Scholar
- Belfort R Jr, de Paula Freitas B, de Oliveira Dias JR. Zika virus, microcephaly, and ocular findings—reply. AMA Ophthalmol. doi:10.1001/jamaophthalmol.2016.1305.
- McCarthy M. Severe eye damage in infants with microcephaly is presumed to be due to Zika virus. BMJ. 2016;352:i855. doi:10.1136/bmj.i855.View ArticlePubMedGoogle Scholar
- Ventura CV, Maia M, Travassos SB, Martins TT, Patriota F, Nunes ME, et al. Risk factors associated with the ophthalmoscopic findings identified in infants with presumed Zika virus congenital infection. AMA Ophthalmol. doi:10.1001/jamaophthalmol.2016.1784.
- Derrington SM, Cellura AP, McDermott LE, Gubitosi T, Sonstegard AM, Chen S, et al. Mucocutaneous findings and course in an adult with Zika virus infection. JAMA Dermatol. 2016;6:691–3.View ArticleGoogle Scholar
- Jimenez Corona ME, De la Garza Barroso AL, Rodriguez Martínez JC, Luna Guzmán NI, Ruiz Matus C, Díaz Quiñonez JA, et al. Clinical and epidemiological characterization of laboratory-confirmed autochthonous cases of Zika virus disease in Mexico. PLoS Curr. 2016; doi:10.1371/currents.outbreaks.a2fe1b3d6d71e24ad2b5afe982824053.
- Furtado JM, Espósito DL, Klein TM, Teixeira-Pinto T, da Fonseca BA. Uveitis associated with Zika virus infection. N Engl J Med. 2016; doi: 10.1056/NEJMc1603618.
- Limb GA, Salt TE, Munro PM, Moss SE, Khaw PT. In vitro characterization of a spontaneously immortalized human Müller cell line (MIO-M1). Invest Ophthalmol Vis Sci. 2002;43:864–9.PubMedGoogle Scholar
- Lanciotti RS, Lambert AJ, Holodniy M, Saavedra S, del Carmen Castillo Signor L. Phylogeny of Zika virus in Western Hemisphere, 2015. Emerg Infect Dis. 2016;5:933–5.View ArticleGoogle Scholar
- Thomas DL, Sharp TM, Torres J, Armstrong PA, Munoz-Jordan J, Ryff KR, et al. Local transmission of Zika Virus—Puerto Rico, November 23, 2015 January 28, 2016. MMWR Morb Mortal Wkly Rep. 2016;6:154–8.View ArticleGoogle Scholar
- Dirlikov E, Ryff KR, Torres-Aponte J, Thomas DL, Perez-Padilla J, Munoz-Jordan J, et al. Update: ongoing Zika virus transmission—Puerto Rico, November 1, 2015-April 14, 2016. MMWR Morb Mortal Wkly Rep. 2016;17:451–5.View ArticleGoogle Scholar
- Bryant P, Morley C, Garland S, Curtis N. Cytomegalovirus transmission from breast milk in premature babies: does it matter? Arch Dis Child Fetal Neonatal Ed. 2002;87:F75–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Nawa M, Takasaki T, Yamada KI, Akatsuka T, Kurane I. Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody. J Virol Methods. 2001;1:65–70.View ArticleGoogle Scholar
- Crill WD, Chang GJ. Localization and characterization of flavivirus envelope glycoprotein cross-reactive epitopes. J Virol. 2004;24:13975–86.View ArticleGoogle Scholar
- Alcendor DJ, Charest AM, Zhu WQ, Vigil HE, Knobel SM. Infection and upregulation of proinflammatory cytokines in human brain vascular pericytes by human cytomegalovirus. J Neuroinflammation. 2012;9:95.View ArticlePubMedPubMed CentralGoogle Scholar
- Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha SA. One-step RT-PCR for detection of Zika virus. J Clin Virol. 2008;1:96–101.View ArticleGoogle Scholar
- Wilkerson I, Laban J, Mitchell JM, Sheibani N, Alcendor DJ. Retinal pericytes and cytomegalovirus infectivity: implications for HCMV-induced retinopathy and congenital ocular disease. J Neuroinflammation. 2015;12:2.View ArticlePubMedPubMed CentralGoogle Scholar
- Djoba Siawaya JF, Roberts T, Babb C, Black G, Golakai HJ, Stanley K, et al. An evaluation of commercial fluorescent bead-based luminex cytokine assays. PLoS One. 2008;7:e2535.View ArticleGoogle Scholar
- Noriyuki K, Shinobu F. Recent advances in ocular drug delivery systems. Polymers. 2011;1:193–221.Google Scholar
- Maharajan MK, Ranjan A, Chu JF, Foo WL, Chai ZX, Lau EY. et al. Zika virus infection: current concerns and perspectives. Clin Rev Allergy Immunol. 2016; doi: 10.1007/s12016-016-8554-7.