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Research Letter |

Presence of Zika Virus in Conjunctival Fluid FREE ONLINE FIRST

Jiufeng Sun, PhD1; De Wu, PhD2; Haojie Zhong2; Dawei Guan2; Huan Zhang2; Qiqi Tan2; Changwen Ke, PhD2
[+] Author Affiliations
1Guangdong Provincial Institute of Public Health, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China
2Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China
JAMA Ophthalmol. Published online September 15, 2016. doi:10.1001/jamaophthalmol.2016.3417
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Published online

Zika virus (ZIKV) is a mosquito-borne flavivirus isolated from the Zika forest of Uganda in 1947.1 The clinical symptoms of ZIKV infection are mostly a mild and self-limited rash, arthralgia, and conjunctivitis in humans. More than 80% of ZIKV infections are asymptomatic. The ZIKV outbreaks in Brazil and French Polynesia indicate that the virus is linked to microcephaly and Guillain-Barré syndrome.2,3 Meanwhile, severe eye damage in infants with microcephaly was associated with ZIKV infection.4 However, it is still unclear whether the eye lesions are the result of microcephaly or directly ZIKV infection.5 Therefore, we wondered whether ZIKV could be detected in conjunctival fluid. In this report, we describe the detection of ZIKV from conjunctival swab samples of laboratory-confirmed ZIKV cases.

Since February 12, 2016, 11 ZIKV infection cases (Chinese travelers) were imported from Venezuela in Guangdong, China (http://www.gdwst.gov.cn/a/yiqingxx). All the cases were confirmed to be ZIKV infection by real-time reverse-transcription polymerase chain reaction (rRT-PCR) (DAAN gene, Guangzhou, China). The virus load was determined using a standard curve with serial dilutions of known plasmid concentrations. The serum and conjunctival swab samples were taken from 6 of 11 cases simultaneously. When evaluated by the institutional review board of the Guangdong province Center for Disease Control and Prevention, this activity was considered a component of the public health response to the imported Zika case in Guangdong; thus, it did not require review. However, each written epidemiology and sampling investigation report was approved by each case.

The results of rRT-PCR for ZIKV in serum of these 6 cases with estimated ZIKV RNA loads ranged from 9.9 × 102 to 2.31 × 106 copies/mL (Table). The conjunctival swab samples from the 6 cases were washed with 800 μL of minimum essential medium. Two hundred–microliter washing solution was used for the rRT-PCR tests. The results demonstrated ZIKV RNA in these samples with a virus load from 1.99 × 103 to 1.788 × 106 copies/mL (Table). The ZIKV RNA was detectable in serum no more than 5 days after symptom onset, but it was detected in conjunctival swab samples until day 7 in case 5. Thereafter, the ZIKV nucleic acid–positive serum and conjunctival swab samples were subjected to virus culture using neonatal mice (BALB/c, aged 1-3 days) brain injection methods. The presence of ZIKV was detected by rRT-PCR as described earlier. Three ZIKV strains, 2 from serum samples (cases 2 and 3, GDZ16006 and GDZ16019) and 1 from conjunctival swab (case 5, GDZ16020), were successfully isolated from neonatal mice brains. A partial ZIKV NS5 gene (approximately 960 base pairs) was successfully amplified and sequenced from these isolations. Phylogenetic analysis was performed to compare with 60 available ZIKV NS5 genes on GenBank using the software Molecular Evolutionary Genetics Analysis version 6.06 (MEGA). Phylogenetic analyses showed that all the strains belong to the Asian lineage of ZIKV and are closely related to those reported from Brazil, Puerto Rico, and Suriname in 2015 (Figure).

Table Graphic Jump LocationTable.  Virus Load Detected Using Real-Time Reverse-Transcription Polymerase Chain Reaction in Serum and Conjunctiva Swab Samples
Place holder to copy figure label and caption
Figure.
Phylogenetic Analysis of Partial NS5 Gene Sequences of Zika Virus (ZIKV) for 3 Imported Cases of ZIKV Infection Returning to China From Venezuela

Maximum-likelihood phylogenetic tree based on a general time reversible substitution model with γ-distributed rate variation and a proportion of invariant sites. The partial sequence of NS5 gene of 3 isolates (red dots) obtained in this study was analyzed against 60 reference strains on GenBank. All positions containing gaps and missing data were eliminated. Scale bar indicates number of substitutions per site.

Graphic Jump Location

Detection of ZIKV RNA is a gold standard of confirmation of ZIKV infection. In this study, we described the direct detection and isolation of ZIKV from conjunctival swab samples. Although isolation of ZIKV in cell culture from urine, semen, saliva, and breast milk has been described,6 to our knowledge, detection and isolation of ZIKV from conjunctiva has not been reported so far. These results, though, are not sufficient to recommend the use of conjunctival swabs as alternative samples for ZIKV diagnosis because of shorter persisting and shedding time of ZIKV in conjunctiva fluid (<7 days) compared with urine and saliva samples (<20 days). It may have implications for transmission of ZIKV, eg, through corneal graft donors, although this report does not provide direct evidence to support that indication. Nevertheless, epidemiological data and experimental studies are needed to assess the further significance of this finding because of increasing complications caused by ZIKV infection in neonates.

Corresponding Author: Changwen Ke, PhD, Guangdong Provincial Center for Disease Control and Prevention, Guangzhou 510300, People’s Republic of China (kecw1965@aliyun.com).

Published Online: September 15, 2016. doi:10.1001/jamaophthalmol.2016.3417

Author Contributions: Dr Ke had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Drs Sun and Wu contributed equally to this work.

Concept and design: Sun, Wu.

Acquisition, analysis, or interpretation of data: Ke, Sun, Zhong, Guan, Zhang, Tan.

Drafting of the manuscript: Ke, Sun, Guan, Zhang, Tan.

Critical revision of the manuscript for important intellectual content: Wu, Zhong.

Statistical analysis: Sun.

Administrative, technical, or material support: All authors.

Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.

Additional Contributions: We thank all the related laboratory and administrative personnel at Guangdong Provincial Center for Disease Control and Prevention and Jiangmen Center for Disease Control and Prevention for their contribution in follow-up investigation.

Dick  GW, Kitchen  SF, Haddow  AJ.  Zika virus, I: isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952;46(5):509-520.
PubMed   |  Link to Article
Mlakar  J, Korva  M, Tul  N,  et al.  Zika virus associated with microcephaly. N Engl J Med. 2016;374(10):951-958.
PubMed   |  Link to Article
Cao-Lormeau  VM, Blake  A, Mons  S,  et al.  Guillain-Barré syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 2016;387(10027):1531-1539.
PubMed   |  Link to Article
de Paula Freitas  B, de Oliveira Dias  JR, Prazeres  J,  et al.  Ocular findings in infants with microcephaly associated with presumed Zika virus congenital infection in Salvador, Brazil [published online February 9, 2016]. JAMA Ophthalmol.
PubMed
Jampol  LM, Goldstein  DA.  Zika virus, microcephaly, and ocular finding—reply [published online June 2, 2016]. JAMA Ophthalmol. doi:10.1001/jamaophthalmol.2016.1307.
Musso  D, Gubler  DJ.  Zika virus. Clin Microbiol Rev. 2016;29(3):487-524.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure.
Phylogenetic Analysis of Partial NS5 Gene Sequences of Zika Virus (ZIKV) for 3 Imported Cases of ZIKV Infection Returning to China From Venezuela

Maximum-likelihood phylogenetic tree based on a general time reversible substitution model with γ-distributed rate variation and a proportion of invariant sites. The partial sequence of NS5 gene of 3 isolates (red dots) obtained in this study was analyzed against 60 reference strains on GenBank. All positions containing gaps and missing data were eliminated. Scale bar indicates number of substitutions per site.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable.  Virus Load Detected Using Real-Time Reverse-Transcription Polymerase Chain Reaction in Serum and Conjunctiva Swab Samples

References

Dick  GW, Kitchen  SF, Haddow  AJ.  Zika virus, I: isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952;46(5):509-520.
PubMed   |  Link to Article
Mlakar  J, Korva  M, Tul  N,  et al.  Zika virus associated with microcephaly. N Engl J Med. 2016;374(10):951-958.
PubMed   |  Link to Article
Cao-Lormeau  VM, Blake  A, Mons  S,  et al.  Guillain-Barré syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 2016;387(10027):1531-1539.
PubMed   |  Link to Article
de Paula Freitas  B, de Oliveira Dias  JR, Prazeres  J,  et al.  Ocular findings in infants with microcephaly associated with presumed Zika virus congenital infection in Salvador, Brazil [published online February 9, 2016]. JAMA Ophthalmol.
PubMed
Jampol  LM, Goldstein  DA.  Zika virus, microcephaly, and ocular finding—reply [published online June 2, 2016]. JAMA Ophthalmol. doi:10.1001/jamaophthalmol.2016.1307.
Musso  D, Gubler  DJ.  Zika virus. Clin Microbiol Rev. 2016;29(3):487-524.
PubMed   |  Link to Article

Correspondence

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