Jpn J Ophthalmol DOI 10.1007/s10384-014-0369-6

CLINICAL INVESTIGATION

Virological and molecular biological evidence supporting herpes simplex virus type 1 corneal latency Shiro Higaki • Masahiko Fukuda Yoshikazu Shimomura



Received: 5 August 2014 / Accepted: 27 November 2014 Ó Japanese Ophthalmological Society 2015

Abstract Purpose Trigeminal and other ganglia are known as sites of latent infection by herpes simplex virus type 1 (HSV-1). In ophthalmology, HSV-1 remains latent in the trigeminal ganglia, and becomes reactivated by several factors, including stress, thermal stimulation, or immunosuppression, and may lead to herpetic keratitis. The purpose of this study was to demonstrate HSV corneal latent infection using molecular biology and virology techniques. Methods Six corneas obtained at penetrating keratoplasty were snap-frozen; three of them were with past history of herpetic keratitis. TaqMan Real-time PCR was used to show positive HSV DNA in the corneas. We proved negative homogenate and positive explant virologically. Using real-time RT-PCR, we showed that only latency-associated transcript (LAT) was detected and no transcriptional products of other virus genes (a, b, c) were detected. Results All three corneas with past history of herpetic keratitis had HSV DNA and showed negative homogenate and positive explant. LAT was detected in all three corneas. However, a, b, or c genes were not expressed. All the results of these corneas were consistent with the conditions of corneal latency. The other three corneas without history of herpetic keratitis showed negative homogenate and negative explant. None of them had LAT.

S. Higaki (&) Department of Ophthalmology, Kinki University Sakai Hospital, 2-7-1 Harayamadai, Minami Ward, Sakai, Osaka 590-0132, Japan e-mail: [email protected] M. Fukuda  Y. Shimomura Department of Ophthalmology, Kinki University Faculty of Medicine, Osaka-Sayama, Japan

Conclusion We have shown a possibility that HSV can latently infect the cornea aside from the ganglion. Keywords

HSV-1  Latency  Cornea  LAT

Introduction In ophthalmology, herpes simplex virus type 1 (HSV-1) primarily infects the eyelids, cornea or conjunctiva, and moves through the axons of the trigeminal nerve to reach the trigeminal ganglia, where it establishes latent infection [1]. HSV-1 may become reactivated by stress, thermal stimulation [2], or immunosuppression [3], and cause herpetic keratitis. In addition to the trigeminal ganglion, the superior cervical, ciliary, spinal, vestibular, and geniculate ganglia [4, 5], as well as other areas, are known as sites of latent infection by HSV-1 [1]. The idea that HSV-1 latently infects the cornea itself has been proposed [6–13]. In 1993, Shimomura et al. [6] showed a possibility of latent infection by HSV-1 in the human cornea using virological techniques; that is, negative homogenate and positive explant. However, molecular biological techniques were not used in that study. Several studies, including ours, have used molecular technique and report that HSV-1 DNA was present in human corneas that were excised several months after herpetic keratitis had healed [7–11]. In addition, some researchers report that latency-associated transcript (LAT) [14–16] was positive in the human cornea [7, 12, 13]. LAT is believed to play an important role during latent infection; therefore, in this state only LAT is expressed abundantly. Other transcriptional products (RNA) or expression products (protein) are not observed.

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Gordon et al. [12] state that the following five points should be shown in the cornea to demonstrate HSV corneal latent infection using molecular biology and virology techniques [12, 13]. 1. 2. 3.

4. 5.

HSV DNA is positive in the human cornea. Negative homogenate, positive explant [6]. LAT is the only transcript abundantly produced. No transcription products of other virus genes (a, b, c) are detected. Absence of intact virions under an electron microscope. No HSV-1 proteins are expressed.

In this study, we examined points 1, 2, and 3. We obtained results that support the possibility of corneal latency.

Table 1 Primer and probe sequences Target gene HSV

50 -ACATCATCAACTTCGACTGG-30 50 -CTCAGGTCCTTCTTCTTGTCC-30 Probe 50 -ATGGTGAACATCGACATGTACGG-30

LAT

16

50 -ACCCACGTACTCCAAGAAGGC-30 50 -TAAGACCCAAGCATAGAGAGCCA-30 Probe 50 -TCCCACCCCGCCTGTGTTTTTGT-30

ICP0

50 -CTACTCTGAGGCGGATACCGAAG-30 50 -GAGTCGTCGTCAATGGTGGTC-30

gB-1

50 -GATCGACAAGATCAACGCCAAG-30 50 -CCAGGTTGTTGCGCACGTA-30

TK

50 -ATGACTTACTGGCGGGTGCTG-30 50 -CCATTGTTATCTGGGCGCTTG-30

GAPDH

50 -GCACCGTCAAGGCTGAGAAC-30 50 -TGGTGAAGACGCCAGTGGA-30

Materials and methods Table 2 PCR conditions

Six corneas obtained at penetrating keratoplasty were snapfrozen; three of them were with a past history of herpetic keratitis and the other three were without. The former three cases had both epithelial and stromal herpetic keratitis. The last episode of case 1 was of the epithelial keratitis type, 1.5 months before the keratoplasty, and case 2 was with the same type about 2 months before the transplant surgery; case 3 had stromal keratitis about 7 months before the surgery. Three control cases without past history of herpetic keratitis and who had been operated for bullous keratopathy were randomly selected. Detailed interviews with these cases revealed no history of herpetic keratitis. The corneas were cut into four pieces. Virological and molecular methods were used to investigate the corneas. TaqMan Real-time PCR was used to detect whether HSV DNA was positive in the corneas. The primers and probe were designed to target within the DNA polymerase region, as we reported in the past [11, 17, 18]. We proved negative homogenate and positive explant virologically [6]. To show that only LAT without any transcriptional products of other virus genes (a, b, c) were present, the following was done. LAT was detected by TaqMan real-time RT-PCR [16]. Total detection of the a, b, and c genes was verified by real-time RT-PCR of the intercalator method using SYBR Green I. We selected infected cell protein 0 (ICP0) as a representative of the a gene; thymidine kinase (TK) as a representative of b; and glycoprotein B1 (gB-1) as a representative of c. Primer and probe sequences targeting LAT [16], ICP0, TK, gB-1 and GAPDH, and temperature and the number of cycles of the real-time RT-PCR method are shown in Tables 1 and 2.

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Target gene HSV

50 °C/2 min, 95 °C/10 min (1 cycle) 97 °C/20 s, 58 °C/s (1 cycle) 96 °C/20 s, 58 °C/60 s (45 cycles)

LAT

95 °C/10 s (1 cycle) 95 °C/5 s, 60 °C/31 s (40 cycles)

TK, ICP0, gB-1

95 °C/30 s (1 cycle) 95 °C/5 s, 55 °C/30 s, 72 °C/34 s (40 cycles) 95 °C/15 s, 60 °C/20 s, 95 °C/15 s (1 cycle)

GAPDH

95 °C/10 s (1 cycle) 95 °C/5 s, 60 °C/31 s (40 cycles) 95 °C/15 s, 60 °C/20 s, 95 °C/15 s (1 cycle)

DNAeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA) was used for DNA extraction, RNeasy Fibrous Tissue Kit (Qiagen) for RNA extraction, and Rever Tra Ace qPCR with gDNA remover (Toyobo Co., Ltd. Life Science Department, Osaka, Japan) for cDNA synthesis. The samples were quantified by an ABI Prism 7700 Sequence Detector (PE Biosystems, Branchburg, NJ, USA). SYBR Premix DimerEraser (Takara, Shiga, Japan) was used for TK, ICP0, and gB-1 quantification. The control plasmids for each transcript were made by Takara Bio, Inc. The quantification of the transcriptional products was performed in duplicate. All the samples were quantified at the same time and place. This study was approved by the Institutional Review Board of the Kinki University Faculty of Medicine, and adhered to the principles of the Declaration of Helsinki. All the patients had given informed consent to this study after they were provided with an explanation of the research.

Corneal latency Table 3 Results of the corneas with past history of herpetic keratitis Case

HSV DNA

Viral culture from homogenate

Viral culture from explant

LAT

ICP0

TK

GB-1

1

2.7 9 107

Negative

Positive

1.3 ± 0.85 9 104

Negative

Negative

Negative

2

3.4 9 10

3

Negative

Positive

2.4 ± 0.25 9 104

Negative

Negative

Negative

3

7.4 9 104

Negative

Positive

6.3 ± 0.42 9 103

Negative

Negative

Negative

The quantitative results are expressed in terms of DNA or RNA copy number per cornea The numbers in the column of LAT show average ± standard deviation

Table 4 Results of the corneas with no past history of herpetic keratitis Case

HSV DNA

Viral culture from homogenate

Viral culture from explant

LAT

ICP0

TK

GB-1

4

1.4 9 106

Negative

Negative

Negative

Negative

Negative

Negative

5

Negative

Negative

Negative

Negative

Negative

Negative

Negative

6

Negative

Negative

Negative

Negative

Negative

Negative

Negative

The quantitative results are expressed in terms of DNA copy number per cornea

Results Table 3 shows the results of the three corneas with a past history of herpetic keratitis. All these corneas had HSV DNA and showed negative homogenate and positive explant, which virologically means corneal latency. LAT was detected in the same three corneas. However, transcripts of ICP0, TK, or gB-1 were not detected in any of the three. This indicates that a, b, or c genes were not expressed. All results for these corneas were consistent with the conditions of corneal latency. Case 1 suffered from herpetic keratitis one year after the keratoplasty and case 3, 4 months after the keratoplasty. They were treated with antivirals and cured. Table 4 shows the results of the corneas without a history of herpetic keratitis. One cornea out of the three had HSV DNA. All three corneas showed negative homogenate and negative explant. LAT was not detected in any of them. GAPDH was positive in all six samples. The above results indicate the possibility of HSV-1 corneal latency in the human cornea.

Discussion This study demonstrates for the first time that latent HSV infection is possible in the human cornea. To prove the latency, virological and molecular biological techniques were used to ensure the following three prerequisites: (1) positive HSV DNA, (2) negative homogenate and positive explant, and (3) LAT as the only transcript abundantly present, with no transcription products of other virus genes (a, b, c) being detected. The number of samples was small. We therefore need future investigations with larger sample

sizes. If, with the help of an electron microscope, we can prove that there are no virus particles, and that there is an absence of protein expression in the same cornea, we can further ascertain the presence of latency [12, 13]. One cornea (case 4) out of the three without a history of herpetic keratitis had HSV DNA detected. In a previous report, there were cases in which HSV DNA was detected in the cornea without a history of herpetic keratitis. We suggest this may be due to spontaneous ocular shedding [19]. In case 4, cultures from homogenate and explant were negative and LAT was not positive. These results are inconsistent with corneal latency. It is now necessary to increase the number of samples of cases without a history of herpetic keratitis, and to examine whether these will demonstrate corneal latency. Sawtell et al. [20] report that early intervention with high-dose acyclovir treatment during primary herpes simplex virus infection reduces latency and subsequent reactivation in the nervous system in vivo. Considering their results, there is a possibility that HSV corneal latency depends on the titer of infectious virus on the cornea. In view of possible corneal latency, we believe that keratoplasty can be useful in decreasing the frequency of recurrence in cases with a history of herpetic keratitis. In other words, we can remove the source of recurrence physically at surgery. To detect the corneal latent infection site, namely, the corneal epithelium, stroma, or endothelium, as the next step, we need to investigate them by in situ hybridization or in situ RT-PCR. The ganglion has been mentioned as a site of HSV latent infection. On the other hand, HHV-6 latently infects monocytes as well as the bone marrow [21], and HHV-7, CD4 ? T cells [22]. We have shown in the current study a possibility that HSV-1 can latently infect the cornea aside

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from the ganglion, although more cases need to be studied to substantiate this hypothesis. Acknowledgements This study was supported by the Michiko Kihara Ophthalmic Medical Fund of the Osaka Community Foundation. The authors wish to thank Mrs. Yukiko Mimuro for the editing of this manuscript and Mrs. Mayumi Mizuno for her technical assistance. Conflicts of interest S. Higaki, None; M. Fukuda, None; Y. Shimomura, None.

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Virological and molecular biological evidence supporting herpes simplex virus type 1 corneal latency.

Trigeminal and other ganglia are known as sites of latent infection by herpes simplex virus type 1 (HSV-1). In ophthalmology, HSV-1 remains latent in ...
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