Application of McCoy cell line for propagation of herpes simplex virus type 1.
Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are double-stranded DNA viruses and are members of the Herpesviridae family. This family includes three subfamilies: alpha-, beta-, and gamma-herpesvirinae. (1) HSV-1 and HSV-2 are capable of causing various diseases, including oral and genital lesions, blindness and encephalitis. (1) Once infected with the virus, it may lead to viral latency in peripheral nerve ganglia. (2)
Unlike other members of the Herpesviridae family, HSV has low specificity to the host; therefore, they have the ability to infect a wide range of hosts. In addition to human cells, herpes viruses could infect animal cells. This feature has led to the successful spread of the viruses throughout the world. It is estimated that the virus has infected 40% to 80% of the world population. (3)
The prevalence of HSV is high in Iran. Studies on pregnant women in Tehran (Iran) showed that the level of neutralizing antibodies against HSV-1 and HSV-2 were 90.75% and 8.25%, respectively. (4) In a study conducted on women in Shiraz, Iran, HSV-2 neutralizing antibodies were found in 28.19% of the studied patients. (5) Studies have shown that HSV-2 infection increases the risk of HIV infection. As a result, development of a new vaccine against HSV infection may also serve in a better controlling the spread of HIV infection. (6) The mechanism of cellular and molecular immunity against recurrent HSV infection is unknown; therefore, studies have been carried out in order to reveal the exact mechanism and develop an effective vaccine. (7)
Although the frequency of drug resistance to HSV is rare in immunocompetent people, it is common in immunocompromised patients and approximately 5% of their isolated viruses are resistant to antiviral drugs; however, this figure is higher (14-30%) in patients receiving bone marrow transplantation. (8)
Mathematical models have shown that antiviral drugs for the treatment of HSV-2 may cause a gradual increase in drug resistant cases. Therefore, administration of antiviral drugs could not reduce the prevalence of this virus. (2)
The high prevalence of HSV in the community, its ability to be latent in infected individuals during their lifetime, and the ability of the virus for causing fatal diseases in immunocompromised patients highlights the increasing need to set up studies to develop vaccines and antiviral drugs. Designing suitable cell lines for the propagation of HSV is required for such studies. McCOY cell line was derived from human synovial fluid in 1957 by Pomerat, but it changed between different laboratories for many years. Nogueira et al. demonstrated that this cell line has human and mouse cell's marker. McCOY cell line is widely used by laboratories for the cultivation of various microorganisms. (9)
The aim of this study was to evaluate the ability of McCoy cell line for the isolation and propagation of HSV.
Patients and Methods
A wild type of HSV-1 was isolated from labial vesicles of a 29-year-old Iranian patient in 2010 and was identified as HSV-1 by PCR using HSV-1 specific primers.
Cell line and Virus Propagation
McCoy cell line was purchased from Pasteur Institute (Iran). The virus was inoculated in McCoy cell monolayer in Dulbecco's Modified Eagle Medium (DMEM) plus 5% heat inactivated fetal calf serum (FCS), 100 IU/ml penicillin and 100 [micro]g/ml streptomycin at 37[degrees]C with an atmosphere of 5% CO2. After the appearance of CPEs in more than 75% of the infected cells, the supernatant of the infected cells containing propagated viruses were collected and stored at 4[degrees]C for one day.
1/log serial dilutions ([10.sup.-1]-[10.sup.-7]) of the stored virus seed were prepared in serum-free DMEM. Its titer was determined by 50% tissue culture infectious dose ([TCID.sub.50]) method as described previously. (10) Tissue culture infectious dose 50% ([TCID.sub.50)] assay is a method used to quantitate infectious virus. This method is useful to determine titer of virus that cause a change in cellular morphology. Serial dilution of virus suspensions should provide in-tissue culture maintenance or growth medium to define virus titers and CPE observed different days after qualifying negative and positive control. [TCID.sub.50] is determined by two methods, namely Reed-Muench and Spearman-Karber. In this study Spearman-Karber method was used to determine [TCID.sub.50].
TCID50 unit volume - 1 = Highest dilution giving
100% CPE + 1/2 - [total number of test units showing CPE/number of test units per dilution]
[TCID.sub.50] [ml.sup.-1] = [10.sup.*][TCID.sub.50] unit [volume.sup.-1]. (11)
Figure 1 shows McCoy monolayer cells. There is not enough information with regard to the origin of these cells in previous studies. Firstly, these cells were isolated from synovial fluid of the knee joint of a patient with degenerative arthritis, however, another sub-line was of mouse origin. These cells are adherent and have fibroblastic appearance. Cytopathic effects of HSV-1 on McCoy cells appeared about 20 hours after the infection of the cells.
As shown in figure 2, HSV-1 CPEs includes ballooning and clustering of infected cells and formation of multinucleated giant cells.
TCID50 was calculated based on the ratio of the CPE positive wells to CPE negative wells (table 1) as described previously, (11) and titer of the propagated virus was [10.sup.-5.25] TCID50/unit [volume.sup.-1].
Several cell lines, such as African green monkey kidney (Vero, CV-1), baby hamster kidney (BHK), fetal rhesus monkey kidney (FRhK), human embryonic lung (HEL), human fetal foreskin (ff), mink lung (ML), primary rabbit kidney (PRK), permanent rabbit kidney (RK13), and HeLa cell line have been used for HSV propagation (12,13). Among the mentioned cell lines, HEL and Vero cell lines showed the highest efficacy for the propagation of HSV. (12) However, Sreedharan Athmanathan and his colleague showed immortalized human corneal epithelial cell line (HCE) was more susceptible compared with Vero cell line and the isolation of HSV-1 in clinical specimens by HCE was premiere to Vero cell line (14) as HSV detection in cell culture is gold standard method but different cell line and cell culture method show distinct sensitivity. (15) Finding more sensitive cell line could improve this method.
McCoy cells have been used in laboratories for about 50 years and are applied extensively for different studies. This cell line can be used for the culture of Chlamydia, studies for the design of vaccines, and as a cellular model for the studies of Chlamydia trachomatis cytotoxicity. (9)
The results of this study showed the high titer of isolated viruses. McCoy cells were used for the first time for the propagation of HSV in our study and high titers of propagated viruses were obtained. Our data showed that the McCoy cells could be used as a suitable cell line for HSV propagation. The clear CPE of the virus on infected cells are important for the detection of the virus. Although it is determined that the mechanism of HSV-1 dependent apoptosis in HEp-2/HeLa cells differ from Vero cells, (16) further studies are required to determine the specific cellular receptors for HSV attachment and absorption and HSV-1 dependent apoptosis. McCoy-Plovdiv is the new generation of McCoy cell line that does not require serum for multiplication. The study of the ability of these cells for HSV propagation has not yet been determined. This cell line may prove useful for serum-free virus cultivation in studies such as vaccine development. (9)
McCoy cell line can support the propagation of HSV-1. High titers of propagated virus could be obtained by culturing of HSV-1 in this cell line.
Maryam Sadat Nabavinia , MSc; Sina Rostami , BSc; Faezeh Ghasemi , MSc; Zahra Meshkat , PhD
 Department of Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran;
 Department of Biology, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran;
 Department of New Sciences and Technology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran;
 Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
Correspondence: Zahra Meshkat, PhD; Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Zip Code: 91967-73117, Mashhad, Iran
Tel: +98 51 38012453
Fax: +98 51 38002287
Received: 22 July 2013
Revised: 4 December 2013
Accepted: 6 January 2014
The authors would like to thank the patient who kindly gave permission to use her sample in this study.
Conflict of Interest: None declared.
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Table 1: Results of virus titration (TCID50 method) in McCoy cells Virus Number of Number of CPE+wells/ dilution inoculated positive total numbers wells Wells for of wells CPE [10.sup.-1] 4 4 1 [10.sup.-2] 4 4 1 [10.sup.-3] 4 4 1 [10.sup.-4] 4 4 1 [10.sup.-5] 4 3 0.75 [10.sup.-6] 4 0 0 [10.sup.-7] 4 0 0
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|Title Annotation:||Brief Report|
|Author:||Nabavinia, Maryam Sadat; Rostami, Sina; Ghasemi, Faezeh; Meshkat, Zahra|
|Publication:||Iranian Journal of Medical Sciences|
|Date:||May 1, 2015|
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