A new isolated local varicella virus: isolation, identification, comparative growth characteristics and immunological evaluation on an animal model

A panel of 4 different cell lines was optimized for isolation, identification, and authentication of a VZV virus from a swab sample of an 8-year-old boy suspected to varicella zoster infection. The system enabled highly efficient and rapid isolation of viruses in 33°C by serial sub culturing to more than 25 passages. The technique relies on isolation of viral genes by increasing the number of particles that are statistically represented in cell culture and verified by CCID50, FAM-RT-PCR, and IE62 antibody in IF test. The viral genes (ORF38, ORF54) confirmed the new isolate as VZV and revealed the amino acid sequence of viral-encoded proteins after 27 passages, identical with positive control virus, in RFLP-PCR test. Utilization of successive serial passages at temperatures lower than the normal body temperature would reduce the virus virulence and directly cause virus attenuation. As a result, the attenuated virus is adapted to growth in vitro and presented higher replication at 33°C. Our goal was to determine if the targeted virus with a large double-stranded DNA genome, varicella virus, is isolated and can be attenuated by cold adapting in vitro, Using vOka as attenuated VZV golden standard, in two quantitative tests, including CCID50 and FAM-RT-PCR. Finally, when compared with the local isolated virus, these results were strongly confirmed. We recorded plaque forming assay to show phenotypic changing which encodes the attenuation regarding size of plaque. Although in plaque forming assay, the size of plaque seemed smaller at first glance, the statistical distribution of the plaque size did not show any change between the virus in the first and last passages. In cell culture, the local VZV isolated viruses formed clear plaques and grew to higher titers compared with lower passages as parental virus.Due to lack of access to human fetal lung cells (MRC-5) and an alternative to vaccine production in the future, a new authenticated local foreskin cell substrate (RFSC) was used for virus cultivation. In comparison to, MRC-5-optimized and cloned-viruses replicated in vitro with kinetics that were similar to those of the RFSC. Laboratory animals that were infected with the optimized virus fluid as vaccine showed a good neutralization antibody against local VZV isolated as compared to vOka as control positive virus. These results demonstrate that the virus isolated from swab sample was authenticated as VZV virus, and this cold adapted attenuated virus may be an applicable candidate for future plan. Author summary We used different cell substrates for isolation, identification, and attenuation of a new local VZV virus from vesicle swab of suspected patient to varicella zoster diseases. The technique involves serial sub culturing of virus in cell culture. Our goal was to determine if the targeted virus with a large double-stranded DNA genome, varicella virus, is isolated and can be attenuated by cold adapting in vitro, using vOka as attenuated VZV golden standard, The virus was cloned and purified by serial dilution cloning and plaque assay. Different techniques were used regarding verification as differentiation from other members in family, potency, and sterility for isolated virus. The virus can grow well in new local foreskin cell substrate and produce good titre as compared with MRC-5. The isolated VZV potentially induced neutralization antibody in animal model. The results of our study imply that local VZV might be an applicable isolate for future plan in research and developing a varicella vaccine.

zoster was performed [1]. In Table 2, the primer set sequence and thermal cycle,        (Table 3).  Infection with each of the three (high and low passages local isolated beside 307 vOKa) viruses proved to be typical syncytium-cytopathic in these cells, which 308 remained persistently infected for the 10 days follow-up period, though virus 309 titers started to increase from 3 DPI onwards. In addition, peak virus titers were categories, where survival rates were 94% and 92%, respectively, which is same 326 as the levels we expected since both cell lines had the same characteristics 327 regarding the growth kinetics ( Fig 3). is sufficient to form a plaque, the virus titer can be presented as a plaque-forming 408 unit (PFU) per milliliter. In addition to titration, we used the plaque method to 409 purify and clone the virus. In the present study, the above method was used to 410 evaluate the titer and clone the isolated virus (after 25 th passage) and also to 411 evaluate the size of the plaque in relation to its attenuation. We determined plaque 412 sizes of the passaged viruses. We did not detect any changes of the virus 413 phenotype. Plaque areas were measured using electronic size counter (Fig 5). The isolated viruses were examined by PCR and restriction fragment length 430 polymorphism (RFLP) analysis, using the ORF38 and ORF54 region. Initially, 431 we performed PCR by designing two pairs of specific primers for these two ORFs 432 (Table 3). Then, via digestion through two restriction enzymes, BglI and PstI with 433 a fully -optimized thermal cycle, digestion was performed on the PCR product.

434
In electrophoresis of PCR and RFLP products on 1.5% agarose, the results were 435 confirmed after safe staining (Fig 6).    passages well adapted and produced significantly more as compared with the 4th 510 passage (Fig 9). This difference was completely significant. The purpose of the 511 above test was to evaluate the rate of ORF62 gene replication, which reported 512 that the increase in gene proliferation could be attributed to limit reduction. Several studies have demonstrated the serological and pathogenesis of VZV virus. 523 We sought to determine the kinetics and optimization of minimum dose of local 524 isolated VZV that can induce antibody production. We found that isolated VZV was injected an ascending trend is also evident in almost all groups in their 534 antibody titters. More animals that received 10 4.7 CCID 50 /dose, 10 4 CCID 50 /dose 535 10 3 CCID 50 /dose or 10 2.7 CCID 50 /dose from high passages (30 th ) developed IgG 536 antibody compared to animals receiving 10 2.7 CCID 50 /dose low passages (5 th : 537 group F). As shown in Fig 10, an increase in Ig M was observed for up to 21 days.

538
Then, a gradual decline from 44 th days to 166 th days after injection was recorded.

539
An increase in Ig G was clearly seen after 44 days and progressed to 162 days 540 post-injection (Fig 11). Note that group H includes animals exposed to vaccinated     The disease begins with symptoms such as lethargy and fever from one to two 620 days before and after the rash. The fever usually lasts for 5-7 days in parallel with 621 the rash. Each rash appears as an itchy lesion within 24 hours of the stage from 622 macular to papule and then to the vesicle and then the pustule and eventually to mainly the trunk, scalp, and even epithelium of the internal organs (endothelium) 625 also become infected [23,24].

626
In the case of VZV, the phenomenon of escape from the immune system as well 627 as escape from binding to antibodies has been observed, which leads to   subsequent experiments were carried out with (P: 5) as the parent isolate (P: 5) 714 that had been passaged fifth on MRC-5 and (P: 30) that we hope was attenuated.

715
The results mainly showed a significant difference between primary characterized 716 isolate (P: 5) and the current passaged virus (P: 30) virus in term of potency and 717 RT-PCR.

718
One feature of the serial sub culturing in human diploid fibroblast that differed 719 from the others was the adaptation of the strain to growth at 300C which we 720 included in our procedure. The idea behind this was to rapidly induce attenuation,   The higher the growth rate, the more cells it can produce over a period of time; 871 thus, the faster the product can be produced and the more efficient it can be.     Czech Republic) containing a protected specific sequence for each Herpes virus 950 such as ORF62 for VZV. The kit generated by an internal control for each virus.

951
The viral gene from infected cells at different passages (4 th , 9 th , 17 th , and 27th)