A new isolated local varicella virus: isolation, identification, comparative growth characteristics and immunological evaluation on an animal model
Authors: Esna-Ashari Fatemeh , Shafyi Abbas , Sabahi Farzaneh , Ravanshad Mehrdad , Sadigh Zohreh-Azita , Mohammadi Ashraf 
Published: December 2020

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.