1. Introduction
Seasonal influenza infection is one of the leading causes of mortality in human communities, especially among at-risk individuals. Nowadays, vaccine design against influenza virus is mainly based on inactivated vaccines, especially vaccines containing broken virions or antigenic subunits (HA and NA proteins) whose efficacy is due to the production of specific antibodies against a particular strain of the virus and cannot provide protection against other influenza virus subtypes [2, 4]. These types of vaccines also require an annual evaluation and modifications [5, 6].
Animal viruses, on the other hand, by exchanging gene fragments, have led to the emergence of new variants of the influenza virus, reducing the protective efficacy of seasonal influenza vaccines in India [5]. Therefore, attention is now focused on the production of complete inactivated vaccines [2].
 Gamma-irradiated influenza viruses (γ-FLU) remain mostly unchanged during treatment. Thus, in this study, we use gamma irradiation to inactivate the human influenza virus strain named A/PR/8/34 [A/Puerto Rico/8/34 (H1N1)],  as a candidate for the production of complete inactivated viral vaccines. 
2. Materials and Methods
This experimental study was performed on Madin-Darby Canine Kidney (MDCK) epithelium cells cultured in sterile and filtered containers with DMEM (Dulbecco’s Modified Eagle Medium), antibiotic, and 10% of Fetal Bovine Serum (FBS). The containers were incubated for 24 hours at 37°C with 5% CO2 and 95% humidity. The virus used was human influenza A.PR.34.34 strain. The appropriate volume of the virus to infect MDCK cells was determined based on the infection coefficient Multiplicity of Infection (MOI). The virus was then inoculated at the optimum dose into cell culture containers. After 48 hours, the cells were collected from the containers and concentrated by ultrafiltration [13, 14].
Concentrated virus titration was performed using a TCID 50% test [15]. Vials containing condensed viruses were irradiated at doses of 5, 10, 15, 20, 25, 28, and 30 kg. Finally, the optimal dose of irradiation for the inactivation of the virus was calculated. The total inactivation of the virus was confirmed by the safety test [17]. Antigenic properties of the irradiated virus were compared with a non-irradiated virus by hemagglutination test.
3. Results
According to the results of 4 passages of irradiated samples at 28 and 30 kg doses (Table 1), as well as results related to the evaluation of the preservation of ability of the surface antigenic properties of irradiated virus (Table 2), the dose of 28 kg was determined as the optimal dose of gamma irradiation for total inactivation of the human influenza virus with an initial titration of 105.75 mL by preserving surface antigenic structures as well as the absence of virus replication symptoms in 4 successive passages.

4. Discussion
Inactivation of the influenza virus by chemical treatments such as formalin or β-propiolactone leads to the induction of cross-linking of viral proteins. Thus, the ability of inactivated viruses to penetrate the cytosol of host cells reduces. Physical treatments such as UV rays also impair the effective uptake of these viruses by antigen-presenting cells through disrupting the activity of the lipid membrane of the virus, thereby decreasing the supply of viral antigens may reduce the efficacy of these vaccines [2, 11, 22].
In contrast, the virucidal effect of gamma irradiation is mainly related to the occurrence of virus nucleic acid damage, including a breakdown in the viral genome, cross-fractures, or the occurrence of nucleotide damage [19]. This method uses frozen viruses to minimize unintended damage to protein structures during the virus inactivation process. This is a significant advantage over other traditional methods of sterilization, especially in vaccine manufacturing [23]. Given these advantages, gamma irradiation can be used to inactivate the human influenza virus.
The results of this study showed that doses of 28 and 30 kg of gamma irradiation could deactivate the human influenza A/PR/34/8 virus, without altering the antigenic properties of the virus surface.
Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Research Ethics Committee of the Islamic Azad University of Tehran Medical Branch (Code: IR.IAU.TMU.REC.1397.309).
Funding
This article was extracted from the PhD. thesis of Ailar Sabaghi approved by  Biology-Microbiology from the Faculty of Science of Islamic Azad University of Qom Branch (No. 508333). 
Authors' contributions
All authors met the standards of writing criteria based on the recommendations of the International Committee of Medical Journal Publishers (ICMJE).
Conflicts of interest
The authors declare no conflicts of interest.


References