Influenza Virus Preservation and its Effect on Infectivity and Viral Load by Lyophilization Technique

Background: Freeze drying (Lyophilization) performed at temperature and pressure below the triple point is being practiced for the preservation of virus stocks for longer periods. The present study is aimed to lyophilize influenza virus strain to study its effects on infectivity and viral load. Material & Methods: In-house Influenza virus reference strain (stock virus) was propagated in MDCK cell line in 25cm2 cell culture flasks. In 24-well plates the serial dilutions of stock virus from 10-1 to 10-7 (100µl inoculum) was inoculated in each well with MDCK cells for TCID50 titer and viral RNA was extracted separately to determine viral load by Real Time PCR. Stock virus was lyophilized in 3 lots and stored at RT (25±2°C) and 4°C separately for 1, 4 and 6 months and subjected to TCID50 (for viral infectivity) and viral load assay (for total viral genome copies). Results: Following lyophilisation and storage of Influenza virus strains at RT and 4°C separately did not affect significantly on the viral stability, infectivity as well as viral copy number till 4 months. However, storage at RT for 6 months resulted in 1 log reduction in viral copy number. Conclusion : Thus, storage of even lyophilized virus stock would necessitate a temperature of at least 4°C for prolonged periods.


Introduction
The structure and function of organisms change and get lost with time, as in laboratory cultures. Attempts to stop the biological clock have been conjured by ancient and modern minds; and the heart of many such schemes has been experiments with temperature and water content. Whereas refrigeration technology provides a means of slowing the rate of deterioration of perishable goods, the use of much lower temperatures has proved a means of storing living organisms in a state of suspended animation for extended periods. Influenza virus infectivity is relatively unstable, and titers drop rapidly and unpredictably onstorage under different conditions. Stability can be improved by simple desiccation or drying bysublimation in vacuum [1][2][3][4][5][6][7][8][9][11][12][13]. However, the most satisfactory method of storage has been in ampoules at -60℃ [6,7,10,14], although Greif (4) reported that the titers of purified suspensions of influenza virus in physiological saline stored at -65℃ for 180 days declined significantly. Since freezers capable of maintaining this low temperature are not available in many laboratories, a method of storing influenza virus at higher freezer temperatures or at refrigerator temperature would offer many advantages. Greif (1,2) reported quantitative data showing that it is possible to freeze-dry influenza virus and still retain significant infectivity. Influenza is a highly contagious viral respiratory infection caused by influenza virus whose epidemic and pandemic has resulted in significant mortality and morbidity. It has been reported that annual epidemic of influenza result in an estimated 3 -5 million cases of severe illness and about 290000 -650000 deaths globally. To preserve maximum infectivity for long periods, cell culture-grown adenovirus must be stored frozen at very low temperatures [15]. However, this makes its handling difficult. The removal of water from viable biological material in the frozen state (freeze-drying) provides another means of arresting the biological clock by withholding water and commencing again by its addition. Lyophilization or freeze-drying is a controllable method of dehydrating labile products by vacuum desiccation [16]. It is considered advantageous to freeze dry viruses and vaccines wherever possible in order to reduce their volume for storage in cold, to enable easy handling and transport, and to enhance their keeping quality [17]. We have studied the feasibility of lyophilizing large numbers of ampoules of several different strains of influenza virus to maintain infectivity for prolonged periods of storage at refrigerator temperature for use as reference reagents.

MDCK cell monolayer was prepared and virus infection was
performed at different dilutions in 10ml sterile tissue culture tubes.
Cell monolayer was observed daily till the cytopathic effect (CPE) appeared, along with the control wells (MEM inoculated) kept under the same condition. TCID50 titer was be calculated by using Reed and Muench [18].

Extraction of Viral RNA
Viral RNA was be extracted from 140μl of tissue culture lysate using RNeasy Minikit (QIAGEN, GmbH, Hilden, Germany) according to the manufacturer's instructions.

Lyophilization of Stock Virus
The virus was lyophilized or freeze dried in 3 batches of 6 vials Each using TFD5503 benchtop lyophilizer using the instrument protocol.

Revival of Lyophilized Virus
Lyophilized virus was revived in sterile conditions using sterile double distilled water after 1,4 and 6 months. The virus ampoule was opened of the ampoule in the bio-safety cabinet.1 ml sterile water was added to the dried virus and mixed gently. The revived virus was kept at 4 degree C for (1-2) Hr and aliquated and subjected to TCID50 and real Time PCR for viral load estimation.

Estimation of Viral Load in lyophilized Virus
To determine the effect of Lyophilization on influenza viral load the lyophilized influenza virus was subjected to real time PCR, the ct values obtained were extrapolated with standard curve to estimate the vial load in samples.

Data Analysis
The viral titre and load between stock and lyophilized virus were compared by a paired sample ttest. The correlation between viral load and TCID50 was studied using Spearman correlation coefficient. Statistical analysis of significance was undertaken using SPSS 16.0 software with a value of (P<0.05) for significance.

National Public Health Laboratory and Nepal Health Research
Council (NHRC ref. no. 1673) before carrying out this study.

Comparison of TCID50 Titer of Lyophilized Virus Stored for Different Time Intervals
The lyophilized virus was stored at RT and 4℃for 1month, 4 months and 6 months. The virus was revived after these fixed

Comparison of Viral Load of Lyophilized Virus Stored for Different Time Intervals
The effect of lyophilization on viral load of Influenza over a period of time was studied. No significant difference was observed in viral load of AdV stored at 4°C for 1, 4 and 6 months. However, in lyophilized virus stored at RT a significant difference in viral load was observed between the virus stored for 1month and that stored for 6 months at RT (P<0.05) but no difference was observed in viral load of Influenza stored at RT for 1 and 4 months ( Figure 5).

Discussion
Freeze-drying will not reverse damage incurred prior to formulation and care must be exercised when selecting an appropriate cell type or technique used to culture or purify the cell or its extracts prior to freeze-drying. To sustain freeze-drying it is necessary to establish a pressure gradient from a sample (highest pressure), to condenser, and finally vacuum pump (lowest pressure) so that water migrates from the sample as drying progresses. In the present study Influenza virus was successfully propagated in MDCK Cell line. The lyophilization cycle was standardized and the virus suspension was lyophilized at temperature below -40°C and under 50m Torr vacuum. The essence of the formulation exercise should be to minimize freeze-drying damage, loss of viability, or activity.
This study considers the stability of Influenza virus after freezedrying and storage and also determines its titer and load. The infectivity of the virus was retained when stored for over 6 months at +4°C or RT. We determined the TCID50 titer of the Influenza virus lyophilized for 1-6 months and observed no significant difference in the infectivity when stored at +4°C or RT.
Thus, the cell culture-grown Influenza virus could withstand the freeze-drying process, and it should be stored at +4°C and RT to retain maximum infectivity. Many culture collections and gene banks insist on high recovery values prior to a protocol being adopted for regular use; 50% viability post thaw has been accepted in some culture collections as a nominal cutoff for adopting maintenance by cryopreservation alone [5]. The ability to determine viral titer rapidly and at high accuracy is one of the most important tools desired when working with viruses in the research laboratory [6,7]. Real-time PCR has been shown to be more sensitive than cell culture-based techniques for detection of viruses in clinical specimens; it was therefore interesting to investigate whether realtime PCR technology could also be an important tool for rapid and efficient estimation of viral titer [8].

Conclusion
The present study could successfully lyophilize Influenza virus and retain its infectivity over a period of 6 months when stored at RT and 4°C. No significant difference in the infectivity or TCID50 titer was observed in the lyophilized virus as compared to the stock virus. However, the viral load was observed to increase with Lyophilization of the virus over 6 months when stored at 4°C which possibly is due to the concentration of the virus on freeze-drying. Competing Interest: The authors declare that they have no competing interests.

Declarations
Availability of data: All necessary data are included in paper.
Remaining data will be provided by corresponding authors on reasonable request.