Download KOZLENKO T

© 2016
Nedosekov V. V., Doctor of Veterinary Science, Professor,
Kozlenko T. G., postgraduate student
National University of Life and Environmental Sciences of Ukraine
OPTIMIZATION OF METHODS OF CLEANING AND INACTIVATION OF FELINE CALICIVIRUS
ACTIVATOR OF CATS
Reviewer – Candidate of Veterinary Sciences, Associate Professor V. V. Mel’nyck
We found that the best method of obtaining a purified preparation of calicivirus is ammonium sulfate
precipitation followed by purification of the antigenic material using gradient sucrose concentration, which
enables to obtain purified preparation pathogen, suitable for the production of antigen. Comparative study modes
formalin inactivation showed that optimal for virus inactivation is the effect of formalin in an amount of 0,2 % at
37 °C and 72 hours of exposure.
Keywords: virus, purification, precipitation, inactivation, formalin, buffers, antigen.
Statement of the problem. As there is a high importance of using efficient diagnostic tools for calicivirus,
scientific and practical interest is the development of a process for high-level and very specific antigen to immunize
animals to obtain hyperimmune serum against the virus and use the latter to manufacture diagnostic products for
serologic reactions.
Analysis of main research and publications where a solution of the problem is initiated. Many foreign
researchers show virus resistance to ether, chloroform, pH 4.0, as well as to heat. Inactivation of the virus occurs at
50 °C for 30 min. At 60 °C – 10 min. (in this case, the isolated RNA is more resistant: it is inactivated at the same
time at 65 °C). At minus 65 °C virus retains infectivity at least 4 years [5].
Feline calicivirus is resistant to freeze, allowing you to use this method of storing strains [1]. At high
temperature 1 M of MgCl2 and MgSO4 solutions not only stabilizes the virus, but even increases the heat
inactivation [8]. However, sodium partially stabilizes the virus against thermal inactivation and pH. Lee K. M. and
Gillespie J. H. (1973) reported that the buffer pH affects the stability of the virus. Most CVC isolates are resistant
to pH 4, but at pH 3 and lower virus titer decreases [2, 5, 9].
Doultree J. C., Druce J. D. et al (1999), studying resistance of Norwalk virus to desinfectant, used as a
model feline calicivirus. The researchers used commercially available disinfectants: glutaraldehyde, iodine, sodium
hypochlorite, ammonia and ethanol. Glutaraldehyde and iodine effectively inactivated feline calicivirus, ammonia
and ethanol had no significant virucide activity. The stability of the virus in suspension and in the dried state was
evaluated by the same authors after exposure at 4 °C, room temperature (20 °C) and 37 °C. With increasing of
temperature stability of virus decreased in suspension and in the dried state. At 37 °C lyophylised virus was
inactivated in 30 days [3].
Through these experiments, Gulati B. R., Allwood P. B., Hedberg C. W. et al (2001) showed that phenolic
compounds 2–4 treatment completely inactivates FCV. The combination of ammonia and soda were effective at
concentrations that twice exceeded conventional. Rinse with clean water reduced the virus titer to 50 CPS 2 lg/cm3 [6].
Kadoi K, Kadoi B. K. (2001) used the same virus as a model for studying resistance of human calicivirus in
seawater. The results showed that the virus remained infectious at 10 °C or below within 30 days [7].
Many researchers recommend to use alkalis, formaldehyde, phenol and chlorine-containing drugs in
conventional concentrations as disinfectants [4].
The aim of our study was to optimize the method of cleaning of specific calicivirus antigen and choose
the best mode of pathogen inactivation of FCV.
Objective: to clear calicivirus suspension of cellular detritus; to find the optimal conditions of inactivation
of the pathogen.
Materials and methods of research. To isolate the work we have used K-2, which took 10 passages in
cell culture CrFK and accumulated in the credits 8,0–8,5 lg 50 CPS/cm3.
Infectious virus activity established by titration in cell culture in 96-well micropannels formed with
monolayer. Pre-poured environment, then made breeding the virus. For this virus prepared Tenfold dilution of 10 -1
- 10-9 in a supportive environment. With each dilution 4 holes were infected by 0,2 cm3 of virus. As a control, cell
culture used in the 4 holes with a supportive environment. The results were taken into account within 3 days. For
positive wells we count wells with specific CPS (rounding cells, their exclusion from the glass, the formation of
voids in a monolayer). In the control wells cell monolayer didn’t change. Infectious titer was calculated by the
method of Reed and Mench.
Precipitation of virus was carried by ammonium sulfate, followed by purification of the antigenic material
using sucrose concentration gradient.
1
As inactivator of the virus we used formalin (an aqueous solution of 38 % formaldehyde) in amounts of
0,1 %, 0,2 % and 0,5 %. We used the exposure from 24 to 120 hours at 37 °C. The completeness of inactivation
determined holding 3 consecutive passages inactivated virus in cell culture CrFK.
Results of research. In the first stage of research we have been tasked to clean FCV suspension obtained
from tissue culture, which is contaminated with cellular components, including cellular membranes of detritus. For
this we conducted low-speed centrifugation of the suspension at 6000 rev/min. 30 min.
Then precipitation by ammonium sulfate was carried. For this sample of ammonium sulfate at the rate of
0,4 g per 1 ml of culture fluid. By ammonium sulfate fill up the culture fluid containing 1 % serum to accelerate the
sedimentation of the virus, and thoroughly stirred to dissolve the crystals. The solution thus became more acid and
slightly turbid. The resulting suspension is centrifuged at 2000 g and 4 °C for 1–2 hours. The supernatant was
carefully poured and the residue was dissolved in the buffer solution, the amount of which was not less than 10 %
of output to ensure adequate dilution of ammonium sulfate. Thus, we were able to concentrate the virus 10 times.
Exit of virus was 96 %.
Concentrated virus was purified using a gradient of concentration of sucrose. To do this, we prepare two
solutions of sucrose in buffer containing 10 mMTris-HCl (pH 7,4) and 50 mMNaCl. The first solution contains 15
g, and the second – 45 g of sucrose in 100 ml of buffer. Using 15 ml of each of these solutions we prepared linear
concentration gradient of sucrose (15–45 %) in volume of 30 ml tubes volume of 35 ml. Prepared 10 % solution of
NP-40 buffer PBS, which was added to the virus. When shaking the virus with detergent solution became clear.
The virus was carefully applied on the gradient layer. Tubes were equilibrated and centrifuged at 80 000 g and 4 °C
for 4 hours. Gradient was fractionated. In fractions we tested contents of the virus.
Our next task was to find the optimal conditions for pathogen inactivation of feline calicivirus. The main
criterion of the study was to preserve the activity of the antigen against calicivirus.
As inactivator we used formalin. For working parameters we studied the effect of inactivation, inactivator
concentration, temperature and duration. For inactivation of virus to virus-containing fluid from the initial
infectious activity CPS 50 8,5 lg/cm3 formalin was added in amounts of 0,1 %, 0,2 % and 0,5 %. We used the
exposure from 24 to 120 hours at 37 °C. The completeness of inactivation determined holding 3 consecutive
passages of inactivated virus in cell culture CrFK.
The effectiveness of inactivation of the virus was assessed in terms of loss of infectivity and maintaining
high activity inactivated antigen preparation.
Changes antigenicity of the virus inactivated in different modes is shown in table.
Antibody titer in blood of
Isolate
Inactivation regime
guinea pigs
(РН, log2)
Temperature,
°С
Formaline
contain, %
Exposure,
hours
Before
injecting virus
21st day after
injection
К-2
0,1
108
≤2
8,0±0,5
0,2
72
≤2
8,3±0,6
0,5
48
≤2
8,5±0,5
Non-inactivated
≤2
8,6±0,5
The experimental results indicate that all variants of inactivation of the virus, it remained antigenicity,
which is slightly different from the original.
Inactivation of the virus when added formalin at a concentration of 0,1 % was longer (108 hours) and
resulted in a slight decrease in activity of the antigen. At the same time, the antigenicity of the virus samples,
formalin inactivated when added in an amount of 0,5 % and 0,2 % (48 and 72 hours, respectively) differed slightly.
Considering the fact that the preferred minimum content of formaldehyde in the product at almost
identical rates antigenicity derived raw materials, can be summarized that the optimal mode of inactivation of virus
is the effect of formalin in an amount of 0,2 % at 37 °C and 72 hours of exposure.
Conclusion. Thus, it was found that the best method of obtaining a purified preparation of feline
calicivirus is ammonium sulfate precipitation followed by purification of the antigenic material using gradient
sucrose concentration, which enables to obtain purified by 96 % and concentrated 10-fold drug pathogen of virus
that is suitable for the production of antigen.
37 °С
2
It is shown that the optimal mode of inactivation of virus is the effect of formalin in an amount of 0,2 %
at 37 °C and 72 hours of exposure. Such conditions show formalin inactivating properties, and does not affect its
antigenicity.
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