Effects of L-lysine-α-Oxidase from Trichoderma In Vitro Experiments on the model of Viruses as Sindbis, Tick-borne encephalitis, West Nile, Tahyna and Dhori

 

V. F. Larichev¹, I. P. Smirnova², S. P. Syatkin², G. I. Myandina², S. M. Chibisov², Е. А. Ryskina²

¹Federal State Budgetary Institution “Research Institute of Epidemiology and Microbiology named after Honorable Academician N. F. Gamaleya”, 16 Gamalei Str, Moscow

²Medical Department of Medical Institute of Peoples’ Friendship University of Russia, 3 Ordzhonikidze Str., Moscow

 

ABSTRACT:

For the first time we studied the effect of the anticancer drug L-lysine-α-oxidase, obtained from culture liquid of Trichoderma harzianum Rifai F-180.

In vitro experiments we established the high activity of L-lysine-α-oxidase in a model of tick-borne encephalitis and the lack of activity against viruses Sindbis, West Nile, Tahyna and Dhori.

 

 

 

1. INTRODUCTION:

The arboviruses are an ecological group of viruses transmitted to susceptible vertebrates by the bites of blood-sucking arthropods: mosquitoes, ticks, sandflies, midges and biting midges.

 

Such infections such as Chikungunya fever and O Nyong-Nyong, Eastern, Western, Venezuelan encephalitis of horses (Togaviridae), dengue fever, yellow fever, encephalitis, Japanese, tick-borne, St. Louis, Murray valley, West Nile (Flaviviridae), Crimean-Congo hemorrhagic fever, Rift Valley fever (Bunyaviridae), and viruses of other families (Reoviridae, Arenaviridae and Rhabdoviridae) are not a complete list of dangerous for human arbovirus infections of great importance for health.

 

For the vast majority of arbovirus infections vaccine preparations have not been developed. In this regard, chemotherapy drugs become critical for treatment and for prevention.

 

The taxonomic principle was taken as the basis for study of antiviral action of drugs (if the product operates on one "model" virus, it can be assumed that it will act on all viruses of this family, as the strategy of the genome of this family is the same) [4].

 

At T. T. Berezov Department of Biochemistry of PFUR for many years we conducted studies of L-lysine-α-oxidase – antivirus and anticancer enzyme from Trichoderma harzianum Rifai F-180. The enzyme L-lysine-α-oxidase is an inhibitor of herpes simplex virus 1-th type, human immunodeficiency virus and other dangerous viruses [1, 2, 5, 6, 7, 8, 11]. This work is devoted to study the possibility of inhibition of homogeneous enzyme L-lysine-α-oxidase viruses of the families Flaviviridae, Bunyaviridae and Orthomyxoviridae.

 

2. Collection of viruses of the family flaviviridae, bunyaviridae, orthomyxoviridae.

We used viruses from the collection of the laboratory of biology and indication of arboviruses (Research Institute of Epidemiology and Microbiology named after honorable academician N. F. Gamaleya): family Togaviridae, genus Alfavirus – virus, Sindbis (strain 574); the family Flaviviridae, genus Flavivirus – tick-borne encephalitis virus (strain 205), West Nile virus (strain AST.986); the family Bunyaviridae, genus Bunyavirus – a virus Tagine (strain 92); the family Orthomyxoviridae, genus Thogotovirus – virus Dhori (prototype strain).

 

3. The study of transplantable cell line of kidney of pig embryo (SPEV) and kidneys of green monkey embryo (VERO clone E6).

Cell line cells:

The cells of the kidney of embryo pig (SPEV) and kidney cells of the embryo of green monkey (Vero clone E6) were grown on media of different composition.. SPEV cells were grown in 96-well plates ("Corning", USA) on a nutrient medium 199 (Institute of Poliomyelitis and Viral Encephalitis named after M. P. Chumakov RAMS) with 5% fetal calf serum ("High Clone", USA). Vero E6 cells were grown in 96-well plates on a nutrient medium Needle with a double set of amino acids (Institute of Poliomyelitis and Viral Encephalitis named after M. P. Chumakov RAMS) with 5% fetal calf serum ("High Clone", USA).

 

The source of infection of cells served virus-containing suspension of brain tissue of infected newborn white mice. To prepare dilutions of brain tissue and drugs we used nutrient medium with 1% fetal calf serum ("High Clone", USA). We prepare 10% brain suspension and then tenfold serial dilutions virus-containing material from 10^-2 to 10^-10.

 

Accounting reactions were performed daily for 7 days, observing the development of cytopathic action (CA) (cell death) of the virus on the cells, looking through the wells under inverted microscope. The titer of the virus was evaluated in lg CA100 (development of the CA in all wells with the same dilution of virus).

 

We used the enzyme L-lysine-α-oxidase from Trichoderma harzianum Rifai F-180. Homogeneous enzyme was obtained by previously developed methods. [5, 6].

 

The activity of L-lysine α-oxidase in culture liquid of Trichoderma was calculated from the increase in H₂O₂, which amount was determined spectrophotometrically orthodeuterium micromethod [6].

 

 

 

Table 1. The scheme of experiment determination of the activity of L-lysine-α-oxidase on transplantable cell lines.

	1**	2	3	4	5	6	7	8	9	10	11	12
*A	-2***	-2	-2	-2	-2	-2	-2	-2	-2	-2	-2	-2
B	-3	-3	-3	-3	-3	-3	-3	-3	-3	-3	-3	-3
C	-4	-4	-4	-4	-4	-4	-4	-4	-4	-4	-4	-4
D	-5	-5	-5	-5	-5	-5	-5	-5	-5	-5	-5	-5
E	-6	-6	-6	-6	-6	-6	-6	-6	-6	-6	-6	-6
F	-7	-7	-7	-7	-7	-7	-7	-7	-7	-7	-7	-7
G	-8	-8	-8	-8	-8	-8	-8	-8	-8	-8	-8	-8
H	Emission control				Emission control				Cells control
	L-lysine-α-oxidase 25 µg/ml				L-lysine-α-oxidase 12.5 µg/ml				Support environment without L-lysine-α-oxidase

Note: * – Number of horizontal rows on the 96-hole tablet

** – Number of vertical rows on the 96-hole tablet

*** – Degree (10^-2, 10^-3...) dilutions of investigated virus

 

4. DISCUSSION OF OBTAINED RESULTS.

Scheme of the experiment (table.1): 10-fold dilutions of virus from 10^-2 to 10^-8 or 10^-4 to 10^-10 (depending on activity pre viral suspensions) sequentially, starting with the top row (A), were added 100 µl into the wells of 96-well plates with monolayer cells. In range (H) brought a supportive environment free of the virus. The contact of virus with cells was carried out for 1 hour at 37 ° C. After incubation virus-containing environment was removed from the tablet. In the wells with the control (comparative) titration – 4 vertical row (No. 9 – 12) – contributed a supportive environment, and in the holes is designed to determine the antiviral activity, added support environment containing L-lysine-α-oxidase: 4 vertical rows (number 1 – 4) at a concentration of 25 µg/ml and 4 vertical rows (No. 5 – 8) of 12.5 µg/ml.

 

In the first phase of the experiment we evaluated the toxic effects of L-lysine-α-oxidase on the cell line cells. We added L-lysine-α-oxidase in concentrations 2.0, 1.0, 0.5, 0.25, 0.1, 0.05, 0.025, 0.012, 0.006, 0.003, 0.0012, 0.0006 mg/ml. To the monolayer of cells in the wells of tablets Toxic effects of the drug were evaluated for the development of the CA. It was found that in both cell lines a marked toxic effect occurs at concentrations of 0.05 mg/ml and above. Concentrations of 0.025 µg/ml of 0.0125 µg/ml (25 and 12.5 µg/ml) are the maximum, although cause an inhibitory effect on the proliferation of cells. To study the antiviral action of the drug was maximal concentrations that do not cause CA.

 

Study of antiviral action of L-lysine-α-oxidase on the West Nile virus, Tahyna, Sindbis and Ghori were performed on Vero E6 cells.

 

In the experiment, it was determined that the titer of West Nile virus in the control titration was 9.0 lg CA₁₀₀, and L-lysine-α-oxidase as at a concentration of 25 µg/ml and 12,5 µg/ml amounted to the same 9,0 lg CA₁₀₀. The titer of virus Tahyna in the control was 7.0 lg CA₁₀₀ with L-lysine α-oxidase in a concentration of 25 µg/ml to 8.0 lg CA₁₀₀, at a concentration of 12.5 µg/ml and 7.0 lg CA₁₀₀. The titer of the virus, Sindbis in control was 6.5 lg CA₁₀₀, and L-lysine α-oxidase in a concentration of 25 µg/ml, and 12.5 µg/ml was 7.0 lg CA₁₀₀. It was found for virus Dhori that the titer in the control was 7.0 lg CA₁₀₀, and L-lysine α-oxidase in a concentration of 25 µg/ml and 12.5 µg/ml and 7.0 lg CA₁₀₀.

 

L-lysine-α-oxidase has no antiviral effect on the viruses of West Nile, Tahyna, Sindbis and Dhori, as no difference in titers of the viruses during their titration with the drug and without [9,10].

 

Experiments on the determination of the activity of L-lysine-α-oxidase relative to tick-borne encephalitis virus (TBE) were conducted on the cell line SPEV, as they develop CA, caused by TBE virus.

 

It was found that the titer of virus in the control was 8.0 lg CA₁₀₀, and L-lysine α-oxidase in a concentration of 25 µg/ml, and 12.5 µg/ml was 5.0 lg CA₁₀₀. L-lysine α-oxidase has a pronounced antiviral effect (index of neutralization – 3.0 lg CA₁₀₀).

 

The negative results of the experiment with other viruses were unexpected, especially with the West Nile virus, which belongs to the same family as the TBE virus (Flaviviridae). However, the experiment with TBE virus were conducted on SPEV cells, and other viruses on Vero E6 cells. The antiviral effect was due to the peculiarities of the cell lines. At the same time, experiments studying the effects of ribavirin on the virus Tahyna and Dhori performed on Vero E6 cells, have shown its ability to inhibit replication of these viruses[3]. And experiments for study antiviral activity of ribavirin in the cells of the SPEV TBE were relatively negative.

 

5. SUMMARY:

We assessed the toxic effect of L-lysine-α-oxidase from Trichoderma on cell line cells. We found that in both cell lines a marked toxic effect occurs at concentrations of enzyme 0.05 µg/ml and above. The study of the antiviral activity of different concentrations of L-lysine-α-oxidase on the West Nile virus, Tahyna, Sindbis and Dhori on Vero E6 cells.

 

We didn’t find any antiviral effect on the viruses of West Nile, Tahyna, Sindbis and Dhori. Experiments concerning tick-borne encephalitis virus conducted on cell lines SPEV L-lysine-α-oxidase showed that the titer of virus in the control was 8.0 lg CA₁₀₀, and L-lysine-α-oxidase in a concentration of 25 µg/ml, and 12.5 µg/ml was 5.0 lg CA₁₀₀. L-lysine α-oxidase has a pronounced antiviral effect (index of neutralization – 3.0 lg CA₁₀₀).

 

6. CONCLUSION:

Thus, in studies conducted in vitro experiments in models of tick-borne encephalitis virus to date, L-lysine-α-oxidase is the first substance that inhibits the CA of TBE virus in transplantable cell line. The mechanism of its antiviral action is not known. We found the lack of enzyme activity against viruses, Sindbis, West Nile, Tahyna and Dhori.

 

7. CONFLICT OF INTEREST:

The authors confirm that the submitted data does not contain conflict of interests.

 

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Accepted on 16.03.2017           © RJPT All right reserved