BHK-21 Cell Cultures Research Articles

Substitutions accrued on Foot-and-mouth disease virus capsid during propagation in cell culture

Three lineages of serotype O of foot-and-mouth disease virus were passaged serially in BHK-21 cell culture without application of any immune pressure, to study the frequency, nature and location of the substitutions accruing on the virus capsid. The viruses showed unusual stability as only 12 substitutions were observed in 13 different regimens and the majority of the substitutions reverted back to the parental genotype very soon after their appearance. Of the 12 substitutions, a maximum of 8 were found in the VP1 region. Some substitutions (81, 147, 152, 203 and 210 in VP1 and 50 in VP3) were observed at the established antigenic sites suggesting that antigenic diversification can occur in the absence of immune selection. The viruses after serial cytolytic infection of BHK-21 cells, demonstrated an ability to infect the integrin-deficient CHO-K1 cell line suggesting an expansion in their receptor usage potential. Even after 25–50 passages in BHK-21 cell system no histidine to arginine switch was observed at the 56th residue of VP3. Amino acid sequence analysis of 141 Indian field isolates for the residues involved in heparin binding sites suggest the importance of net positive charge in the HS-binding pocket or elsewhere on the capsid for interaction with the alternative receptors and cell culture adaptation rather than acquisition of positive charge at any particular position for all serotype O strains.

Toxicity Test on Taro Leaf Extract (Colocasia Esculenta L. Schoot) as Mouthwash to BHK-21 Fibroblast Cell Culture in Denture Users

Background: One of requirements for a material to be used in dentistry is that the material must be non toxic and non irritant, as well as have biocompatibility properties, not triggering a detrimental effect on the biological environment, both locally and systemically. The first-line tests that must be conducted is in vitro tests, one of which is MTT Assay. Samples used in this research were fibroblast cells (cultured BHK-21 cells) since fibroblasts are the most important and largest components in pulp, periodontal ligament, and gingiva. Purpose: This research aimed to determine toxicity effect of taro leaf extracts (Colocasia esculenta L. Schoot) on BHK-21 fibroblast cells. Methods: This research is an experimental laboratory research using Post Test Only Control Group design. The research treatment are the administration of Taro leaf extract (Colocasia esculenta L. Schoot) with 100%, 80%, 60%, and 40% concentration on BHK-21 fibroblast cell. Results: The fibroblast cell life percentage in 100%, 80%, 60%, and 40% concentration respectively are 106,3%, 50,9%, 36,2%, and 30,1%. The toxicity results are obtained using MTT assay technique after 24 hours. The optical density absorbency values are read by ELISA reader and represent life cell viability. Conclusion: Taro leaf extract (Colocasia esculenta L. Schoot) at a concentration above 80% is considered as non-toxic material to BHK-21 fibroblast cells

Detection and Quantification of Bluetongue Virus Serotype 23 by Evagreen Based Real Time PCR

Bluetongue, an economically important arthropod-borne viral disease of livestock especially sheep. Presently, there are 27 serotypes of bluetongue virus prevalent worldwide. Traditional bluetongue virus typing methods such as virus isolation and serum neutralization tests (SNT) are cumbersome, time-consuming, and many times inconclusive. The other diagnostic methods such as antigen-capture enzyme-linked immunosorbent assay (ELISA), dot immunobinding assay, and immuno-electron microscopy, have been developed. All these techniques have limited sensitivities and thus are unable to detect the virus at a sub-clinical level. Serotype specific RT-PCR and qRT-PCR based amplification of BTV Seg-2 are most reliable, less time consuming and high specificity for serotype typing. In the present study, standardization of EVA-green based qRT-PCR was done for amplification and quantification of BTV-23 serotype from infected BHK-21 cell culture; the results showed high sensitivity as well specificity of realtime PCR for diagnosis of BTV-23.

Protective antibody response of Balb/c mice to Bali rabies virus isolate propagated in BHK-21 cells.

The protective antibody response of Balb/c mice to Bali rabies virus (RABV) in BHK-21 cells was studied. The virus was isolated from a rabid dog and was adapted to replicate in BHK-21 cell culture for seven passages. The BHK-21-adapted Bali RABV (BHK-Bali RABV) was inactivated with binary ethylenimine and 24 mice were immunized twice at 21-days intervals with the inactivated virus and Rabisin® vaccine. Virus replication was detected using indirect immunofluorescence, immunocytochemistry, and western blotting assays. Enzyme-linked immunosorbent assay examination 2 weeks after the first immunization revealed RABV antibody titers that were mostly below the minimum protective level (<0.5 equivalent unit, EU). Antibody titers increased sharply after the second immunization. Antibody titers in serum of mice induced by inactivated BHK-Bali RABV one week after the second immunization were slightly lower (0.8–3.8 EU) than those induced by Rabisin vaccine (0.9–6.3 EU). RABV antibody titers were stable for at least 6 weeks after the second immunization. Both Rabisin vaccine and inactivated BHK-Bali RABV induced neutralizing antibodies with neutralization titers (50% protective dose per ml) of 29.84 for 0.1 ml Rabisin, 211.41 for 0.2 ml Rabisin, 27.41 for 0.1 ml BHK-Bali RABV, and 28.25 for 0.2 ml BHK-Bali RABV. Thus, inactivated BHK-Bali RABV induces a protective immune response in Balb/c mice, but at lower levels compared to induction by Rabisin vaccine.

Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India.

Aim:Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates.Materials and Methods:The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established.Results:The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains.Conclusion:Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

First Report of Isolation of Bluetongue Virus 23 from Culicoides Peregrinus Vector from India

Bluetongue (BT) disease is caused by bluetongue virus (BTV) of Reoviridae family. BTV is transmitted by several species of biting midges (Culicoides sp.) to domestic and wild ruminants. BT is non-contagious but infectious disease. BTV isolate was isolated from Culicoides peregrinus vector from Tamil Nadu state. After appearance of 75% cytopathic effect in BHK-21cell culture, viral nucleic acid (dsRNA) was extracted. RNA-PAGE analysis showed the BTV specific characteristics migration pattern (3: 3: 3: 1) of viral nucleic acid. Furthermore, viral nucleic acid was subjected to cDNA synthesis for ns1 and vp2 gene based RT-PCR analysis. The ns1 gene RT-PCR, specific cytopathic effect and migration pattern in RNA-PAGE confirmed the sample as BTV. The vp2 gene serotype specific RT-PCR identified the isolate as BTV serotype 23. The study suggested that Culicoides peregrinus could be a potential vector which plays important role in BTV transmission in southern India.

The study of the properties of the novel virucidal disinfectant

Prevention measures are crucial in actual production, whereas the outbreak of a disease requires immediate detection and elimination of the source of infection as well as complex veterinary and sanitary measures. Here the critical role is attributed to disinfection, which breaks the epizootic chain due to the elimination of pathogenic microorganisms on the objects under veterinary surveillance and involves the application of various disinfectants. Aim. The study of virucidal properties of the novel disinfectant DZPT-2. Methods. The culture technique was used at the fi rst stage of studies to determine the embryotoxic effect of the preparation on chicken embryos and the cytotoxic effect of the disinfectant on BHK-21 cell culture. The second stage of experiments envisaged the study of virucidal effect of the disinfectant using both the suspension method, test- objects (batiste, wood, glazed tile, metal, glass) and the bioburden. Results. The experiments using the agent of bovine viral diarrhea demonstrated that the disinfectant DZPT-2 in the concentration of 1.0 % of the active ingredient (AI) when exposed for at least 30 min and in the concentration of 1.5 % of AI when exposed for 15– 60 min disinfects all the test-objects, contaminated by the virus, completely. When exposed for up to 15 min, the disinfectant DZPT-2 does not demonstrate its virucidal effect on Newcastle disease virus, but it disinfects all the test-objects, contaminated by the mentioned virus, when used in the concentration of 0.5–1.0 % of AI and exposed for at least 30 min. Conclusions. It was determined that the preparation DZPT-2 demonstrates its virucidal properties in the concentration of 0.5 % of AI (Newcastle disease virus) and 1.0 % of AI (agent of bovine viral diarrhea) when exposed for 30 min. The new disinfectant DZPT-2 is a promising preparation to be used in practical veterinary medicine to prevent and fi ght viral diseases of farm livestock and poultry.

Serotyping and Adaptation of Foot and Mouth Disease Virus in BHK-21 Cell Line towards the Development of Vaccine Candidate

This research work was conducted for isolation, identification and serotyping of Foot and Mouth Disease (FMD) virus from the field isolates of Gouripur Upazila of Mymensingh district, Bangladesh. Samples were collected from infected cattle in virus transportation medium and transported at the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh with maintain proper cool chain duringOctober to April 2012. Inocula were prepared from the collected samples and treated with antibiotics-antimycotics, preserved at -20°C for isolation and serotyping of the virus by antigen detecting ELISA method. After serotyping, the prepared inoculum was inoculated into 24hours confluent BHK-21 cell culture separately and incubated at 37°C for 48hours for development of CPE. Within 48hours of post infection, complete development of cytopathic effects (CPE) in BHK-21 cell culture were observed under inverted microscope which included the rounding and flattening of the cells, breaking down of the intracellular bridges and finally cell death which indicated the presence of FMD virus. Clear BHK-21 cell culture fluid was collected and preserved at -80°C as seed. Of the 20 samples, 15(75%) samples were found positive for FMDV in cell culture of which 13 samples of tongue epithelia (86.67%) and 2 samples of foot tissues (40%) were positive on the basis of cytopathic effects (CPE). Again the prepared inoculum was taken for serotyping the virus by ELISA method. On the basis of the result of ELISA, it was concluded that the serotype of the FMD virus of the collected field isolate was serotype “O”.

Efficacy assessment of an inactivated Tembusu virus vaccine candidate in ducks

Duck Tembusu virus (TMUV) is a recently identified pathogen that causes severe egg drop and neurological disease in domestic duck and goose flocks. The infection has spread across the China mainland since its outbreak in 2010. Effective vaccines are needed to fight the disease. In this work, we describe the development and laboratory assessment of a cell culture-derived, inactivated duck TMUV vaccine. The TMUV-JXSP strain was successfully propagated on a baby hamster kidney cell line (BHK-21), inactivated with beta-propiolactone (BPL) and emulsified with mineral oil. The efficacy of different vaccination schedules was assessed in laying ducks and table ducks using virus challenge experiments. Two doses of vaccine provided efficient protection against the virus challenge to avoid the egg production drop in laying ducks. An ELISA demonstrated that 97% (39/40) of ducks seroconverted on day 21 after one dose of the inactivated vaccine and that significant increases in antibody titers against the virus were induced after the second immunization. For table ducks, a single dose of vaccine immunization resulted in a protection index of 87% and significant reduction of viral loads in tissues. Sterilizing immunity can be attained after second immunization. Our results demonstrate that BHK-21 cell culture is suitable for duck TMUV propagation and that BPL-inactivated TMUV vaccine can provide a high level of protection from virus challenge in laying ducks and table ducks. These data provide a scientific basis for the development of an inactivated vaccine for the prevention of duck TMUV infection.

RT-PCR based detection and adaptation of foot and mouth disease virus serotype “A” in BHK-21 cell line

The economic consequences of an outbreak of Foot and Mouth Disease (FMD) in Bangladesh is devastating. A sensitive, reliable and rapid diagnosis is crucial for the effective control of FMD. The present research was conducted for quick molecular detection and adaptation of FMD virus (FMDV) in BHK-21 (Baby Hamster Kidney) cell line. A two-step RT-PCR method was applied for the detection of the FMDV. Without prior adaption into BHK-21 cell culture, it is difficult to detect FMDV directly from the field samples by RT-PCR method. Samples were collected from the tongue epithelium (N=19) and inter digital tissues (N=4) of the suspected animals in Sujanagar, Bera, Santhia of Pabna district and Kotowali, Trishal of Mymensingh districts of Bangladesh during the year 2014. BHK-21 cell subculture was done from a previously cultured bottle containing BHK-21 cells. Prepared inocula were inoculated into BHK-21 cell culture and incubated at 37ºC for 24 h. After 36 h, cytopathic effects (CPE) were observed in BHK-21 cell line characterized by rounding and flattening of the cells, multinucleated giant cells formation, breaking down of the intracellular bridges and finally cell death indicated the presence of FMDV. Clear infectious BHK-21 cell culture fluid was collected and preserved at -20ºC temperature for virus detection by RT-PCR with serotype specific primers. Viral RNA was extracted from the clear infectious cell culture fluid for cDNA synthesis and used for PCR. Out of 23 samples tested, 3(13.04%) were positive for FMDV serotype A. The findings of this study can be helpful for the selection of vaccine having specific FMDV type, and it may help in controlling FMD in Bangladesh.Progressive Agriculture 27 (1): 64-69, 2016

Genetic Characterization of Foot and Mouth Disease Virus Serotype A Circulating in Eastern Egypt

Currently, three serotypes of Foot-and-Mouth Disease virus (FMDV), A, O and SAT2 were circulating among different provinces of Egypt. The study was planned to isolate and characterize FMDV type A from recent outbreaks in 4 governorates of Egypt (Ismailia, Suez, Sharkia and Port Said) in order to formulate an effective control strategies. A total of 82 pooled tongue epithelia and vesicular fluids samples were collected from cattle and buffaloes in the period of March 2013 to December 2014. Thirty nine and twenty nine pooled samples showed positive to FMDV type A when examined by RT-PCR and ELISA respectively. A total of 48 pooled samples showed FMDV like CPE when isolated and propagated in BHK21 cell culture and then identified by VNT and confirmed by PCR. The samples give a suspected bands at 600 bp when PCR products examined by agarose gel electrophoresis. PCR products of three isolates were selected for nucleotide sequencing representing 2 cattle from Suez, Sharkia and one buffalo from Port Said. When the VP1 sequence of the current strains compared to other strains circulates in the middle east region of Asian and Egyptian topotypes submitted to gene bank showed a close relation to FMDV-A Iran 5-2006 and FMDV-A Iran 53-2005, with identity percentages ranged between 94.9 and 97.1 %, indicating that the FMDV type A virus circulating in the area of study may evolved from the FMDV circulating in Iran (FMDV-A Iran 53-2005).

Genetically modified rabies virus ERA strain is safe and induces long-lasting protective immune response in dogs after oral vaccination

Oral immunization in free-roaming dogs is one of the most practical approaches to prevent rabies for developing countries. The safe, efficient and long-lasting protective oral rabies vaccine for dogs is highly sought. In this study, rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain wild-type (rERA) and a genetically modified type (rERAG333E) containing a mutation from arginine to glutamic acid at residue 333 of glycoprotein (G333E) were generated by reverse genetic. The recombinant virus rERAG333E retained growth properties of similar to the parent strain rERA in BHK-21 cell culture. The G333E mutation showed genetic stability during passage into neuroblastoma cells and in the brains of suckling mice and was significantly reduced the virulence of rERA in mice. rERAG333E was immunogenic in dogs by intramuscular inoculation. Mice orally vaccinated with rERAG333E induced strong and one year longer virus neutralizing antibodies (VNA) to RABV, and were completely protected from challenge with lethal street virus at 12months after immunization. Dogs received oral vaccination with rERAG333E induced strong protective RABV VNA response, which lasted for over 3years, and moderate saliva RABV-specific IgA. Moreover, sizeable booster responses to RABV VNA were induced by a second oral dose 1year after the first dose. These results demonstrated that the genetically modified ERA vaccine strain has the potential to serve as a safe and efficient oral live vaccine against rabies in dogs.

Quality Control of Biotechnological Inputs DetectingMycoplasma

The aim of this work was to study the Polymerase Chain Reaction (PCR) as a tool of quality control of bovine sera and cellular cultures used in the biotechnological industry. A total of 46 samples of bovine sera derived from two slaughterhouses and 33 samples of BHK21 cells derived from two biotechnological industries were evaluated using the primers GPO-3 (sense) and MGSO (antisense). The PCR technique sensibility analysis showed that 280 bp were amplified for the quantities of 50 ng to 0.006 ng of Micoplasma DNA. The primers specificity was confirmed in the test using Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Candida albicans; except by the positive control, none of the samples showed amplification. The presence of Mycoplasma in bovine sera and in the cultures of BHK21 cells showed that 56.5 and 15.2%, respectively, were contaminated. Thus, it was possible to conclude that PCR was a fast and confident technique to detect mycoplasma and that it could be used to control the quality of immunobiological products and inputs, such as sera and cultures of BHK21 cells.

IN VITRO EFFECT OF DIFFERENT INACTIVATORS ON RABIES VIRUS

ascorbic acid, Beta propilacton and binary ethylenamine (BEA) were evaluated as inhibitors of rabies' virus (Evely- Retkitincki-Abeleseths skauis) in optimized BHK21 cell cultures. Ascorbic acid inactivated rabies virus at concentrations of 0.1, 0.5 and1mg/ ml after 22,18 and 14 hours at 37o C respectively, while BPL inactivated the virus after 4,6,8 hours with concentrations of 1/500,1/10000,and1/20000 respectively at 37oC. BEI inactivated the virus after 6 hours when used with 0.3 M. These Inactivators did not affect the antigincity of rabies virus in three prepared experimental vaccine batches. Ascorbic acid appears to be the most preferable one as safe natural noncarcinogenic agent when compared with BPL and BEI.

Genotoxicity test of propolis extract, mineral trioksida aggregat, and calcium hydroxide on fibroblast BHK-21 cell cultures

Background : Health industry has always used natural products as an alternative. Propolis, a natural antibiotic, is a resinous yellow brown or dark brown substance derived from honey bees (Apis mellifera). The main chemical compounds contained in propolis are flavonoids, phenolics and other various aromatic compounds. Flavonoids are well known plant compounds that have antibacterial, antifungal, antiviral, antioxidant and anti-inflammatory proprieties. Propolis is expected to be an alternative used for root canal treatment with lower toxicity compared to calcium hydroxide (Ca(OH) 2 . Over the last decade, a new material, mineral trioxide aggregate (MTA) was developed, and has been used as the gold standard. All materials used in mouth should be biocompatible. The initial level of material biocompatibility evaluation involves toxicity and genotoxicity tests. Purpose : This research is aimed to conduct comparison test of genotoxicity effect of propolis extract, MTA and Ca(OH) 2 on fibroblast BHK-21 cell culture. Methods : This research was conducted with single-cell gel electrophoresis method. Results : The results indicate that propolis extract cannot cause DNA damage, while MTA can cause apoptosis and Ca(OH) 2 can cause neucrosis. Conclusion : It can be concluded that propolis extract has genotoxicity effect lower than MTA and Ca(OH) 2 , but MTA has lower effect on fibroblast BHK-21 cell culture.

Isolation and characterisation of foot-and-mouth disease virus from a captive Indian elephant (Elephas maximus)

Foot-and-mouth disease (FMD) is a highly contagious viral disease of economic importance affecting cloven footed animals as well as more than 70 wild species including elephant. A natural case of FMD in a captive Indian elephant (Elephas maximus) was recorded in the year 2013. The FMD virus (FMDV) was isolated from the clinical sample in BHK-21 cell culture and found to be serotype O. Genetic analysis revealed that the grouping of the isolate from the affected elephant was within Ind2001 lineage. Further the isolate was found to be in close genetic relationships with contemporary isolates sampled from domestic animals in nearby areas. Herewith we provide the direct evidence for virus transmission from the infected domestic animal to captive elephant during the height of FMD outbreak.

A positively charged lysine residue at VP2 131 position allows for the enhanced adaptability of foot-and-mouth disease virus serotype A in BHK-21 cells

Field outbreak strains of foot-and-mouth disease virus (FMDV) infect host cells through certain Arg-Gly-Asp (RGD) dependent integrin family of cellular receptors. In contrast, FMDV adapted in non-host cell cultures are reported to acquire the ability to infect cells via heparin sulphate (HS) or other unidentified cell surface molecules. It has been reported that during the serial passage of FMDV serotype A in BHK-21 cell culture, VP2 E131K (E2131K) substitution was fixed within the heparin sulphate binding site. The fixation of positively charged residue at position VP2 131 of serotype A is considered to associate with the ability to utilise alternative receptor. In this study, an infectious full-length cDNA clone for Indian FMDV vaccine strain A IND 40/2000 was constructed. Through site-directed mutagenesis on the cDNA clone, recombinant virus containing positive charged amino acid residue at position VP2 131 was rescued. The recombinant mutated virus was shown to have specific and strong affinity for HS and demonstrated an enhanced infectivity in BHK-21 cell line. The introduction of lysine residue at VP2 131 position that allows cell culture adaptation of FMDV serotype A could be exploited for the generation of vaccine seed stocks with improved growth properties in BHK-21 cell line.

Preparation and identification of a single-chain variable fragment antibody against Newcastle diseases virus F48E9

This article describes a proposed method for convenient and efficient detection of Newcastle diseases virus (NDV) that uses the fusion of single-chain variable fragment (scFv) and pOPE101 vector. In order to select the single chain variable fragment (scFv) against NDV F48E9, the total RNA was extracted from the spleen of immunized chicken, and then was converted into cDNA via the reverse transcription. The scFv was spliced by using splice-overlap extension polymerase chain reaction (SOE-PCR). The scFv gene was cloned into a pOPE101 vector and expressed in E. coli. Under the optimized conditions, antibody affinity was studied by indirect ELISA. One positive clone was selected by ELISA screening, named ZL.6. Based on the positive clone and the germline sequence, the results of sequence analysis showed that there are more variation in CDR of VH and VL. In addition, BHK21 cell culture was conducted to examine the potential antiviral activity of ZL.6. The experimental result demonstrated that ZL.6 was able to neutralize NDV F48E9 which infected BHK21 cells. So ZL.6 will be proved useful for further characterization of NDV as potential diagnostic tool and therapeutic agent.

English

This study was conducted in five regional states of Ethiopia from January 2011 to March 2012 with the objective of identifying the serotypes of foot and mouth disease by molecular technique in Ethiopia. Epithelial tissue samples were collected from cattle and swine found in the foot and mouth disease outbreak areas of the country and submitted to the National Veterinary Institute, DebreZeit, Ethiopia and World Reference Laboratory for Foot and Mouth Disease, Pirbright, UK. Thus, virus isolation and serotype identification were performed. From a total of 59 samples, cytopathic effect was observed in 43 (72.88%) samples in BHK-21 cell culture. Serotyping of foot and mouth disease viruses were done by applying agarose gel-based RT-PCR at the National Veterinary Institute, and by cell culture ELISA at World Reference Laboratory for Foot and Mouth Disease. Serotype O was recorded throughout the country where outbreaks occurred. Regular investigation of foot and mouth disease outbreaks is important to have more detailed information on the serotypes and topotypes circulating in Ethiopia and for effective vaccine development. Key words: Ethiopia, foot and mouth disease (FMD), serotype.

INVESTIGATION OF PHYSICAL AND CHEMICAL PROPERTIES OF POTATO LEAFROLL VIRUS ISOLATES IN CULTURES OF MAMMALIAN CELLS

The paper presents the research data of physical and chemical properties of potato leaf rolling virus isolates in the PEKV and BHK-21 cell cultures. It was shown that in mammalian cell cultures the isolates of potatoes leaf rolling virus were resistant to the action of lipid dissolving agents, cultural media with pH from 4.0 to 10.0 and were thermoresistant at 50 °C in the absence of divalent ions of magnesium. The virus inactivation temperature point along with the optimum temperature for its reproduction were established.