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Goat milk is commonly used as a “grandmother's drug” in traditional therapy of oral ulcers and lesions caused by Herpes Simplex Virus type 1 (HSV-1) infection without any basic scientific study. The aim of this study is to characterize and quantify the anti-viral effect of goat milk and its components against different viruses and to gain understanding of the mechanism responsible for these effects.
Our experiments reveal that goat milk has a clear antiviral effect, that pasteurization of the milk reduce but does not eliminate this effect. Goat milk had a greater antiviral efficacy than bovine milk. Similarly, the goat milk also inhibited in addition to HSV-1 other viruses (Coxsackievirus A9 as well as the pseudo virus SARS-CoV-2).
Interestingly, the antiviral effects of the goat milk are more pronounced when the virus is pre-incubated with the goat milk casein, than when the casein is added to cell-virus mixture. Preliminary data suggest that the mechanism is non-specific, mediated by a component of the casein fraction. This fraction probably consists of one or several different substances that interact with the membrane or capsid receptors of different viruses, thereby inhibiting cell entry and replication. This putative mechanism should be further studied on COVID- 19 in-vitro study.
Viral diseases are still serious threats to public health all over the world. Herpes Simplex Virus Type-1 (HSV-1) is a large, enveloped virus, which belongs to the Herpes viridian family. Following infection of the virus, a latency period ensues with occasional recurrent reactivations that last for a lifetime. HSV-1 infection may result in a large variety of mild to severe symptoms, including lesions, aphtha's ulcers, vesicles (mainly around the mouth) and even encephalitis and death [
]. Common believes (“grandmother drug”), consider fresh un-pasteurized goat milk straight from the udder to be a natural effective treatment for this oral viral infection. Yet transmission of Brucellosis by the unpasteurized milk still exists [
]. Therefore, the use of goat milk for this purpose deserves establishment of its anti-viral effect on scientific ground.
The increasing use of cytotoxic drugs and immunosuppressant, and along with the growing numbers of Acquired Immune Deficiency Syndrome (AIDS) patients, result in an increase of the number of infections caused by HSV-1. Periodic reactivation in immunocompromised might cause serious and life-threatening complications [
High prevalence of multiple human herpesviruses in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral therapy.
]. Some of the Herpes viruses are sensitive to antiviral drugs. Acyclovir and its derivatives are the drug of choice for treatment of HSV-1 infection. Since viral resistance to acyclovir has been reported [
]. Milk proteins are composed of approximately 80% caseins and 20% whey proteins.: Caseins (αs1-, αs2, β-, and κ-CN) and whey proteins; α-lactalbumin (α-LA), β-lactoglobulin (β-LG), serum albumin (SA) and immunoglobulins [
]. Goat milk proteins are similar to the major cow milk proteins in their general classifications, but they differ from cow milk proteins in their population [
]. We think that the major difference between goat and caw caseins is the different self-assembly of these amphiphilic proteins in aqueous solutions. Specifically, in goat milk, casein resides in small micelles, suspended in the aqueous goat milk, whereas in cow milk β-casein is more soluble and the existing micelles are smaller, less solvated, and less heat stable [
]. Compared to cow milk, goat milk is usually higher in calcium, short and medium chain fatty acids and vitamins A and D but lacks folate and vitamins C and B12 [
This work addresses the antiviral properties of goat milk against HSV-1 and the effect of goat milk and several fractions of milk on different stages of the virus life cycle. First, we studied the influence of crude, pasteurized and fractionated goat milk on HSV-1 epithelial cells, to explore the possibility of cytotoxic activity. Then, we examined the influence of crude, pasteurized and fractionated goat milk on HSV-1 replication, we examined in infected cells under conditions of no cytotoxic effects. The knowledge acquired in this study enables us to propose a working hypothesis for more detailed mechanistic studies.
Materials and methods
Milk samples
Milk samples from normal lactating goats were collected from the commercial goat farm in Shadmot Dvora, Israel, in 3 different occasions. Goat milk was kept at +4 °C after morning milking.
The milk was defatted by centrifugation at 3000×g for 15 min at 4° C followed by precipitation of caseins by adjusting the medium pH to 4.6 using 1 M HCl. Subsequently, the precipitated caseins were collected by centrifugation at 19,000×g for 1 h at 4° C. Then, the crude milk, skim milk, pasteurized milk, and fractionated goat milk (casein, whey) were all kept at – 700C for further studies.
Cell cultures
Inhibition of HSV-1 replication was examined in epithelial cell culture (Human Carcinoma Cell Line – A549) using a plaque assay method [
]. A549 (Human, Caucasian, Lung, Carcinoma (, BHK (Baby Hamster kidney), Hep2 (Human, Caucasian, Larynx, Carcinoma), HuKid (Human Kidney) cells were obtained from American Type Culture Collection (ATCC), VA, USA - CCL185, CRL6282, CCL23 and were also participate in the study.
The cells were grown in 75-cm2 Falcon plastic flasks, in RPMI 1640 medium, containing 15% fetal calf serum, 10 U of mycostatin per ml and 100 μg each of penicillin, streptomycin, and neomycin per ml.
The cultures were incubated at 37° C in a humidified, 5% CO2 atmosphere. The cells were subcultured weekly, and for the present study, cells from the 3rd to 12th passage were used. Plastic Microtest II tissue culture plates (Falcon), with 96 circular flat-bottom wells (7 mm in diameter), were used for the preparation of microcultures. Plastic petri dishes (Falcon, 35 mm in diameter) were used for macrocultures. Freshly trypsinized OMK cells were seeded at a density of 2 × 104 cells (in 0.2 ml) per microculture and 2 × 10′ cells (in 2.0 ml) per macroculture. All cultures were incubated at 37 C in a humidified, 5% CO2 atmosphere for 3–4 days. At this time, the cells approached confluency and were used for infectivity studies.
Culture medium
Eagle Minimum Essential Medium (E. MEM) was prepared according to Eagle H [
]. with Hanks salt base and contained Non-essential Amino Acids (NAA) and 3% Glutamine. M199 was made according to Morgan et al.. Both mediums contained antibiotics (PSMY – Penicillin - 100 U/ml, streptomycin - 100 μg/ml and mycostatin - 200 μg/ml) and 10% or 2% Fetal Bovine Serum (FBS) for uninfected or infected cells, respectively. 3% Carboxy Methyl Cellulose (CMC) (Sigma, C-5678) [
The antiviral effect of milk was measured by plaque formation
The crude goat milk viral inhibitory effect on Coxsackievirus A9 (CVA9) was examined according to 3 protocols of neutralization tests: 1. In-vitro viral neutralization by goat milk prior to infection (infection in presence of goat milk) 2. Cell treatment by goat milk prior to infection (Cell washout prior to infection and infection without presence of goat milk) 3. Treatment of infected cells with goat milk following viral infection of cells (infection without goat milk and treatment of infected cells). We seeded BGM cell culture in 100 × 20 mm agar plates. After creating a uniform cell layer, we incubated CVA9 virus (100 pfu) with and without crude goat milk with a final concentration of 50% in 45 min duration in 37 °C. We infected the cells with the mixture of virus with and without the milk. After 1hr of adsorption in 37oC, we removed the surface layer liquid and added a medium of M199 + 2% FCS which contains 0.4% agar. We incubated the experimental plates in 37oC at 5% CO2 for 72hr until plaques were clearly observed. We fixed the agar plates and stained them in GC solution overnight. The next day, following separation of the agar layer, we calculated the plaques and counted the percentage of plaque inhibition by goat milk in comparison to the number of plaques that were created on the agar plates, without the goat milk treatment.
Scanning electron microscope and phosphotungstic acid (PTA) staining
The HSV virus was concentrated twice for 10 min at 1000 rpm in 4oc followed by ultracentrifugation for 2 h at 6000 rpm in 4oc. The concentrated virus was incubated with and without crude goat milk in a final concentration of 50% for 45 min at 37 oc. Samples from the mixture were stained with PTA in a negative staining technique and a Jeol Transmission electron microscope (TEM) was used to view the samples at a ×40,000 magnification.
Statistical analysis
Data are expressed as mean values ± standard deviation of four or five replicates. Unpaired t-test was used to compare the mean values of different groups. p < 0.05 was considered significant.
Results
This research was conducted in an in-vitro cell culture system. Prior to investigating the antiviral effect of milk and its fractions, we studied the effect of goat milk, pasteurized and fractionated (casein, whey) on A549 cells. The whey fraction was significantly (p < 0.05) toxic to the A549 cells at low concentrations compared to the cell culture medium as seen in Fig. 1 and therefore was not useful for our study. The toxic effect of the raw milk on A549 cells is depicted in Fig. 2. Not only that crude goat milk did not have a cytotoxic effect on the cells, a 20% growth in its presence was observed compared to the culture medium control (p < 0.05) (Fig. 2).
Fig. 1The cytotoxic effect of the whey fraction on A549 cells is apparent from a concentration of 1.25%. SD, standard deviation in five replicates: P < 0.05.
The antiviral effect of goat milk caused 41% ± 11% viral inhibition. Similarly, pasteurized goat milk caused 48% ± 22%, while the most effective inhibition was achieved by the casein fraction 84% ± 12% by treating the free HSV-1 virus with casein from goat milk before cell infection. All these effects were significant P < 0.01 compared to the treatment with the cells (Fig. 3).
Fig. 3The cytotoxic effect of raw goat milk, pasteurized milk, and casein fraction, according to the protocol. The treatment in the tube of the virus was significantly higher (P < 0.01) than the treatment with the cells.
To examine whether the inhibition ability of goat milk is unique, bovine milk was also examined. The results revealed that cow milk was toxic to the A549 cell culture and did not inhibit the virus replication (Fig. 4, Fig. 5). Furthermore, we studied the toxicity of cow milk on other cell cultures – Hep2 and Hukid cells, which showed that cow milk remained toxic to the cells at low concentrations of 1% and 2.5% cow milk. In addition, virus replication inhibition was absent and an increase in the virus replication ability was observed at 1% cow milk concentration, thus had the opposite effect from goat milk. We used a Plaque essay to calculate % inhibition by cow milk compared to plaques formed without cow milk treatment as the control group. The results are described as mean ± SD values of 4 replicates. (Fig. 5).
Fig. 4The cytotoxic effect of cow milk on A549 cells is apparent from a concentration of 2.5% p < 0.05.
The results described above show that goat milk inhibits virus replication. The question remains is to explore the mechanisms involved. Three different mechanisms have been considered:
1)
Viral inhibition by antibodies found in goat milk. This assumption has been ruled out through Immunofluorescence Assays (IFA) [
] for detecting anti HSV-1 antibodies in goat milk which were all negative.
2)
The possibility that antiviral activity of goat milk is caused by high concentration of chloride ions has been ruled out based on our finding of a lower chloride concentration (<50 mEq/l) in goat milk than in human blood (98–110 mEq/l).
3)
Reduced viral infectivity of HSV-1 might have been caused by surface blockade of HSV-1 by goat milk active components. This possibility is compatible with the results described above (Fig. 3) of maximal inhibition by neutralization of the free virus before infection by goat milk. This is strengthened by scanning electron microscopy observations: Specifically, goat milk treatment of the free HSV-1 before infection revealed an aura around the virus, which was remarkably like the “decoration” seen after specific neutralizing antibodies treatment. This observation supports the hypothesis that casein fraction of goat milk interacts with the viral envelope and inhibits replication (Fig. 6).
Fig. 6Scanning electron microscope of A) HSV-1 with medium and B) HSV-1 with crude goat milk, A: Control virus mixture + medium, B: Test virus mixture + medium + crude goat milk. Both images(A&B) show a particle and an infected cell, following PTA staining. The control image (A) depicts the virus without treatment where the PTA crystals can be seen. Conversely, the test image (B) depicts a halo surrounding the cell (treated with goat milk).
This possibility of non-specific antiviral inhibition is further supported by the finding of anti-viral activity of goat milk on different viruses: HSV-1, CVA9 and SARS-CoV-2 Pseudo virus. The effect of crude goat milk as an inhibitor of viruses other than HSV-1 was examined on the CVA9 of the enterovirus family. Crude goat milk appears to have up to 50.8% inhibitory effect on CVA9 as seen in test tube neutralization: Thirty-one plaques following treatment with goat milk compared to 63 plaques in the control (Fig. 7).
Fig. 7In-vitro viral inhibition – test tube neutralization, We calculated the plaques and counted the percentage of plaque inhibition by goat milk in comparison to the number of plaques that were created on the agar plates, without the goat milk treatment. The experimental plates were photographed under light table by digital camera (A) CVA9 virus with crude goat milk, (B) CVA9 only, (C) medium only.
Preliminary effect of goat milk casein on SARS-CoV-2 Pseudo virus was tested in a separate study by measuring fluorescence to determine if the virus penetrates the cell. More than 75% inhibition of the virus entrance was found (Fig. 8).
Fig. 8The antiviral effect of the casein fraction of goat milk on SARS-CoV-2 Pseudo virus.
The observed antiviral effect of goat milk in vitro accords with the common belief of its effect in children. The similar protection of the cells against other viruses indicates that the antiviral effect occurs via a non-specific mechanism and the observation of maximal protection when the virus is first incubated with the milk, indicates that the goat milk protects the cells by covering the virus. This possibility is consistent with the electron microscopic observation of an aura around the free HSV-1 virus before but not after infection, which, is remarkably like the “decoration” seen after specific neutralizing antibodies treatment.
The assumption is that the anti-viral effect of goat milk against all virus studied, is mediated by a kind of a component-peptide casein that assembly to the viral membrane and disrupts the virus ability to attach and penetrate the cell [
]. Therefore, the anti-viral effect was observed in more than one virus strain examined. Then, the influence of crude, pasteurized and fractionated goat milk on HSV-1 replication was examined in infected cells under conditions of no cytotoxic effect. HSV-1 replication inhibition was examined according to three different protocols as described above. Each protocol was used with crude, pasteurized and casein fraction of un-pasteurized goat milk. As expected, the better results was found in the first protocol where the virus was first incubated with the milk and was not found in the cell.
The plaque assays proved that there was probably a second antiviral component in the whey or fat fraction, which we could not test due to technical and toxicity reasons.
We have attempted to develop an accurate, quantitative assay of goat milk viral inhibitory effect, based on the highly sensitive molecular assay Real-Time PCR (TaqMan), which has a wide dynamic range. Since the TaqMan results are presented on a logarithmic scale, and the standard deviation is more than ×2, we concluded that this assay was not suitable for detecting inhibition by 2–10 folds as the plaque assay can, and therefore was not used.
In order to examine whether the inhibition of goat milk is unique, bovine milk was also examined. As our results show, bovine milk did not inhibit the virus replication, at non-toxic concentrations [
]. The difference between goat milk and bovine milk may be due to the antiviral potency of goat milk that results from the self-assembly of its caseins. However, there is no study related to their potential antiviral effects in the literature.
The effect of crude goat milk as an inhibitor of viruses other than HSV-1 was examined on the CVA9 of the enterovirus family which presented an inhibitory effect on the virus propagation in cell culture. Viral inhibition of CVA9 was chosen for comparison to HSV-1 because it acts as a model for viruses that are non-enveloped and have a single strand of RNA. This small RNA virus, a member of the Enterovirus group in the Picornaviridae family, is very similar to the Coxsackievirus A16, which causes mouth ulcers (the “foot and mouth disease”) in children.
The results described above revealed a neutralization ability of different viruses by goat milk, with no cytotoxic effect with the same mechanism. Reduced viral infectivity of HSV-1 and other viruses is caused by surface blockade of the virus by goat milk active components as described above, which probably led to inhibition of replication.
In conclusion, goat milk appeared to be an efficient antiviral agent. Thus goat milk is not merely a “grandmother drug”, this activity is due to the casein in the goat milk and pasteurization reduces this effect. In view of the non-specific nature of the antiviral effect of the goat milk, and the finding of in vitro effect on SARS-CoV-2 Pseudo virus, it is of interest, to study the possibility effect of casein of goat milk on COVID-19 [
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
Thanks to Boaz Rubin for his helpful assistance.
References
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Kimberlin D.W.
Prober C.G.
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Chapter 32 Pathogenesis and disease Unique biologic properties of HSV that influence pathogenesis.
High prevalence of multiple human herpesviruses in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral therapy.
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