BEE VENOM FOR PARVOVIRUS AND BOCAVIRUS
What is Parvovirus B19?
It is also called B19V, which is a single-stranded virus belonging to the family Parvoviridae. The type B19 only affects humans and the symptoms differ according to overall health and age. It has been found that 2 out of 10 people are infected with this virus and it is not even necessary that they would have any apparent symptoms.
What are the Symptoms of Parvovirus?
It most commonly leads to fifth disease – mild rashes (observed mostly in children). Common symptoms of this virus are:
How Is It Transmitted?
Usually, it spreads through the respiratory secretions of an infected person like nasal mucus, sputum, and saliva. It can also spread through infected blood products.
How Is It Diagnosed?
Normally a blood test sample is obtained from a possible subject to determine if the virus has been activated or not.
How It Can Be Prevented?
Currently, there is no allopathic medicine or vaccine available that can help prevent this virus. But the chances of being affected or affecting others, with parvovirus B19, can be contained through the following measures:
Now, let’s have a look at the Bocavirus.
What is Bocavirus?
It is also called human bocavirus or HBoV, which is a non-enveloped and a single-stranded virus belonging to the family Parvoviridae. It is a newly discovered virus that affects children mostly. HBoV is also termed as 'an emerging viral pathogen', due to the reason that it is associated with other infections. It has to be yet proven that, if this virus manifests on its own or is caused due to other viruses or exists in parallel with other viruses.
What are the Symptoms of Bocavirus?
The following are the signs and symptoms associated with Bocavirus.
Children having these symptoms can get very ill and often require hospitalization.
How Is It Transmitted?
According to the scientific findings, it can be declared that bocavirus is mostly spread through respiratory secretions. Traces of this virus can be detected from blood samples and stools. It is most commonly found in young children, who often remain sick.
How Is It Diagnosed?
Normally a blood test sample is obtained from a possible subject to determine if the virus has been activated or not.
How It Can Be Prevented?
Bocavirus strains are not solely responsible for the symptoms stated above. And there is no vaccine currently available that helps in controlling this virus. General hygiene and protection are recommended to prevent this virus from spreading and being transmitted.
How Can Parvovirus and Bocavirus Be Treated?
Conventional medicines that are being used to treat symptoms of these viruses are not effective or have some sort of adverse effects. Researchers are currently making use of natural therapies like honey bee venom to treat Parvovirus and Bocavirus.
Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 CellsAbstract
B19 and HBoV are human pathogens. Both VP1u of HBOV and B19 demonstrated an enzymatic activity similar to that of sPLA2 and were found to take part in the pathogenesis of the lower respiratory tract illness. But it has not been addressed that how VP1u from HBoV and B19 affects the tight junction.
This clinical study is based on the idea of determining how VP1u from HBoV and B19 infects the tight junction of airway epithelial A549 cells. This process was determined by examining the effectiveness of immunoblotting analysis, transepithelial electrical resistance, and phospholipase A2 activity.
Clinical results show that transepithelial electrical resistance is decreased in the A549 cells through a treatment done by TNF-alpha (10ng), 2 doses of HBoV-VP1u and B19-VP1u (4000ng and 400ng), respectively and bee venom(10ng) of the control group.
An increase in caludin-1 and a decrease in occludin have been detected in the A549 cells after treating them with TNF alpha or combined doses of HBoV-VP1u than that of the control group. Adding further, a significant decrease in NA+/K+ ATPase has been observed in the A549 cells after treating them with TNF-alpha and a high dose of B19-VP1u combined with both doses of HBoV-VP1u than that of the control group.
These findings establish that not B19 VP1u, but HBoV VP1u plays an important role in disrupting the tight junction present in the airway tract.
Introduction
Epithelium present in the respiratory system along with other organs acts as a barrier between the elements of the external environment and the underlying tissues. The epithelial cells become polarized after the formation of specific cell-cell junctions, they are known as apical junction complexes like TJs (tight junctions) and AJs (adherent junctions). Tight junctions are very close cell-cell connections that result in paired strands that seal the empty gaps between different neighboring cells and help control the interactive permeability of the small molecules.
Tight junctions also act as a barrier against potential pathogens and various foreign particles and this helps in avoiding tissue injury and infection. TJs are made up of a protein complex that contains ZO (zona occludens), occluding and tetraspanin claudins. Due to its important role, the epithelium present in the respiratory tract is highly vulnerable to different molecules having proteolytic activity like sPLA2. Making use of A549 cells, quite known in the Vitro model of tight junctions, this clinical study examines how HBoV-VP1u and B19-VP1u affect the tight junction modules.
Method
381-bp DNA fragment including nucleotides 3056-3442 of HBoV strain, obtained from the Center for Disease Control, Taiwan, was augmented with the help of a polymerized chain reaction. Primers used in this reaction were 5′-GCGTCGACTGAGGTTCCTG G-3′ (reverse) and 5′-GCGAATTCATGCCTCCAATTAAG-3′ (forward). They were subjected to an EcoRI site at 5’ end and Sal I site at the 3’ end to be cloned into pET-32a.
HBoV-VP1u and B19-VP1u proteins were considered for the sPLA2 activity with the help of a colorimetric assay kit obtained from Cayman Chemical. This process was performed according to the instructions provided by the manufacturer, through dynamic colorimetric measurements (optical density was maintained at 414mm) carried out after every 10 minutes. Results obtained are interpreted as micromoles per minute per milliliter.
Epithelial cells (A549) were acquired from the ATCC (American type culture collection) and were developed in DMEM (Dulbecco's Modified Eagle Medium) and were supplemented with a 10% (FBS) fetal bovine serum at 37 degrees centigrade and 5% carbon dioxide incubator. To conduct a clinical experiment, epithelial cells were seeded and grown fully in dishes (100cm2). The effect of HBoV-VP1u and B19-VP1u at different doses on the TJs of the epithelial cells was carried out.
To examine the epithelial barrier integrity, epithelial cells were first plated and then cultured for some time of 48hours and after that exposed to the TNF-alpha (10ng/ml), bee venom PLA2 in three different doses (4000ng/ml, 400ng/ml and 1ug/ml) recombinant HBoV-VP1u or B19-VP1u proteins for 1 day, respectively. To perform the clinical study, epithelial cells were seeded at a very high density on the Snapwell inserts from Costar, USA and kept at 37 degrees centigrade in 5% carbon dioxide and 95% air atmosphere. The TEER values were acquired after subtracting the blank filter resistance from every reading.
The cultured cells were gathered through centrifugation at 800g for 5 minutes and then washed with freezing PBS for two times. Collected cells were again suspended in 600ul of a PRO-PREP buffer obtained from iNtRON Biotech, Korea and kept on ice for an hour. These samples were then again centrifuged at 13,000rpm for five minutes at 4 degrees centigrade. The supernatant labeled as a crude extract was shifted into a fresh Eppendorf and kept at minus 20 degrees centigrade. The protein concentration of obtained samples was analyzed through Bradford's assay with the help of a Japanese spectrophotometer at 595nm and BSA was maintained as a standard.
Protein samples were divided into 10% or 12.5% of the SDS PAGE and then electrophoretically shifted to a nitrocellulose membrane obtained from Amersham Biosciences, USA. Statistical analyses were managed with the help of GraphPad Prism 5 software through one way analysis of the variance followed by the Tukey multiple comparison test. The data gathered was verified thrice through independent experiments.
Result
Both HBoV-VP1u and B19-VP1u play an important role in the pathogenesis and infectivity of different diseases, due to their PLA2 activities. This study was conducted to confirm that HBoV-VP1u and B19-VP1u proteins demonstrated PLA2 activity by purifying and constructing recombinant HBoV-VP1u and B19-VP1u proteins.
Under positive control, bee venom PLA2 displayed PLA2 activity having a value of 0.235µmol/min/mL, while no sort of PLA2 enzymatic activity was recorded in the TNF-alpha. A Significant bvPLA2 activity was seen in HBoV-VP1u and B19-VP1u proteins at 0.065µmol/min/mL and 0.035µmol/min/mL, respectively. And a more significant increase in sPLA2 activity was detected in a high dosage of HBoV-VP1u and B19-VP1u proteins having an activity of 1.362µmol/min/mL and 0.765µmol/min/mL, respectively.
Effects of HBoV-VP1u and B19-VP1u on the TJs in epithelial cells was further investigated by analyzing claudin-1 and occludin – two crucial indicators of the TJs through immunoblotting analysis. Results acquired showed that caludin-1 increased in the epithelial cells after treating them with TNF-alpha and both doses of the HBoV-VP1u when compared with the control. On the other hand, claudin-1 did not show any variation in the epithelial cells after being treated with BVPLA2 and both of the doses of B10-VP1u. Similar results were experienced in the expression of occludin. The protein level of occludin in the epithelial cells was further decreased when subjected to TNF-alpha or the HBoV-VP1u treatment when compared to the control, while no sort of variation was observed in the epithelial cells after being treated with BVPLA2 or B19-VP1u.
The expression of Na+/K+ ATPase has a close relationship with the polarity and cell tight junction of the epithelial cells. Hence, this clinical study examined the expression of Na+/K+ ATPase in the epithelial cells after being treated with the recombinant B19-VP1u and HBoV-VP1u proteins through immunoblotting analysis. A significant decrease of the expression Na+/K+ ATPase in epithelial cells was detected after being treated with TNF-alpha, both doses of HBoV-VP1u and 4000ug/ml of B19-VP1u. On the other hand, Na+/K+ ATPase expression in the epithelial cells did not record a considerable variation after being treated with BVPLA2 and 400ug/ml of B19-VP1u when compared to the value of the controls.
Discussion
sPLA2 consists of a complex family of different enzymes that are spread throughout the epidermis. It has been found that these enzymes help hydrolyze glycerophospholipid ester bond at the sn-2 position to create lysophospholipid and free fatty acid. The mechanism of sPLA2 is involved in different important epidermal processes. And the most thoroughly investigated action of sPLA2 is its role in inflammation. It is to be noted that there are different types of sPLA2s in the lung tissues that yield numerous eicosanoids that lead to pulmonary edema.
According to the results of this clinical study, bee venom PLA2, with both doses of HBoV-VP1u and B19-VP1u demonstrated enzymatic action of sPLA2 and reduced the transepithelial electrical resistance, indicating that there is increased permeability of the TJs in the epithelial cells. Though a high dose of B19-VP1u could help reduce Na+/K+ ATPase expression, only HBoV-VP1u and TNF-alpha increased expression of accludin-1 and decreased expressions of the occludin and Na+/K+ ATPase. According to these findings it can be declared that HBoV-VP1u instead of B19-VP1u impacted disruption of TJs in the epithelial cells and this process is not associated with sPLA2 enzymatic activities.
Conclusion
As we know that HBoV and B19 are closely related to injury and infection caused in the air tract, but the pathogenic mechanism of these viruses remains unknown. The clinical study discussed here clearly demonstrated that the HBoV-VP1u demonstrated a disruptive effect on the TJs in the epithelial cells while B19-VP1u portrayed a low capacity to disrupt the TJs. According to these findings, it is certainly believed that HBoV-VP1u and B19-VP1u have different roles in treating respiratory infections. Hence, honey bee venom can be used to treat Parvovirus and Bocavirus without any sort of complications.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182432/
It is also called B19V, which is a single-stranded virus belonging to the family Parvoviridae. The type B19 only affects humans and the symptoms differ according to overall health and age. It has been found that 2 out of 10 people are infected with this virus and it is not even necessary that they would have any apparent symptoms.
What are the Symptoms of Parvovirus?
It most commonly leads to fifth disease – mild rashes (observed mostly in children). Common symptoms of this virus are:
- Severe anemia
- Swollen or painful joints
- Redness of skin
How Is It Transmitted?
Usually, it spreads through the respiratory secretions of an infected person like nasal mucus, sputum, and saliva. It can also spread through infected blood products.
How Is It Diagnosed?
Normally a blood test sample is obtained from a possible subject to determine if the virus has been activated or not.
How It Can Be Prevented?
Currently, there is no allopathic medicine or vaccine available that can help prevent this virus. But the chances of being affected or affecting others, with parvovirus B19, can be contained through the following measures:
- Covering nose and mouth completely while sneezing or coughing
- Washing hands thoroughly with a disinfectant soap and water
- Resting and staying at home while sick
- Avoiding close contact with those who are suffering from this virus
Now, let’s have a look at the Bocavirus.
What is Bocavirus?
It is also called human bocavirus or HBoV, which is a non-enveloped and a single-stranded virus belonging to the family Parvoviridae. It is a newly discovered virus that affects children mostly. HBoV is also termed as 'an emerging viral pathogen', due to the reason that it is associated with other infections. It has to be yet proven that, if this virus manifests on its own or is caused due to other viruses or exists in parallel with other viruses.
What are the Symptoms of Bocavirus?
The following are the signs and symptoms associated with Bocavirus.
- High fever
- Persistent coughing
- Acute respiratory tract infection
- Rhinorrhea
- Cyanosis
- Vomiting
- Diarrhea
Children having these symptoms can get very ill and often require hospitalization.
How Is It Transmitted?
According to the scientific findings, it can be declared that bocavirus is mostly spread through respiratory secretions. Traces of this virus can be detected from blood samples and stools. It is most commonly found in young children, who often remain sick.
How Is It Diagnosed?
Normally a blood test sample is obtained from a possible subject to determine if the virus has been activated or not.
How It Can Be Prevented?
Bocavirus strains are not solely responsible for the symptoms stated above. And there is no vaccine currently available that helps in controlling this virus. General hygiene and protection are recommended to prevent this virus from spreading and being transmitted.
How Can Parvovirus and Bocavirus Be Treated?
Conventional medicines that are being used to treat symptoms of these viruses are not effective or have some sort of adverse effects. Researchers are currently making use of natural therapies like honey bee venom to treat Parvovirus and Bocavirus.
Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 CellsAbstract
B19 and HBoV are human pathogens. Both VP1u of HBOV and B19 demonstrated an enzymatic activity similar to that of sPLA2 and were found to take part in the pathogenesis of the lower respiratory tract illness. But it has not been addressed that how VP1u from HBoV and B19 affects the tight junction.
This clinical study is based on the idea of determining how VP1u from HBoV and B19 infects the tight junction of airway epithelial A549 cells. This process was determined by examining the effectiveness of immunoblotting analysis, transepithelial electrical resistance, and phospholipase A2 activity.
Clinical results show that transepithelial electrical resistance is decreased in the A549 cells through a treatment done by TNF-alpha (10ng), 2 doses of HBoV-VP1u and B19-VP1u (4000ng and 400ng), respectively and bee venom(10ng) of the control group.
An increase in caludin-1 and a decrease in occludin have been detected in the A549 cells after treating them with TNF alpha or combined doses of HBoV-VP1u than that of the control group. Adding further, a significant decrease in NA+/K+ ATPase has been observed in the A549 cells after treating them with TNF-alpha and a high dose of B19-VP1u combined with both doses of HBoV-VP1u than that of the control group.
These findings establish that not B19 VP1u, but HBoV VP1u plays an important role in disrupting the tight junction present in the airway tract.
Introduction
Epithelium present in the respiratory system along with other organs acts as a barrier between the elements of the external environment and the underlying tissues. The epithelial cells become polarized after the formation of specific cell-cell junctions, they are known as apical junction complexes like TJs (tight junctions) and AJs (adherent junctions). Tight junctions are very close cell-cell connections that result in paired strands that seal the empty gaps between different neighboring cells and help control the interactive permeability of the small molecules.
Tight junctions also act as a barrier against potential pathogens and various foreign particles and this helps in avoiding tissue injury and infection. TJs are made up of a protein complex that contains ZO (zona occludens), occluding and tetraspanin claudins. Due to its important role, the epithelium present in the respiratory tract is highly vulnerable to different molecules having proteolytic activity like sPLA2. Making use of A549 cells, quite known in the Vitro model of tight junctions, this clinical study examines how HBoV-VP1u and B19-VP1u affect the tight junction modules.
Method
381-bp DNA fragment including nucleotides 3056-3442 of HBoV strain, obtained from the Center for Disease Control, Taiwan, was augmented with the help of a polymerized chain reaction. Primers used in this reaction were 5′-GCGTCGACTGAGGTTCCTG G-3′ (reverse) and 5′-GCGAATTCATGCCTCCAATTAAG-3′ (forward). They were subjected to an EcoRI site at 5’ end and Sal I site at the 3’ end to be cloned into pET-32a.
HBoV-VP1u and B19-VP1u proteins were considered for the sPLA2 activity with the help of a colorimetric assay kit obtained from Cayman Chemical. This process was performed according to the instructions provided by the manufacturer, through dynamic colorimetric measurements (optical density was maintained at 414mm) carried out after every 10 minutes. Results obtained are interpreted as micromoles per minute per milliliter.
Epithelial cells (A549) were acquired from the ATCC (American type culture collection) and were developed in DMEM (Dulbecco's Modified Eagle Medium) and were supplemented with a 10% (FBS) fetal bovine serum at 37 degrees centigrade and 5% carbon dioxide incubator. To conduct a clinical experiment, epithelial cells were seeded and grown fully in dishes (100cm2). The effect of HBoV-VP1u and B19-VP1u at different doses on the TJs of the epithelial cells was carried out.
To examine the epithelial barrier integrity, epithelial cells were first plated and then cultured for some time of 48hours and after that exposed to the TNF-alpha (10ng/ml), bee venom PLA2 in three different doses (4000ng/ml, 400ng/ml and 1ug/ml) recombinant HBoV-VP1u or B19-VP1u proteins for 1 day, respectively. To perform the clinical study, epithelial cells were seeded at a very high density on the Snapwell inserts from Costar, USA and kept at 37 degrees centigrade in 5% carbon dioxide and 95% air atmosphere. The TEER values were acquired after subtracting the blank filter resistance from every reading.
The cultured cells were gathered through centrifugation at 800g for 5 minutes and then washed with freezing PBS for two times. Collected cells were again suspended in 600ul of a PRO-PREP buffer obtained from iNtRON Biotech, Korea and kept on ice for an hour. These samples were then again centrifuged at 13,000rpm for five minutes at 4 degrees centigrade. The supernatant labeled as a crude extract was shifted into a fresh Eppendorf and kept at minus 20 degrees centigrade. The protein concentration of obtained samples was analyzed through Bradford's assay with the help of a Japanese spectrophotometer at 595nm and BSA was maintained as a standard.
Protein samples were divided into 10% or 12.5% of the SDS PAGE and then electrophoretically shifted to a nitrocellulose membrane obtained from Amersham Biosciences, USA. Statistical analyses were managed with the help of GraphPad Prism 5 software through one way analysis of the variance followed by the Tukey multiple comparison test. The data gathered was verified thrice through independent experiments.
Result
Both HBoV-VP1u and B19-VP1u play an important role in the pathogenesis and infectivity of different diseases, due to their PLA2 activities. This study was conducted to confirm that HBoV-VP1u and B19-VP1u proteins demonstrated PLA2 activity by purifying and constructing recombinant HBoV-VP1u and B19-VP1u proteins.
Under positive control, bee venom PLA2 displayed PLA2 activity having a value of 0.235µmol/min/mL, while no sort of PLA2 enzymatic activity was recorded in the TNF-alpha. A Significant bvPLA2 activity was seen in HBoV-VP1u and B19-VP1u proteins at 0.065µmol/min/mL and 0.035µmol/min/mL, respectively. And a more significant increase in sPLA2 activity was detected in a high dosage of HBoV-VP1u and B19-VP1u proteins having an activity of 1.362µmol/min/mL and 0.765µmol/min/mL, respectively.
Effects of HBoV-VP1u and B19-VP1u on the TJs in epithelial cells was further investigated by analyzing claudin-1 and occludin – two crucial indicators of the TJs through immunoblotting analysis. Results acquired showed that caludin-1 increased in the epithelial cells after treating them with TNF-alpha and both doses of the HBoV-VP1u when compared with the control. On the other hand, claudin-1 did not show any variation in the epithelial cells after being treated with BVPLA2 and both of the doses of B10-VP1u. Similar results were experienced in the expression of occludin. The protein level of occludin in the epithelial cells was further decreased when subjected to TNF-alpha or the HBoV-VP1u treatment when compared to the control, while no sort of variation was observed in the epithelial cells after being treated with BVPLA2 or B19-VP1u.
The expression of Na+/K+ ATPase has a close relationship with the polarity and cell tight junction of the epithelial cells. Hence, this clinical study examined the expression of Na+/K+ ATPase in the epithelial cells after being treated with the recombinant B19-VP1u and HBoV-VP1u proteins through immunoblotting analysis. A significant decrease of the expression Na+/K+ ATPase in epithelial cells was detected after being treated with TNF-alpha, both doses of HBoV-VP1u and 4000ug/ml of B19-VP1u. On the other hand, Na+/K+ ATPase expression in the epithelial cells did not record a considerable variation after being treated with BVPLA2 and 400ug/ml of B19-VP1u when compared to the value of the controls.
Discussion
sPLA2 consists of a complex family of different enzymes that are spread throughout the epidermis. It has been found that these enzymes help hydrolyze glycerophospholipid ester bond at the sn-2 position to create lysophospholipid and free fatty acid. The mechanism of sPLA2 is involved in different important epidermal processes. And the most thoroughly investigated action of sPLA2 is its role in inflammation. It is to be noted that there are different types of sPLA2s in the lung tissues that yield numerous eicosanoids that lead to pulmonary edema.
According to the results of this clinical study, bee venom PLA2, with both doses of HBoV-VP1u and B19-VP1u demonstrated enzymatic action of sPLA2 and reduced the transepithelial electrical resistance, indicating that there is increased permeability of the TJs in the epithelial cells. Though a high dose of B19-VP1u could help reduce Na+/K+ ATPase expression, only HBoV-VP1u and TNF-alpha increased expression of accludin-1 and decreased expressions of the occludin and Na+/K+ ATPase. According to these findings it can be declared that HBoV-VP1u instead of B19-VP1u impacted disruption of TJs in the epithelial cells and this process is not associated with sPLA2 enzymatic activities.
Conclusion
As we know that HBoV and B19 are closely related to injury and infection caused in the air tract, but the pathogenic mechanism of these viruses remains unknown. The clinical study discussed here clearly demonstrated that the HBoV-VP1u demonstrated a disruptive effect on the TJs in the epithelial cells while B19-VP1u portrayed a low capacity to disrupt the TJs. According to these findings, it is certainly believed that HBoV-VP1u and B19-VP1u have different roles in treating respiratory infections. Hence, honey bee venom can be used to treat Parvovirus and Bocavirus without any sort of complications.
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182432/