Ultrasonic Applications in MonkeyPox Virus
Ultrasonication is an important processing method for the isolation of the monkeypox virus (MPXV) from analytical samples, for virus DNA fragmentation as well as in monkeypox vaccine manufacturing. For sample preparation before diagnostics and analysis (PCR, ELISA etc.) ultrasonication is used to lyse cells in order to release the monkeypox virus from the cell interior and / or to fragment DNA. In vaccine production, the applications range from virus particle / DNA preparation, encapsulation in drug carriers and formulating the inocula.
Below you can find detailed information about ultrasonic monkeypox virus sample preparation as well as ultrasonically assisted MPXV vaccine production.
What you will find on this page:
- Ultrasonic Lysis for Monkeypox Virus Extraction
- Ultrasonic Fragmentation of Monkeypox Virus DNA
- Ultrasonic Applications in MPXV Vaccine Production
Ultrasonic Lysis and DNA Fragmentation before Polymerase Chain Reaction (PCR)
A monkeypox virus infection is detected by nucleic acid amplification testing (NAAT), using real-time or conventional polymerase chain reaction (PCR), for detection of unique sequences of viral DNA. The sample (e.g. from nasopharyngeal swap or skin biopsies) contains the virus in cells.
For analysis, the virus must be released from the cells ans the viral DNA must be fragmented for PCR.
Ultrasonic cell disruption / lysis is a reliable and efficient method to isolate viruses from cell samples and thereby a preferable technique over chemical agents, such as lysozyme, proteinase K and different detergents which are alternatively used to achieve cell lysis and release of DNA.
However the use of such chemical reagents requires time-consuming sample preparation in several steps before PCR analysis to prevent inhibition of the PCR reaction. Since PCR inhibitors affect the efficiency of amplification, small variations of the amount of inhibitors that are not removed can lead to large variations in the PCR product amplification (Diaco, 1995).
The advantage of ultrasonication for lysis is that complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation are desirable. (cf. Fykse et al., 2003)
Ultrasonic DNA fragmentation is simple and reliable as is produces DNA fragments tunable in length (basepairs, bp). Using ultrasound, DNA can be effectively fragmented to the targeted DNA length. Precise control over ultrasonic parameters and sophisticated cooling options prevent DNA degradation.
Read more about ultrasonic DNA fragmentation!
Vaccines used against the monkeypox virus are currently live virus vaccines. Newly developed vaccines might use other platforms such as DNA-based The currently contains a modified Vaccinia Ankara viurs, an attenuated, non-replicating orthopoxvirus. It also contains Tris (tris-aminomethane) and sodium chloride. The vaccine may also contain small amounts of DNA and protein from the Chicken Embryo Fibroblast cells, which are used to grow the vaccine virus, benzonase and antibiotic compounds such as gentamicin and ciprofloxacin.
Ultrasonic homogenization is used in the production of live-attenuated virus vaccines, DNA vaccines, multi-valent DNA cocktails, mRNA vaccines, recombinant protein vaccines, virus-like particle vaccines, etc.
- dispersion of virus particles and agents
- inactivation of viruses
- drug carrier formulation (NLC, SLN)
- dissolving of agents
- preparation of adjuvants
- degassing / de-aeration
Protocol for Ultrasonic Isolation of the Monkeypox Virus
The research team of Stittelaar (2005) used ultrasonic lysis to release the monkeypox virus from the growth host cell culture as well as from cells obtained via throat swaps from infected primates.
Monkeypox Vaccine Preparation:
Monkeypox viruses were grown on specific-pathogen-free chicken embryo fibroblast cells. Following incubation for 1 to 2 days, the virus-cell suspension was harvested by one round of freeze-thawing and then concentrated by centrifugation. The pellet was resuspended and subjected to several rounds of ultrasonic homogenization.
Monkeypox Virus Isolation from Vaccinated Macaques:
Samples were freeze-thawed three times and sonicated in an ultrasonic CupHorn. Two dilutions (1:10 and 1:100) in transport medium supplemented with 1% fetal bovine serum were used to inoculate Vero cell monolayers in six-well plates. After 1 h of incubation at 37°C, the inocula were removed and replaced with culture medium supplemented with 1% fetal bovine serum. The monolayers were cultured for 5 days at 37°C and stained with a crystal violet solution.
(cf. Stittelaar et al., 2005)
Ultrasonicators for Virus Analysis and Vaccine Production
Hielscher Ultrasonics broad product portfolio offers the ideal ultrasonicator for research and analytical laboratories as well as for industrial vaccine manufacturing.
Hielscher Ultrasonics is specialised in the design, manufacturing and distribution of high-performance ultrasonicators and sono-bioreactors for the use in research and analytical laboratories as well as for implementation in industrial vaccine production (e.g. vaccines, APIs).
Sonication can be applied to open vessels, closed continuously-stirred reactors and continuous flow-through reactors. All parts of the ultrasonic systems, which get in contact with the liquid medium, are made from stainless steel, titanium, or glass. Autoclavable parts and sanitary fittings ensure the production under pharma-grade conditions.
Automatic Data Recording: Intelligent software records the parameters of the sonication process automatically on the integrated SD memory card. The precise control of all process parameters ensure the reproducibility, standardisation of production, and facilitates the fulfilment of pharmaceutical product safety standards.
Hielscher Ultrasonics’ ultrasonic processors are highly reliable and can be precisely controlled. All industrial ultrasonicators can adjusted to deliver the full range from lower to very high amplitudes. The robustness of Hielscher’s ultrasonic systems allows for 24/7 operation under heavy duty and in demanding environments.
The table below gives you an indication of the approximate processing capacity of our ultrasonicators:
|Batch Volume||Flow Rate||Recommended Devices|
|multi-well / microtiter plates||n/a||UIP400MTP|
|1 to 500mL||10 to 200mL/min||UP100H|
|10 to 2000mL||20 to 400mL/min||UP200Ht, UP400St|
|0.1 to 20L||0.2 to 4L/min||UIP2000hdT|
|10 to 100L||2 to 10L/min||UIP4000hdT|
|n/a||10 to 100L/min||UIP16000|
|n/a||larger||cluster of UIP16000|
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Literature / References
- Stittelaar, Koert; Amerongen, Geert; Kondova, Ivanela; Kuiken, Thijs; Lavieren, Rob; Pistoor, Frank; Niesters, Hubert; Doornum, Gerard; Van der Zeijst, Bernard; Mateo, Luis; Chaplin, Paul; Osterhaus, Albert (2005): Modified Vaccinia Virus Ankara Protects Macaques against Respiratory Challenge with Monkeypox Virus. Journal of Virology 79, 2005. 7845-51.
- Shah Purvin, Parameswara Rao Vuddanda, Sanjay Kumar Singh, Achint Jain, and Sanjay Singh (2014): Pharmacokinetic and Tissue Distribution Study of Solid Lipid Nanoparticles of Zidov in Rats. Journal of Nanotechnology, Volume 2014.
- J. Robin Harris, Andrei Soliakova, Richard J. Lewis, Frank Depoix, Allan Watkinson, Jeremy H. Lakeya (2012): Alhydrogel® adjuvant, ultrasonic dispersion and protein binding: a TEM and analytical study. Micron Volume 43, Issues 2–3, February 2012, 192-200.
- Doron Melamed, Gabriel Leitner, E. Dan Heller (1991): A Vaccine against Avian Colibacillosis Based on Ultrasonic Inactivation of Escherichia coli. Avian Diseases Vol. 35, No. 1 (Jan. – Mar., 1991), 17-22.
- Huang C-F, Wu T-C, Wu C-C, Lee C-C, Lo W-T, Hwang K-S, Hsu M-L, Peng H-J. (2011): Sublingual vaccination with sonicated Salmonella proteins and mucosal adjuvant induces mucosal and systemic immunity and protects mice from lethal enteritis. APMIS 119, 2011. 468–78.
Facts Worth Knowing
Monkeypox (MPV, MPXV, or hMPXV) is a species of double-stranded DNA virus, which causes a viral zoonotic disease. This disease is known as monkeypox infection and can occur in humans and animals. It belongs to the genus Orthopoxvirus in the family Poxviridae. Monkeypox virus is one of the human orthopoxvirus along with variola (VARV), cowpox (CPX), and vaccinia (VACV) viruses. Orthopoxviruses are characterized by large brick-shaped virus particles, containing a double-stranded DNA genome of approximately 200,000 bp.
ELISA stands for Enzyme-Linked ImmunoSorbent Assay and is a labeled immunoassay that is considered the gold standard of immunoassays. ELISA is an immunological test, valued for its high diagnostic sensitivity, is used to detect and quantify molecules, including antibodies, antigens, proteins, glycoproteins, and hormones. The principle of ELISA is based on an antigen-antibody interaction. By this antigen-antibody interaction, the specific antibodies bind to its target antigen. Only when the interaction takes place, the substrate can bind to the enzyme and a subsequent substrate conversion can be observed, which means a positive result is obtained.