Animal Inoculation for Virus Cultivation

Virus Cultivation

• Used to grow and propagate viruses in a lab by infecting a host cell culture with the virus of interest.
• Involves tissue culture, embryonated eggs, and live animals.
• Used for understanding virus behavior, developing vaccines, antiviral drugs, and studying viral evolution.
• Blood, tissue, saliva, urine, stool, swabs, and fluids are specimens for culture.

Methods of Cultivation of Viruses: Animal Inoculation

• Isolates specific viruses and studies the pathogenesis of specific viral diseases.
• Lab mice (white mice), especially nursing mice, are the preferred animal.
• Toga and Coxsackie viruses are cultured using rodents younger than 48 hours old.
• Hamsters, guinea pigs, and chimpanzees are used as substitutes.
• Mimics human infection, and produces larger quantities of virus.
• Enables study of disease progression, and facilitates the development of treatments.
• Can help identify new viruses, and provides a means of viral research.
• Ethical concerns, limited availability of animal models and the variation in response to infection.
• High costs and limited relevance to human infection.

Methods of Cultivation of Viruses: Embryonated Eggs

• Goodpasture first used embryonated chicken embryos for virus cultivation techniques in 1931.
• Burnet's technique is used to cultivate viruses in numerous embryonated cell locations.
• Viruses are cultured in 8-11-day-old chick embryos.
• Viruses can be cultivated in the chorioallantoic membrane, allantoic cavity, amniotic sac, and yolk sac.
• The culture/inoculation of avian embryos is a cost-effective and practical method.
• Popular technique for isolating viruses from clinical samples.
• Embryonated egg is an optimal medium for growth and replication.
• Antibodies in the yolk of eggs from vaccinated flocks may inhibit the proliferation of certain microorganisms.
• Salmonella, Mycoplasma, and other microbes can be transmitted from an infected fowl to its eggs.
• The chorioallantoic membrane (CAM) is a thin, highly vascularized membrane that surrounds the developing embryo and serves as a primary site for virus replication in the egg.
• The allantoic cavity is a fluid-filled space within the egg that contains the allantoic fluid, which can be used for virus cultivation.
• The amniotic sac is a protective membrane that surrounds the developing embryo and contains the amniotic fluid.
• The yolk sac is a membranous sac that surrounds the yolk of the egg and provides nutrients to the developing embryo.

Methods of Cultivation of Viruses: Tissue Culture

• Introduced by Steinhardt in 1913
• Assay was conducted using cell culture, which is most commonly used in diagnostic virology.
• Tissue culture utilized rabbit corneal tissues.
• Organ culture studies the growth and replication of viruses in a specific organ or tissue.
• Advantages of organ culture include the ability to study virus replication in a more physiologically relevant environment, but it is a time-consuming technique.
• Explant culture studies the growth and replication of cells and tissues outside of the body.
• In explant culture a small piece of tissue or organ (called an explant) is removed from an organism and placed in a culture dish containing a nutrient-rich medium.
• Examples of tissues that can be used for explant culture include lung tissue, liver tissue, and skin tissue.

Comparing Tissue Culture Methods

• Explant Culture: Retains some tissue organization, cells migrate out from explant, suitable for viruses requiring structured tissues and is used in specialized research.
• Tissue Culture: Mostly single-cell or monolayer growth, cells grow in monolayers or suspension, suitable for most viruses, especially for large-scale production and is used in routine virology, vaccine production.

Cell Culture Method

• Most frequently employed.
• Cells are grown in vitro on glass or a treated plastic surface in a suitable growth medium.
• At first growth medium, usually balanced salt solution containing 13 amino acids, sugar, proteins, salts, calf serum, buffer, antibiotics and phenol red are taken and the host tissue or cell is inoculated.
• Cell culture is the process by which cells are grown under controlled conditions and on incubation divide and spread out on the glass surface to form a confluent monolayer.
• Primary Cell Culture occurs when cell culture is the process by which cells are grown under controlled conditions.
• Diploid Cell Culture the cells contain the same number of chromosomes as the parent cells used and for the isolation of some fastidious viruses and production of viral vaccines.
• Heteroploid cultures come from cancer cells and can be serially cultured indefinitely so named as continuous cell lines, are not useful for vaccine production and an example is HeLa (Human Carcinoma of cervix cell line), HEP-2 (Humman Epithelioma of larynx cell line).
• Advantage of Cell Culture is relative ease, broad spectrum, cheaper and sensitivity.
• Disadvantages of Cell Culture is the process requires trained technicians with experience in working on a full-time basis, State health laboratories and hospital laboratories do not isolate and identify viruses in clinical work, and Tissue or serum for analysis is sent to central laboratories to identify virus.
• Applications of virus culture include Virus Detection and Identification, Vaccine Production, Host-Pathogen Interaction Research and Viral Structure and Replication Studies.
• Virus culture provides accurate and timely identification of viruses, is cost-effective and allows for rapid production of vaccines and diagnostic reagents.

Virus Purification

• It isolates the virus from host tissues and cell organelles.
• Due to virus features such as larger size than proteins, more stable than cell components, and presence of surface proteins, techniques used for virus purification are similar to those used for isolating and purifying proteins and cell organelles.

Centrifugation

• It applies centrifugal force to separate particles from a solution based on size, shape, density, viscosity, and rotor speed.
• Used in environmental science for wastewater treatment.
• Used in molecular science to extract biomolecules like DNA, RNA, and protein.
• Used in medical research to separate components from urine and blood serum.
• Used in chemical science for uranium enrichment.
• Used in food science for skimmed milk production by removing fat.

Types of Centrifugation

• Differential Centrifugation: Alternates low-speed centrifugation, where most of the virus remains in the supernatant to high-speed centrifugation where there virus will be in the pellet.
• Density Gradient Centrifugation: Centrifuges particles (like virions) or molecules (like nucleic acids) in a solution of increasing concentration and, therefore, density; uses high solubility solutes like sucrose. Two types:
  • Zonal: Centrifuged in a pre-established density gradient using a high-density sucrose solution of 5-20% to create the gradient, and the particle moves though the gradient at a rate determined by its sedimentation coefficient; low speed for 1-3hours; Homogeneous and will move as a sharp band that can be harvested after moving part-way-sedimentation coefficent is a measure of how quickly a particle settles in a centrifuge, depending on its size and shape
    • Sample Loading
    • Centrifugation
    • Zone Formation
    • FractionCollection.
    • Since dense particles occurs at the bottom and the light particles remain at the top. The zone in the gradient is stable due to absence of diffusion and convection currents and isolating zones by collectong from the bottom of the tube.
  • Isopycnic: Is used for concentrating such particles possessing the same size but different density; High speed for 48 hr with 20-70% sucrose.
    • Sample Loading
    • Centrifugation
    • Zone Formation
    • FractionCollection.

Ultracentrifugation

• Technique to concentrate viruses from aqueous solutions via a simple pelleting step.

Electrophoresis

• Based on change and/or size, and change on virus particles contribute to electrophoretic mobility.
• In a density gradient column (a liquid medium, usually sucrose) is typically used.
  • Gel Electrophoresis is for a small sample quantity.
  • Zonal Electrophoresis is for a large sample quantity.

Chromatography

• Final purification of partially purified samples and proteins, depending on surface properties.
• Can be used for large scale virus purification protocols and for both enveloped and non-enveloped viruses; such as the influenza virus.
• Purity can be improved by 30 to 100 fold; further improvement can be made byemploying an additional chromatography column which can improve the concentration by 10 to 30 fold.

Virus Yield

• Virus yield is the total weight of purified virus, varying with virus and plant; ranges from 0.05 ug/g leaf with Barley yellow Dwarf-virus (BYDV) to 2000 µg TMV/g tobacco.
• Virus yield reduction assay measures how effective antiviral compounds are, involving infecting cells with a virus, then adding test compounds and measuring the virus titer.
• Cumulative virus production curve shows the number of viruses produced over time where the slope represents rate of production.

Maintenance/Storage of Virus

• Lyophilization: Freeze drying through sublimation.
• Deep Freeze: Store in Na azide, chlorobutanol to prevent microbial growth and stabilize virus.
• Extracted or purified virus is stored in small quantities with equal volume of glycerol and 3 or 4 crystals of thymol or sodium azide at 4°C or frozen protect microbial growth.
• Add liquid nitrogen in equal volume as storage buffer.

Virus Assay

• Measures viruses.
• Investigates viral replication, enzymes, and cell entry mechanisms.
• Detects, quantifies, and characterizes viruses.
• Diagnostics
• Research
• Vaccine development
• Environmental monitoring.
• Viral Titer: Quantity of virus present in a sample.
• Infectivity: A pathogen's capacity to infect a susceptible host.

Types of Virus Assay

• Infectivity Assay: Measures virus infectivity, replication, and vaccine efficacy.
• Plaque Assay: Quantifies viral particles, assesses antiviral drug effectiveness, and aid in understanding viral pathogenesis. a scientific method for determining the quantity of infectious viruses present in a sample and uses cell culture to identify infectious viruses in concentrates; plaques form to show the presence of the virus after three to five days. Also estimates waterborne enteroviruses.
• Culture is incubated to allow plaques to form, the cell monolayer is stained to visualize the plaques, and count the number of plaques that form to calculate the titer (PFU/ml).
• Plaques are areas of infected cells that form when a virus-infected cell lyses.
• Related Assay:
• Plaque Reduction Neutralization Assay
• Diagnostic test that quantifies the presence of neutralizing antibodies in a sample, applicable to serum and antibody solutions
• Tissue Culture Infectious Dose 50 (TCID50)
• Measures viral infectivity, the dilution to infect 50% of cells in tissue culture
• Visible cytopathic effect in each well
• Involves Cell culture, serial dilution, and observation of cells using statistical methods
• Nucleic Acid Amplification Assay (NAATS) -Detects and amplifies DNA RNA sequences even in low amounts
• Polymerase Chain Reaction
• Detects viral DNA with DNA polymerase
• Reverse Transcription PCR (RT-PCR)
• Amplifies the RNA sequences then detect RNA viruses like influenzas and COVID
• Real-Time PCR (QPCR)
• amplifies target information and uses probes to provide data for load monitoring and genetic expression.
• Loop-Mediated Isothermal Amplification (LAMP)
• The rapid testing method for amplification which ideal for diagnostics
• Serological Assays
• Detects and amplifies specfic sequences of rna dna bacteria
• ELISA
• test used to look for antibodies hormones or antigens
• used in diagnosis and infection also used to gather responses from immune systems.
• Hemagglutination
• Diagnose the red blood cells and uses interaction
• interaction with red blood cells for the bottom part with lattice. Test measures with the antibody against the neutralize of virus , it aids a lot with measurement asessment and immunity for protection. The serum uses a mixture and the cell disease also the fluid which involves example reduction test Virus Characterization Viral isolation and characterisation are essential steps in identifying viral pathogens, understanding their biology, and developing treatments or vaccines. This process is crucial in clinical diagnostics, research, and public health. This is done in order to accurately identify viruses from biological samples, analyze their characteristics, and assess their impact on human health

General Concept of Viruses

• Helical
• Polyhedral
• Spherical
• Complex

Virus Host Interaction

• Attachment
• Entry of phage DNA
• Syntheis of viral genomes and proteins
• Assembly
• Release

Type of Viral Infections

• Acute Infections are where the virus replicates rapidly, often leading to the destruction of the host cell and noticeable symptoms in the host (e.g., influenza, COVID-19).

• Chronic Infections occur when the virus persists in the host for a long period, often replicating at a slower rate without killing the host cells immediately (e.g., hepatitis).

• Latent Infections are some that remain dormant within host cells for long periods, only reactivating under certain conditions (e.g., herpesvirus). Steps in Viral Isolation

• Sample Collection

• Viral Culture

• Use of host systems

• Soil sample and the digestion along with dispersion

• Techniques For Isolation : Centrifugation is a simple way to concentrate with virus while it’s spinning the virus and then separates based on size. Filtration : It can help filter the particles for better analysis , the pores can hep separate form cell as well. UltraCentrifugation is going to help more purify and used by the samples in high speeds .It will also have the gradeant centrifucation for viral density.

• Characterisation Electron Microspores is used to give visuals on particles , provides image and the morophory aids to the virus type and structure.

• *Advantages , It can visualise the virus and hard tp identify based by appeancer like hairlacal.

• Molecular techniques Techniques like Polymerase Chrain reactions it widely used to dectect viral the dna or rna amplidies the furthr analysis . High sensitive and specfic amounts that enable for detection

Real Time Pcr (Qpcr)

A variation of PCR that allowed for the real time measurements of Rna or dna the Viral the loading to agnositing is being used. Sequencibg is like using to to anylsi the entire virus .and it helps identifies mutations and varations serorgly Test to detech antiboidiers and it can determne the virus or to meausre the respobse for test

• ELISA (Enzyme-Linked Immunosorbent Assay) is a common method used to detect viral antigens or antibodies.

Characterization of a fieldable Process for Airborne Virus Detection

• The air particles like Sampling using electropacstics. Then the rna extractions the probe is heates in the waater
• rna Capillary electrophoresis concentration the rna in small amounts .With Electrifcation increases that rna
• en the detection the richded rna and it test the molecular

Challenges and consideration

• The isolation or viarl susceptible or contaicment with microorganisms And to uuse the steril technies That lpw viarli load makes different with pcr rna for more reauqired .and Host that hardes to grpw with conditopns hsi to b easily replocatred to easy setting

Applications

• Clincia Diagrnosics used isolate viraul and cahcrterisgation of infections or the causative .
• For develpment idenifity with the virus. .
• And it help ids with viral stfrians , and tcan track wuth the spread for monitor the emergrinf virus like coronais and idnfulaz.