Viral Culture and Growth - Comprehensive Notes

Viral culture and growth: comprehensive notes

• Purpose of culturing viruses

  • To study organisms, scientists culture viruses in the laboratory using host cells.
  • A complication: viruses must be grown within a host cell.
  • In vitro viral production often uses batch culture, an enclosed vessel with liquid media.
  • Batch culture enables growth of a large population of viruses for study.
  • A viral sample is inoculated into a population of growing cells, typically in a culture tube or culture flask.

• Batch culture and sampling

  • The culture media is sampled over time and assayed or tested for the presence of viral particles.
  • The culture fluid is then sampled over time to monitor viral production.
  • Viruses may be produced in batch culture, a type of culture in an enclosed vessel of liquid media.

• One-step growth curve: overview

  • Growth typically follows a one-step growth curve when observing one cycle of viral replication.
  • To observe one cycle, four phases of the viral growth curve are analyzed.
  • The four phases are observed in the context of an inoculated culture of host cells.

• Four phases of the viral growth curve (one-step)

  • Inoculation phase
  • The virus is recognized by receptors on the surface of the host cell and is taken in (entry).
  • Eclipse phase
  • A short period following inoculation during which the virus is undetectable in the culture medium.
  • Rise period
  • Viral particles begin to appear in the liquid media.
  • The rise period ends when the viral particles have been liberated from the host cell.
  • Burst phase (also called lysis or release phase)
  • Infected cells lyse and release viral particles.
  • Important quantities related to the cycle
  • The number of viruses that inoculate or infect other host cells is initially small.
  • Burst size (also called birth size): the number of virus particles produced per infected host cell.

• Terminology nuances in the growth curve

  • Inoculation phase: virus entry via host cell receptors.
  • Eclipse phase: time when virions are intracellular and not detectable in the medium.
  • Rise period: appearance of virions in the culture fluid as they are released.
  • Burst size / birth size: number of virions produced per infected cell; two terms used in the literature.

• Growth in liquid media

  • Viruses can be grown in a suspension of liquid media.
  • A viral sample is mixed with host cells and grown in a batch culture.
  • The culture fluid is sampled over time and assayed for viral particles.
  • The culture medium may be replaced or supplemented as necessary to maintain cell viability.

• Purification and isolation of virions from culture

  • A culture sample may be filtered to remove larger particles and debris.
  • Filtration step example: a 0.2 μm filter is used to remove anything larger than the virion.
  • Purified virions (the filtrate) can be used for further culturing or experimentation.
  • Mathematical note: filtration efficiency and pore size are critical for removing contaminants while allowing virions to pass through.
  • In formulas: the filtration cutoff can be denoted as 0.2 \ \mu \mathrm{m} for readability.

• In vivo culture and embryonated eggs

  • In vivo culture involves growing viruses in a whole living organism, such as plants or animals, to study immune responses or for diagnostic isolation.
  • Ethical and practical considerations apply; some human viruses cannot be cultured in animals or do not produce signs/symptoms in the animal model.
  • Growing a virus in an embryonated egg is convenient and inexpensive.
  • Procedure: Drill a hole in the shell and insert a viral suspension or infected tissue into the egg fluid.
  • Viral growth is signaled by death of the embryo, embryonic damage, or formation of pox/lesions on the egg membrane.
  • This method is still used to grow some viruses for vaccines, though embryonated eggs have largely been replaced by cell culture.

• Cell culture and tissue culture

  • Cell culture involves growth of human or animal cells in a monolayer on a solid substrate in liquid media.
  • The tissue culture is inoculated with viral suspension; after attachment, unattached virions are removed, and gelatin media may be used to slow viral dispersal and allow plaque formation.
  • Viruses can be grown in primary cell lines or continuous cell lines.

• Primary vs continuous cell lines

  • Primary cell lines: derived from tissue slices and tend to die after only a few generations.
  • Continuous cell lines: transformed or cancerous cells that can be maintained through many generations (nearly infinite) and are routinely used for viral work.

• Plate cultures and plaque formation

  • Plate culture of colonies enables isolation of a population descended from a common progenitor.
  • On solid media, viruses do not form a visible mass like cells, but they form plaques in a plaque assay.
  • Plaque: a clear area formed when virions from a single progenitor lyse host cells.
  • Plaque assays involve mixing diluted virus with a host cell and embedding in soft agar, then pouring over a solid agar plate.
  • With no virus, host cells form a continuous lawn; where virus is present, infection and lysis create plaques.
  • Plaques are counted to calculate viral concentration in terms of plaque forming units (PFU).

• Plaque assay procedure and calculations

  • Step 1: Mix a diluted suspension of virus with an appropriate host cell in soft agar.
  • Step 2: Overlay the soft agar with a solid agar plate to constrain the spread of infection.
  • Step 3: Incubate to allow infection and plaque formation.
  • Step 4: Count plaques; use counts to calculate the concentration of infectious particles.
  • Outcome: plaque-forming units (PFU) per unit volume.
  • Formula for PFU per mL (typical convention):
    \text{PFU/mL} = \frac{N}{V \, d}
    where:
  • N = number of plaques counted on the plate,
  • V = volume of diluted virus plated (in mL),
  • d = dilution factor (the dilution of the plated sample expressed as a decimal; e.g., for a 1:10^6 dilution, d = 10^{-6}).
  • This formula yields the concentration in the original stock.

• Tissue culture and plaque formation in monolayers

  • In tissue culture, host cells are grown as a monolayer in flasks or plates.
  • After inoculation with virus, cells are observed for cytopathic effects and plaque formation.
  • Gelatin or similar semi-solid media can slow viral dispersal and help visualize plaques.

• Types of viral infections

  • Acute infection
  • Symptoms develop rapidly and are typically short-lived.
  • Example: influenza virus infection.
  • During an acute infection, the virus is in an active lytic phase with continuous biosynthesis, assembly, and shedding of infectious viral particles.
  • The infected host may clear the infection with or without medical support; recovery can occur within days to weeks, depending on host immunity and virus.
  • Characteristics: rapid infection, rapid onset, and typically rapid host response.
  • Influenza example: destructive mucosal cell infection with rapid immune response; if effective, clearance within about one to two weeks.
  • Latent infection
  • Symptoms may develop after an acute period; virus persists in the host without active disease.
  • Example: Varicella zoster virus (chickenpox/shingles).
  • The virus can go dormant in nerve cell ganglia and is not actively replicating during latency.
  • Reactivation may occur later, causing disease symptoms.
  • Mechanisms: viral genes stabilized within the cell; can be located in the nucleus or cytoplasm; proviral form may be integrated into the host genome.
  • Chronic infection
  • Symptoms develop gradually over weeks or months and may persist for a long time.
  • Outcomes depend on host health and virus type; may be slow to resolve or persist.
  • Example: HIV can produce a chronic infection; the integrated viral genome is called a provirus.
  • Consequences: continuous immune system engagement and progressive immune damage if untreated.
  • Provirus concept
  • Integration of the viral genome into the host cell genome can create a provirus, which may remain latent or drive persistent infection.
  • HIV and proviral integration
  • Integration is critical to establishing a permanent infection; HIV damages the immune system and can progress to AIDS if untreated.

• Oncogenic viruses and cancer development

  • Some viruses can contribute to cancer by integrating their genome into the host chromosome and disrupting cell growth regulation (transformation).
  • Transformation: a process by which a virus induces carcinogenesis in a host cell, expanding the population of infected cells and producing more viral particles.
  • Oncogenic viruses: viruses capable of inducing cancer; they can persist in the host via proviral integration.
  • Human papillomavirus (HPV) is an example of an oncogenic virus; HPV virions can cause latent infection but may persist for months or years.
  • Latent viral genomes can drive abnormal growths such as warts or cancer while not always expressing active disease.

• Practical and ethical implications

  • Biosafety and containment: viral culture requires appropriate biosafety levels and containment to protect researchers and the environment.
  • Animal and embryonated egg use: in vivo culture and embryonated eggs raise ethical considerations; alternatives (e.g., cell culture) are increasingly used where possible.
  • Vaccine development: embryonated eggs have historically supported vaccine production but are being complemented or replaced by more controlled cell culture systems.
  • Diagnostic and therapeutic relevance: understanding growth curves, infection types, and replication cycles informs vaccine design, antiviral strategies, and disease management.

• Connections to foundational principles and real-world relevance

  • Host-virus interactions depend on receptor binding, entry, and intracellular replication; the inoculation and eclipse phases illustrate early infection steps.
  • The one-step growth curve models how a single infection cycle proceeds and yields insight into burst size and progeny production.
  • Plaque assays connect virology with quantitative microbiology, enabling estimation of infectious titers (PFU/mL) and virus quantification in samples.
  • Distinguishing acute, latent, and chronic infections helps explain clinical presentations, transmission dynamics, and long-term outcomes for diseases such as influenza, varicella-zoster, and HIV.
  • Proviral integration and oncogenesis illustrate how viruses can interact with host genetics to influence cancer risk and disease progression.