6.1–6.4 Acellular Pathogens: Viruses and Related Agents

6.1 Viruses

  • Learning objectives

    • Describe the general characteristics of viruses as pathogens
    • Describe viral genomes
    • Describe the general characteristics of viral life cycles
    • Differentiate among bacteriophages, plant viruses, and animal viruses
    • Describe the characteristics used to identify viruses as obligate intracellular parasites

  • Historical context and discovery

    • Viruses are ultramicroscopic and were first identified as filterable agents smaller than bacteria
    • Tobacco mosaic disease (TMD) research led by Dmitri Ivanovski (1892) using porcelain Chamberland filters (pore size ≈ 0.1 μm0.1\,\mu m)
    • Chamberland filters remove bacteria ≥0.2 μm0.2\,\mu m; the TMD extract passed through, suggesting a smaller agent
    • Ivanovski initially suspected a very small bacterium or bacterial spore; Beijerinck (1899) proposed it might be a toxin-like chemical
    • The term virus (Latin for poison) was adopted; Ivanovski is credited as the original discoverer of viruses and a founder of virology
    • Modern methods (electron microscopy) now allow visualization of viruses (Fig. 6.2)

  • General characteristics of viruses

    • Infectious, acellular pathogens
    • Obligate intracellular parasites with host- and cell-type specificity
    • Genome: DNA or RNA, never both
    • Genome is enclosed by a protein capsid; some viruses have a phospholipid membrane (envelope) studded with viral glycoproteins
    • Many genes required for replication are lacking; viruses rely on host-cell machinery
    • New virions form by assembly of viral components inside the host cell
    • After assembly, virions transport the genome to new host cells to continue infection

  • Taxonomy and origins

    • Viruses are not included in the tree of life because they are acellular
    • Viral classification and nomenclature (ICTV) are based on genetics, chemistry, morphology, and replication mechanisms
    • Virus numbers are large: seven orders, 96 families, 350 genera (examples: family names end with -viridae; genus names end with -virus)
    • The Baltimore classification system groups viruses by genome type and replication strategy into seven groups
    • In addition to formal systems, viruses are informally grouped by: naked vs enveloped structure, genome type (ssDNA, dsDNA, ssRNA, dsRNA), positive-sense vs negative-sense RNA, segmented vs nonsegmented genomes, etc.
    • Examples: herpesviruses (dsDNA enveloped); HIV ( +ssRNA enveloped ); tobacco mosaic virus ( +ssRNA virus )
    • ICD (International Classification of Diseases) coding links ICTV taxonomy to disease classification; ICD codes are used for treatment, billing, epidemiology, and vital records (death certificates)
    • Clinical Focus 2 highlights the rabies workup and the role of diagnostic tests in management (immunofluorescent antibody testing, RT-PCR)

  • Viral structure and morphology

    • Virions are typically smaller than cells; size range roughly 20 nm20\,\text{nm} to 900 nm900\,\text{nm}; some giant viruses approach bacterial cell size
    • Capsid shapes: helical, polyhedral (icosahedral), or complex (as seen in some bacteriophages and poxviruses)
    • Naked viruses: nucleic acid + capsid (no envelope)
    • Enveloped viruses: nucleic acid + capsid + envelope from host cell membrane
    • Spikes (glycoproteins) extend from capsid or envelope and mediate attachment; examples include influenza HA (hemagglutinin) and NA (neuraminidase) spikes
    • Viral envelope origin: can be intracellular or cytoplasmic; spikes facilitate entry and exit
    • Bacteriophages (viruses that infect bacteria) frequently exhibit complex shapes (head, tail sheath, tail fibers, tail pins) and infect bacteria
    • The structure and composition determine entry, replication, and host range

  • Key historical milestones in virology

    • Wendell Stanley (1935): crystallized TMV and showed it is composed of RNA and protein; contributed to understanding that viruses are not cellular life
    • 1943: Stanley isolated Influenza B virus; his work contributed to influenza vaccine development
    • 1946: Nobel Prize for Stanley’s virus work

  • Viral envelope and utility of spikes

    • Spikes enable binding to host receptors; envelope-spikes can be targets for neutralizing antibodies
    • Influenza virus examples: H and N spikes; H1N1 (1918, 2009), H2N2 (1957), H3N2 (1968)

  • Common pathogenic viruses by genome type (illustrative examples)

    • dsDNA, enveloped: Poxviridae (Orthopoxvirus, Parapoxvirus), Herpesviridae (Simplexvirus)
    • dsDNA, naked: Adenoviridae (Atadenovirus), Papillomaviridae (Papillomavirus)
    • dsRNA, naked: Reoviridae (Rotavirus)
    • +ssRNA, naked: Picornaviridae (Enterovirus C – Poliovirus; Rhinovirus; Hepatovirus – Hepatitis)
    • +ssRNA, enveloped: Togaviridae (Alphavirus, Rubivirus – Rubella)
    • −ssRNA, enveloped: Filoviridae (Zaire Ebolavirus), Orthomyxoviridae (Influenzavirus A,B,C), Rhabdoviridae (Lyssavirus – Rabies)

  • Host range and transmission concepts

    • Host range: viruses can infect many host types, but most have a narrow host range; some infect multiple species
    • Tissue tropism: specific tissues or cell types within a host that a virus targets (e.g., poliovirus in brain/spinal cord; influenza in respiratory tract)
    • Transmission routes: direct contact, indirect contact via fomites, vectors (mechanical vs biological)
    • Zoonoses: viruses that jump from animals to humans; reverse zoonoses: human-origin virus infects animals

  • Bacteriophages and therapy

    • Phage therapy revisits bacteriophages to treat bacterial infections; advantages over antibiotics include specificity to a single bacterium and preservation of normal microbiota
    • FDA-approved phage preparations for reducing Listeria in ready-to-eat meats (2006); regulation and labeling considerations
    • Controversies and consumer concerns about phages in foods

  • Practical implications and connections

    • Phage therapy highlights the importance of alternative antimicrobial strategies amid rising antibiotic resistance
    • The concept of phage conversion demonstrates how phage genes can alter bacterial virulence (lysogenic conversion)
    • Understanding viral evolution and mutation is crucial for vaccine design and public health responses

  • Clinical Focus 1: Rabies diagnostic approach (case excerpt)

    • Symptoms: prickling/itching at bite site, fever, weakness; concern for rabies after dog bite
    • Diagnostics used: immunofluorescent staining for rabies antibodies in skin (nape of neck nerves) and RT-PCR on saliva; blood antigen testing for rabies
    • Treatment: prophylaxis with human rabies immunoglobulin (HRIG) and rabies vaccine series
    • Conceptual questions:
    • Why search for antibodies via immunofluorescence rather than the virus itself in tissue?
    • What is prognosis if rabies infection progresses after symptom onset?

6.2 The Viral Life Cycle

  • Learning objectives

    • Describe the lytic and lysogenic life cycles
    • Describe the replication process of animal viruses
    • Describe unique characteristics of retroviruses and latent viruses
    • Discuss human viruses and their virus-host cell interactions
    • Explain the process of transduction
    • Describe the replication process of plant viruses

  • General principles of viral replication

    • All viruses rely on host cells for replication; they do not carry out all metabolic processes themselves
    • In most cases:
    • DNA viruses replicate in the nucleus (with exceptions)
    • RNA viruses replicate in the cytoplasm (with noted exceptions such as influenza)
    • Some large DNA viruses (e.g., poxviruses) replicate in the cytoplasm
    • Exceptions and notes:
    • Influenza virus replicates in the nucleus (uncommon for RNA viruses)

  • Life cycles in bacteriophages (viruses that infect bacteria)

    • Lytic cycle (virulent phages): leads to host cell death and release of new phages
    • Five stages: Attachment, Entry (penetration), Biosynthesis, Maturation, Release
    • Attachment: phage binds specific bacterial surface receptors (e.g., lipopolysaccharides, OmpC)
    • Host range is typically narrow; can be exploited for phage therapy or typing
    • Entry: tail sheath contracts; genome injected into bacterial cell; phage head remains outside
    • Biosynthesis: viral genome replication and transcription/translation; host chromosome degraded via viral endonucleases; early genes (polymerases) expressed first; late genes (capsid, tail) expressed later
    • Maturation: assembly of new virions
    • Release: lysis of host cell via phage-encoded holin or lysozyme
    • Lysogenic cycle (temperate phages): phage genome integrates into host chromosome as a prophage and is replicated with the host genome
    • Lysogen: a bacterial cell carrying a prophage
    • Lysogeny: infection by a temperate phage with potential latent/indirect effects on host phenotype
    • Lysogenic conversion (phage conversion): prophage can confer new traits to host (e.g., toxin genes increasing virulence)
      • Examples: Vibrio cholerae with cholera toxin gene; Clostridium botulinum with botulinum toxin genes
    • Prophage excision and induction: environmental stress can trigger excision; phage can re-enter the lytic cycle
    • Induction allows the prophage to switch to lytic growth and produce progeny
    • Transduction
    • Generalized transduction: random piece of bacterial DNA is packaged into a phage head during lytic cycle and transferred to a new host
    • Specialized transduction: occurs at the end of lysogeny when prophage excises; nearby bacterial DNA adjacent to integration site is packaged and transferred
    • Result: horizontal gene transfer; contributes to genetic variation and adaptation (e.g., antibiotic resistance, metabolic traits)

  • Life cycle of animal viruses

    • Entry mechanisms differ from bacteriophages: endocytosis or membrane fusion (fusion of viral envelope with host membrane)
    • Tropism and tissue specificity (tissue tropism): viruses typically infect specific tissues or cell types
    • Examples: poliovirus shows neural tropism (brain/spinal cord); influenza targets respiratory tract
    • Genome replication strategies by RNA/DNA types
    • +ssRNA genomes: can be directly translated into proteins by host ribosomes
    • −ssRNA genomes: must be copied to +ssRNA by viral RNA-dependent RNA polymerase (RdRP) before translation; RdRP is packaged with the virus
    • dsRNA genomes: copied to +ssRNA by RdRP, which is used as template for translation
    • Retroviruses (e.g., HIV): +ssRNA viruses that carry reverse transcriptase; reverse transcriptase synthesizes cDNA from RNA; cDNA integrates into host genome as a provirus; provirus remains latent and cannot be excised; chronic infection may result
    • Persistent infections
    • Latent infections: virus remains dormant in cells; may reactivate later (e.g., herpesviruses, varicella-zoster)
    • Chronic infections: continuous or intermittent viral expression with ongoing disease (e.g., HIV, hepatitis C)
    • Immune evasion strategies include minimizing viral antigen expression, altering immune cell function, mutational antigenic drift, and latency

  • Plant viruses

    • Similar to animal viruses in basic replication; can be enveloped or non-enveloped; genomes can be DNA or RNA; most plant viruses have +ssRNA genomes acting as mRNA
    • Transmission often via mechanical means, insect vectors, fungi, nematodes, or wounds; host range can be narrow or broad
    • Life cycle includes entry, uncoating, genome replication using host machinery, movement through plant vascular system (phloem), systemic infection, and transmission to new plants
    • Can establish latent or persistent infections without necessarily killing the host

  • Viral growth curve and one-step growth dynamics

    • Bacteriophage infections follow a one-step growth curve (not sigmoidal like bacterial growth curves)
    • Key phases:
    • Inoculation: virions attach to host cells
    • Eclipse: genome entry and virion components are synthesized; no mature virions detectable yet
    • Burst: mature virions are released in large numbers after host lysis
    • Burst size: the maximum number of virions produced per bacterium
    • If no viable hosts remain, virions decay over time
    • Figures (e.g., Fig. 6.14) illustrate the one-step curve and the eclipse/burst phases

  • Ethics and case-based discussions

    • Ebola ethics in 2014 highlighted compassionate use of unregistered drugs (ZMapp) and balancing against safety and allocation concerns
    • Case timelines (Duncan, 2014) illustrate incubation, transmission risk, and public health responses

  • Links to learning

    • Interactive resources on current virus taxonomy and classifications (ICTV website)
    • Case studies and ethical discussions accessible through OpenStax resources

6.3 Isolation, Culture, and Identification of Viruses

  • Learning objectives

    • Discuss why viruses were originally described as filterable agents
    • Describe the cultivation of viruses and specimen collection and handling
    • Compare in vivo and in vitro techniques used to cultivate viruses

  • Filtration and isolation basics

    • Porcelain Chamberland filters (0.1 μm\mu m pore size) allowed viruses to pass while removing bacteria (≈0.2 μm\mu m and larger)
    • Modern filtration uses membrane filters to separate cells from virions; virions pass through finer pores while cells are retained
    • Filtration yields a virion-rich filtrate suitable for downstream analysis

  • Cultivation of viruses

    • Viruses require living host cells for replication
    • In vivo cultivation: whole organisms or embryonated eggs; embryos serve as incubation sites
    • In vitro cultivation: cell culture systems (primary cultures and continuous cell lines)

  • In vivo host sources

    • Embryonated eggs (e.g., chicken or turkey eggs) used for vaccine production (e.g., influenza vaccines)
    • Location within eggs is important due to tissue tropism (amniotic cavity, chorioallantoic membrane, yolk sac)
    • Embryo viability and tissue integrity are critical for successful viral replication

  • In vitro cultivation

    • Primary cell culture: cells freshly derived from tissues; attached to a surface; finite lifespan; require anchorage for growth
    • Secondary cultures: when primary cultures reach contact inhibition, they are subcultured to expand
    • Continuous cell lines: derived from transformed cells or tumors; can be subcultured indefinitely; often grow in suspension and may lack anchorage dependence and contact inhibition (e.g., HeLa cells)
    • HeLa cell line: famous immortal cell line derived from Henrietta Lacks’s cervical cancer; widely used in research; raises ethical considerations about consent and ownership
    • Immortal cell lines may form clusters or piles, unlike primary cultures that require careful maintenance to avoid overconfluency

  • Detection and identification of viruses after growth

    • Cytopathic effects (CPEs): observable changes in host cells due to infection (loss of adherence, rounding, nuclear changes, vacuoles, syncytia, inclusion bodies, lysis)
    • Cytopathic effects vary by virus type and are used as a screening/diagnostic clue
    • Hemagglutination assays (HA): some viruses have surface proteins (hemagglutinins) that cause red blood cell agglutination
    • Hemagglutination inhibition (HAI): antibodies block HA-mediated agglutination; a positive result indicates presence of virus-specific antibodies
    • Serological assays often involve antibody layers on membranes and colorimetric readouts (EIAs/ELISAs)

  • Serology and molecular detection

    • Hemagglutination assay matrix (HA) can be qualitative or semi-quantitative based on observed agglutination patterns
    • HAI assays detect presence of virus-specific antibodies in patient serum to infer infection history or current infection
    • Nucleic acid amplification tests (NAATs): detect viral nucleic acids
    • PCR: detects viral DNA; primers bind specific sequences to amplify target DNA
    • RT-PCR: detects RNA viruses; reverse transcriptase converts RNA to cDNA, which is then amplified
    • Enzyme immunoassays (EIAs / ELISAs): detect viral antigens or antibodies using enzyme-labeled antibodies and colorimetric substrates; used for screening and confirmation

  • HPV case study (HPV scare)

    • Pap smear + HPV DNA test used to screen for abnormal cervical cells and HPV presence
    • Vaccination recommended if tests are negative, to reduce future exposure risk
    • Two different tests provide complementary information: cytology for cellular changes and DNA test for viral presence

  • Key figures and concepts

    • Fig. 6.16: filtration in virus isolation; membranes allow virions to pass while removing larger cells
    • Fig. 6.17: in vitro cell culture, primary cultures, and bacteriophage/plaque assay on bacterial lawns
    • Fig. 6.18: embryo-based viral replication sites within chicken eggs
    • Fig. 6.19: growth characteristics of primary vs continuous cell cultures; anchorage dependence and contact inhibition
    • Fig. 6.20–6.21: HeLa cells and cytopathic effects illustrating transformation and ethics of cell line origin
    • Fig. 6.23: EIAs for viral antigens using antibody-antigen interactions
    • Fig. 6.22: outcomes of hemagglutination inhibition tests

  • Detection, identification, and case-based questions

    • Common methods to detect viruses in clinical samples: CPEs, PCR/RT-PCR, NAATs, EIAs, serology (HA/HAI)
    • Ethics and case discussions emphasize consent, access, and equitable use of diagnostic and research resources

  • Practical notes

    • For filters and viruses: pore sizes for filtration matter greatly when separating viruses from cells
    • Cultivation choices (in vivo vs in vitro) depend on the virus and application (diagnosis, vaccine production, research)
    • Primary vs continuous cell cultures differ in lifespan, anchorage requirements, and growth dynamics; HeLa cells are a landmark example of immortal lines

6.4 Viroids, Virusoids, and Prions

  • Learning objectives

    • Describe viroids and their unique characteristics
    • Describe virusoids and their unique characteristics
    • Describe prions and their unique characteristics

  • Viroids

    • Discovered in 1971 by Theodor Diener
    • Viroids are acellular particles consisting only of a short circular RNA genome
    • They are able to self-replicate using host-cell machinery
    • First viroid described: potato spindle tuber viroid (PSTV)
    • Viroids lack a protein coat (no capsid)
    • Like viruses, viroids rely on host cellular machinery to replicate their RNA genome
    • PSTV exemplifies how viroids can cause plant diseases (deformities, slow sprouting)

  • Virusoids and prions

    • The excerpt introduces virusoids and prions as part of this section, but detailed characteristics are not provided in the text excerpt
    • Viroids, virusoids, and prions represent subviral or nontraditional infectious agents distinct from classical viruses

  • Summary and context

    • Viroids demonstrate that RNA alone (without a protein coat) can be infectious and replication-competent in plants
    • The study of viroids, virusoids, and prions expands understanding of infectious disease agents beyond conventional viruses

  • Case and ethics notes

    • The chapter’s ethics discussions (e.g., HeLa cells) highlight ongoing debates about consent, ownership, and the use of biological materials in research

  • Cross-cutting takeaways for exams

    • Distinguish viruses from cellular life by acellularity and reliance on hosts
    • Recognize the major genome types and their replication implications (DNA vs RNA; + vs − strands; ds vs ss; presence or absence of envelope)
    • Differentiate lytic and lysogenic life cycles in bacteriophages and relate analogous processes in animal viruses (latent vs active infections)
    • Understand the practical detection and identification methods: CPEs, HA/HAI, NAATs (PCR/RT-PCR), EIAs/ELISAs
    • Recall the historical milestones and classic examples (TMV, TMV structure, H and N spikes, Baltimore classification, ICTV taxonomy)

  • Quick conceptual recap

    • Virions are ultramicroscopic, acellular particles that require host cells to complete replication
    • Viral genomes can be DNA or RNA, single- or double-stranded, with or without envelopes
    • The life cycle varies by host (bacteriophage vs animal vs plant viruses); transduction in bacteria and latency in animals illustrate the diversity of strategies
    • Subviral agents like viroids challenge the notion that a protein coat is necessary for infectivity; prions illustrate protein-only infectious agents

  • Practice prompts (to review concepts)

    • Explain why the tobacco mosaic virus was initially thought to be a toxin rather than a virus
    • Compare the lytic and lysogenic cycles in bacteriophages with latency in animal viruses
    • Describe how +ssRNA and −ssRNA genomes are used to produce viral proteins in animal cells
    • Outline the steps of the one-step growth curve for a bacteriophage and the meaning of burst size
    • Differentiate between generalized and specialized transduction and their genetic consequences
    • Explain how hemagglutination and hemagglutination inhibition assays work and what they measure
    • Summarize how EBV/HSV/HIV achieve persistent infections and immune evasion strategies

  • Note on expression format

    • All mathematical expressions and genome-related references are presented in LaTeX format where applicable, e.g.,
    • Pore size: 0.1 μm (porcelain filters)0.1\,\mu m\, (porcelain\ filters) and 0.2 μm (bacterial exclusion)0.2\,\mu m\, (bacterial\ exclusion)
    • Virion size range: 20 nm≤virion size≤900 nm20\,\text{nm} \leq \text{virion size} \leq 900\,\text{nm}
    • Burst size: defined as the maximum number of virions produced per bacterium