Maciej Przybylski Chair and Department of Medical Microbiology

I. Basic characteristics of

1 Basic dogma in biology

DNA RNA PROTEIN REPRODUCTION

Virus definition

1. Viruses are acellular structures

2. Multiplication of viruses undergoes only inside living host cells

3. Viruses are completely dissimilar to any other organism

4. Viruses present traits both of living organism (genetics) and abiotic organic matter (metabolism, structure)

5. Single virion contains only DNA or RNA (never both)

6. Virions’ reproduction is a result of intracellular process of de novo synthesis, and mature virion doesn’t demonstrate growth nor direct division

8. Virion doesn’t posses genetic information concerning synthesis of the system responsible for energy production and protein synthesis, what prejudges its strict parasitic character

2 ssRNA (+) dsDNA ssRNA (-)

RNA Viruses DNA

dsRNA ssDNA ssRNA (+) dsDNA RT RT

Baltimore classification of viruses

4 Group I: Double-stranded (ds) DNA viruses 4 Group II: Single-stranded (ss) DNA viruses 4 Group III: ds RNA viruses 4 Group IV: ss RNA viruses - Positive-sense (+) 4 Group V: ss RNA viruses - Negative-sense (-) 4 Group VI: (+)ss RNA viruses that replicate through a DNA intermediate 4 Group VII: ds DNA viruses that replicate through a ss RNA intermediate

3 Classification of DNA viruses

Classification of RNA viruses

4 Sizes of RNA viruses

Taxonomy of viruses

(Order) -virales 4 Family -viridae – Subfamily –virinae • Genus – – Species » Type

10th Report of International Commitee on Taxonomy of Viruses (2016, updated) ICTV Master Species List 2016 v. 3 (March 2017) http://ictvonline.org/virusTaxonomy.asp

5 Example

Syncytial Respiratory Virus (RSV)

4 Order:

4 Family:

4 Subfamily: (-)

4 Genus:

4 Species: Orthopneumovirus (Respiratory Syncytial Virus – it is a virus name, not a species name!)

4 Types: human respiratory syncytial virus A2, B1, S2 (HRSV-A2, HRSV-B1, HRSV-S2)

Viral families – criteria of classification 4 nucleic acid (DNA/RNA, single-/, double-stranded polarity +, - or +/-) 4 genome configuration (linear, circular, segmented) 4 genome length [bases, base pairs] 4 morfology (ikosahedral, helical, composite) 4 enveloped/non-enveloped 4 size (diameter) of viral particle 4 range of susceptible hosts

6 Example of viral family description

Rhabdoviridae: 4 single-stranded RNA with negative polarity 4 linear genome, non-segmented 4 genome size: 11 000 – 15 000 bases 4 helical symmetry of capsid 4 enveloped 4 virion length: 100-450 nm, diameter: 50-100nm 4 infectious for vertebrates, invertebrates and plants

Viral species classification criteria

4genetic similarity 4antigenic properties 4physical and chemical properties of virions 4tissue and cellular tropism 4pathogenesis and cytopathology 4host range 4routes of transmission

7 Virus structure

4Nucleic acid (DNA or RNA, never both) 4Capsid 4Core and matrix proteins (only selected families) 4envelope (certain families) 4surface proteins, incorporated into envelope (certain families)

Genome organisation

Linear Circular Segmented

8 Genome organisation

4 coding and non-coding sequences 4 non-coding sequences: up to 10% of genome 4 tiny viruses: 1600-2300 bases, the largest: ~200 000 bases 4 single sequence may encode more than one protein

Capsid – composed of subunits (capsomers)

Morphology: with clear symmetry axes: icosahedral or cubic (20-wall-polyhedron) helical

or composite structure

Nucleic acid + capsid = nucleocapsid

9 Icosaedral capsid symmetry

Helical capsid symmetry

10 Deltavirus (Hepatitis D Virus)

4 Diameter: 22 nm 4 Genome: 1,6 kilobases (1 ORF)

Picornavirus

4 Diameter: 28 nm 4 Genome size: 7,8 kilobases (13 ORFs)

11 Adenovirus

4 Diameter: 70 nm 4 Genome size: 35 kilobase pairs, 35 ORFs)

Herpes simplex virus 1

4 Diameter: 200 nm 4 Genome size: 152 kilobase pairs (79 ORFs)

12 Variola virus

4 Diameter: 250 x 400 nm 4 Genome size: 197 kilobase pairs (223 ORFs)

Virus replication in the cell

4 adsorption 4 penetration 4 uncoating 4 synthesis of early proteins 4 genome replication 4 synthesis of late proteins 4 assembly and maturation of virions 4 release of infectious offspring virions

13 Adsorption

Virus entry into cell (penetration)

14 Early proteins

4 they may be present within the virion or they are synthetised immediately after uncoating 4 those are functional proteins (enzymes): – DNA or RNA polymerases – regulatory proteins – modyfying enzymes (e.g. phosphotransferases) – reverse transcriptase

Nucleic acids replication

4undergoes due to activity of – virus-encoded DNA or RNA polymerases – cellular DNA polymerases (ss DNA viruses)

4nucleic acids replication requires different strategies in various viral families

15 Late proteins

4mostly structural: – capsid subunits – surface proteins – core and matrix proteins 4also certain types of functional proteins (latency, oncogenesis)

16 Viral particle assembly

helical capsid (e.g. )

Viral particle assembly

icosahedral capsid (Picornaviridae )

17

Release of viral particles from infected cell

Budding particles

Cell lysis

and direct cell-to-cell spread

18 Consequences of viral infection of the cell

19 II. Methods of culture

Isolation and cultivation of viruses

2. Cultivation techniques: V in laboratory animals V chicken embryos V cell cultures

20 Isolation and cultivation of viruses

1. Cell culture types: V primary V semicontinuous V continuous 2. Sources of cells for cell culture development: V embrional tissues V neoplasm tissues V mature tissues

Isolation and cultivation of viruses

Scheme of primary cell culture set-up

21 Isolation and cultivation of viruses

Scheme of continuous cell culture

Isolation and cultivation of viruses

Cell culture flasks

22 Virus cultivation: Ï laboratory diagnosis of clinical disease Ï scientific investigations Ï antigen production

monolayer – single layer cell culture growing of solid surface

Vero – monolayer (epithelial cell line derived from monkey kidney)

23 Media for cells:

- sugars - aminoacids - vitamins - mineral compounds - buffering componetnt (pH 7,2 - 7,4) - pH colour indicator - antibiotics/antimycotics serum (usually fetal bovine serum) •in concentration 10 – 20% growth medium •in concentration 2 - 5% maintenance medium

Culture conditions:

• temperature 37 OC

• CO 2 concentration 5% • humidity

24 diagnosis

4 clinical symptoms 4 theoretical knowledge of virus properties, its pathogenesis, diseases caused by particular virus, course of infection and clinical outcome 4 knowing, if laboratory diagnosis is needed 4 chose of proper clinical material 4 chose of adequate laboratory method 4 taking and transport of clinical specimen 4 virological laboratory examination of clinical specimen, sending the result 4 interpretation of the result, introduction of therapy

When viral disease should be suspected? 4 in case of well-known, specific symptoms: – chickenpox, cold sores, shingles, rubella etc.

4 in case of difficulties with isolation of non-viral pathogens: – meningitis, diarrhoeas, etc.

4 in case of symptoms of dangerous viral infections: – hepatitis, AIDS, tick-borne encephalitis, haemorrhagic fevers etc.

4 non-specific symptoms persisting for a long time: – infectious mononucleosis (EBV), cytomegaly (CMV), aplastic crysis ()

25 Symptoms which indicate the need for laboratory virological diagnosis:

– any symptoms of CNS infection (meningitis, encephalitis, palsies) – hepatitis – myocarditis, cardiomyopathies – diarrhoea in children < 2 y.o. – increased body temperature persisting over 2 weeks w/o specific symptoms – suspection of in utero infection (TORCH = to xoplasma – not a virus !, rubella, CMV, HSV) – suspection of HIV infection – haemorrhagic fevers

Other indications for laboratory diagnosis:

4 infections of respiratory tract (influenza, parainfluenza, adenoviruses, coronaviruses) 4 diarrhoeas (, , adenoviruses, ) 4 infections in pregnant women with viruses other than TORCH group (VZV, echoviruses, influenza, measles) 4 immunosuppressed patients 4 blood/organ donors 4 monitoring of antiviral therapy 4 control of epidemics 4 antibody surveys

26 Types of specimens

4swabs 4throat aspirates 4sputum 4blood 4vesicle fluid 4CSF 4urine 4biopsy 4stool 4serum

Specimen collection

4 acute phase of the disease (symptoms!) 4 aseptic collection 4 sterile containers 4 transport medium 4 request form

27 Transport of specimen

Fast! 4°C!

Viral pathogenesis

28 Diagnostic methods Detection of:

– infectious viral 4specific antibodies particles – nonspecific – viral antigens antibodies – viral nucleic acids – viral enzymes Direct methods Indirect methods

Electron microscopy

4method practically not used in today’s diagnostic labs 4diagnostic usefulness: in infections, when similar symptoms are caused by the viruses belonging to various families (distinct morphology) 4very useful in scientific investigations

29 Virus isolation in cell culture

4 allows to obtain high titres of infectious virus present in clinical specimen

4 requires long time 4 leads only to initial identification of isolated virus 4 some important viruses cannot be cultured 4 there is always risk of laboratory infection with cultured virus 4 experienced personnel is needed 4 expensive

Virus isolation specimen processing

4inoculation as soon as possible 4low temperature 4antibiotics 4avoid freezing and thawing 4avoid drying and pH changes

30 Cytopathogenic effect

Set of morphologic changes of the cells as an effect of cell culture infection with the virus

31 CPE

32 In standard cell lines we can isolate:

4 HHV-1, HHV-2, VZV 4 CMV 4 rabies virus 4 adenoviruses 4 bunyaviruses 4 4 hantaviruses 4 rotaviruses 4 poxviruses 4 paramyxoviruses 4 orthomyxoviruses 4 arenaviruses 4 astroviruses 4

Untill now, there isn’t a possibility to culture:

4 hepatitis viruses B, C, D and E

4 papillomaviruses (HPV)

4 caliciviruses

33 Non-cytopathogenic effects

1. hemadsorption

2. viral interference

Hemadsorption in cell line infected with parainfluenza virus

34 Plaque forming units (PFU)

4 After infection of cell line, semiliquid layer is applied on the monolayer surface, so viruses are able to infect only adjacent cells 4 Focal areas of cells damaged by viruses derived from single infectious viral particle 4 Used for virus titration (1 PFU = 1 infectious virion)

Direct detection and identification methods

4 identification based on viral antigens with use of monoclonal antibodies 4 identification based on detection of specific sequence of viral nucleic acids

Identification of virus isolated in cell lines or identification/detection directly in clinical sample

35 Antigen detection

4Methods based on immunological complexes formation (antigen-antibody)

4One of the most popular type of identification methods – number of techniques is available: – Using labelled antibodies – Using other methods for the visualization of Ag/AB complexes

Antibodies labelling

4 fluorochromes (fluorescence level) – fluoresceine, rhodamine, auramine • Direct fluorescence assays (DFA) 4 enzymes (quantity of the product of enzymatic reaction) – horse radish peroxidase (HRP), alkaline phosphatase (ALP) • ELISA technique 4 isotopes (radioactivity level) – isotopes of carbon, iodine or phosphorus

36 Other methods of visualization

4 immunochromatography 4 latex agglutination 4 precipitation 4 immunodiffusion 4 viral hemagglutination 4 neutralization assay

37 Nucleic acids detection

4 each virus posseses unique sequences of nucleotides, detection of these sequences is both detection and identification of the virus 4 possibility of nucleic acids amplification (DNA, RNA or cDNA in case of RNA viruses) 4 application of labelled nucleotides or oligonucleotides 4 DNA and RNA can be isolated from any clinical specimen

Methods of nucleic acids detection

4 PCR, RT-PCR 4 nested PCR 4 multiplex PCR 4 real-time PCR (qPCR) 4 Transcript-mediated amplifictaion (TMA) 4 DNA or RNA hybridization

38 Real-time (qPCR)

4 PCR product binds to dsDNA- binding fluochrome (non- specific binding) or to fluorochrome-labelled probe (specific binding) 4 active fluorochrome levels are measured in each cycle of the reaction

39 In situ hybridization

Applications of in situ hybridization

4detection of viruses in cell cultures or histological slides 4detection of particular viral genes 4localization of viruses in particular cells/tissues 4detection of mRNA transcripts = active replication process

40 Interpretation of the results of direct methods

Detection of the virus in material which normally is sterile is considered identification of an agent responsible for symptoms (cerebrospinal fluid, blood , material from lower respiratory tract , bioptates )

4 false-negative results 4 false-positive results – low number of the virus in sample – detection of the virus – inadequate method present in clinical material, – alteration of virus, its antigen or but not responsible for nucleic acids due to wrong symptoms conditions of transport or storage – contamination of sample of clinical material – methodological errors

Serological methods – detection of specific antibodies

4 detection of all antibodies in single serum sample 4 paired sera examination 4 detection of immunoglobulins in classes 4 detection of antibodies against particular viral antigens 4 IgG avidity testing 4 detection of secreted antibodies in mucosal exudates

41 Detection of all antibodies in single serum sample

4 gives only the information about past contact with viral antigens in practical applications used as a first step of serological diagnosis of infections with: – HIV – hepatitis viruses – haemorrhagic fevers viruses – measles virus – rubella virus – CMV in graft recipients/donors

4 used for serological populational investigations 4 used for estimation of vaccinations efficacy

Paired sera examination

42 Detection of IgM class antibodies

4 is it acute or past infection? (use of quantitative tests and comparison to IgG level) 4 application for diagnosis of perinatal and congenital infections (IgMs don’t penetrate through placenta) 4 application for acute infections of CNS (IgMs don’t penetrate blood-CSF bareer)

43 Laboratory methods for antibodies detection

4 are based on (mentioned in antigen detection methods) specific antigen-antibody reaction

– latex agglutination – ELISA – Western blot – indirect immunofluorescence assay – complement fixation assay – neutralization assay – haemagglutination-inhibition assay

Western blot - detection of antibodies against various antigens of the same virus

– very sensitive and specific method – used as a confirmation test in serological diagnosis of HIV infections – reference method for other serological techniques

44 Western-Blot

45