Searching for microbes Part X Virology Part

Searching for microbes Part X. Virology – Part One Ondřej Zahradníček To practical of a. VLLM 0421 c zahradnicek@fnusa. cz

Content of this slideshow Viruses Hepatitis viruses HIV Diagnostics of hepatitis and HIV infection Herpesviruses (including diagnostics) Practical diagnostic approaches Check-up questions Story

Story • Buy the peach, Ma‘am! cried a seller on an exotic market. Mrs. Green bought them and took them to the hotel. • In the hotel room she wanted to wash them, but oops – water supply is out of order. What to do now? Mrs. Green was unsure, but the peaches were SO nice… So, she decided to eat several fruits. • After coming back from abroad, Mrs. Green became yellow…

Who was the criminal? • Hepatitis A virus. But it could be also hepatitis E virus – both of them are transmitted by dirty hands and unwashed nutrients. • Besides these types of hepatitis there also other, transmitted by blood or sexually • There are various hepatitis types, and the viruses causing them are different. But one problem is common: the diagnostic should respect the fact that they are viruses, not bacteria.

Viruses

How viruses look like http: //vietsciences. free. fr/khaocuu/nguyenlan dung/virus 01. htm

What are viruses? • Viruses are acellular particles, it is unsure, whether they are organisms at all • On viral origin there are several theories, it is not sure that all of them have the same origin • Like cellular organisms, they attempt to „keep the gene“, but they need an alien cell to it • Besides human viruses we have animal viruses, plant and bacterial viruses (bacteriophages) • Prions – „mistakes in protein“ are not viruses, but they use to be described in virology.

Classifying viruses • According to nucleic acid: DNA viruses and RNA viruses • According to number of DNA/RNA strands: single stranded (ss) and double stranded (ds), in single stranded also according to "plus" or "minus" strand • According to presence of viral envelope both DNA, and RNA viruses are classified into enveloped and unenveloped ones • Further they are classified into families and genera, like bacteria or animals; species names are usually not used.

Viral particle – virion • Virion is not a cell. Virions are smaller than majority of cells, including bacterial cells: mostly 20– 300 nm • Composition of a virion – nucleocapsid or nucleus + capsid – envelope (in enveloped viruses) – in some viruses atypical composition (VHB) When a virus is just present in a host cell, its structure and arrangement is different from virus in outer environment

Nucleocapsid • is present in all viruses • is composed of nucleic acid (DNA, RNA) and protein capsid • capsid may have helicoidal, cubic or other symmetry. • viruses with cubic symetry form so named pseudocrystals – regular formations (individual virions might have shape of for example icosaedre)

Lipoprotein envelope • only enveloped viruses have it. • it is a lipidic bi-layer, coming from host cell (originally cytoplasmic membrane, nuclear membrane, etc. ) with incorporated viral proteins • sometimes the membrane is connected by a specific protein to the nucleocapsid. • enveloped viruses are less resistant (they die at envelope damage e. g. by drying) • More about viruses: see Bonus info

Hepatitis viruses

Hepatitis viruses • There are five main types of hepatitis VHA to VHE, caused by viruses HAV to HEV. Each belongs to another family, majority are RNA viruses, but hepatitis B virus is a DNA virus • VHA and VHE (mnemotechnic help: vocals) are transmitted by faecal oral route (hands), they do not become chronic • VHB, VHC and VHD (consonants ) – transmission by blood, or sexual (in VHC rather unimportant), they may become chronic

Survey of hepatitis types Virus Relationship Transmission HAV Picornaviridae faecal-oral HBV Special group of DNA viruses sexual, blood HCV (+ HGV) Flaviviridae blood HDV sexual, blood HEV Delta agens – viroid Related to caliciviruses faecal-oral

http: //www. epidemic. org/cgibin/hepcglossary. cgi? query=Hepatitis. A&caller=the. Facts/viruses/viral Replication. html Hepatitis A virus www. faqs. org/health/Sick. V 2/Hepatitis. html.

Hepatitis B virus www. uct. ac. za/depts/mmi/stannard/hepb. html.

Virus of hepatitis B Complete virion (Dane particle) 42 nm Only HBs. Ag 22 nm HBs. Ag, inside it delta agens (VHD) 35 nm

Hepatitis C virus http: //www. pcswn. com/Hep%20 C%20 Awareness%20 Month/cut %20 model%20 of%20 Hepatitis% 20 C%20 virus. gif

Hepatitis D virus virology-online. com/viruses/Hepatitis. D. htm

Hepatitis E virus http: //vietsciences. free. fr/khaocuu/nguyenl andung/virus 01. htm

Hepatitis • They are infectious liver inflammations, with jaundice as a typical symptom. Not all jaundices mean a hepatitis: in some inborn diseases, or in cholelithiasis (stones in bile ways) jaundice is present, too. • Patient has fever, intestinal problems, yellow colour of sclera or skin, changed urine and stool colour etc. Hepatitis B, C and D may become chronic, and they may act as precanceroses.

http: //www. nursing. uiowa. edu/site s/LRS_equip_photos/Index-7. htm Diseased organs

Patients with jaundice http: //www. gihealth. com/images/img. Jaundice. Big. jpg medicine. ucsd. edu/Clinicalimg/skin-jaundice. html.

Mostly hepatitis B is a classical health problem • turns to chronicity, possibility of cirrhosis or hepatal carcinoma • sooner transmission in hospitals, today only sexual transmission and i. v. drug abusers • screening of hepatitis B common in many situations (before operations, in pregnancy etc. )

www. pegasys. com/hcp/efficacyhepatitis-b. aspx

www. pegasys. com/hcp/ efficacy-hepatitis-b. aspx

pathmicro. med. sc. edu/virol/hepatitisdisease 2. htm Up: patient with jaundice at hepatitis B. Right: woman from Thai refugee camp, with hepatoma after hepatitis, later died because of her disease

Liver changes at VHB pathmicro. med. sc. edu/virol/hepatitis-disease 2. htm

Risk factors at hepatitis B pathmicro. med. sc. edu/virol/hepatitis-disease 2. htm

Development of disease in the USA pathmicro. med. sc. edu/virol/hepatitis-disease 2. htm

Prevention and treatment of hepatitis • Vaccination against hepatitis B is part of normal vaccination calendar in many countries • Vaccination against hepatitis A is available and recommendable in people travelling to various countries, including Mediterranean. It is also possible to get vaccinated against hepatitis A + B • In some hepatitis types, treatment by interferons is used • Otherwise hepatoprotective drugs are used, and other symptomatic therapy

HIV

HIV Human Immunodeficiency Virus • It is a retrovirus, it has reverse transcriptase (an enzyme transcripting RNA into DNA) • HIV virus exists in two types, majority of infections are caused by Type 1 virus • Transmitted by blood, sexually and mother-to-child transmission • There is a lot of drugs against HIV, but their effectiveness is limited

Retrovirus http: //fc 09. deviantart. net/fs 34/f/2008/301/4/8/Retrovirus_by_Velica. jpg

Diseases caused by HIV • Virus attacks mainly cellular immunity • After a non-specific primary infection there is a long latent period, where no symptoms • Then generalized lymphadenopathy occur, step by step opportune infections are found, and the disease comes to complete AIDS • In AIDS its own symptoms are not leading. The main finding in patients is occurrence of opportune infections (toxoplasmosis, pneumocystosis, various mycoses etc. ) and tumours

http: //vietsciences. free. fr/khaocuu/nguye nlandung/virus 01. htm

Diagnostics of hepatitis and HIV infection

And now diagnostics. Repeating first. • Aim of microbiological methods: to detect a pathogen, eventually its antibiotic susceptibility (not yet common for viruses) • Direct methods are all methods of – detection of a whole microbe as morphological of physiologic unit – detection of its part (antigen, nucleic acid) – detection of its product (e. g. a toxin) • Indirect methods: detection of antibodies against the microbe. Positivity = the microbe met the host IN HISTORY (weeks / months / years)

Survey of methods – and what will be used in J 10: • Direct methods (work with a sample or a strain) – Microscopy – in viruses complicated, see J 11 – Culture – in viruses complicated, see J 11 – Biochemical identification – not in virology – Antigen detection (using antibody) – Animal experiment (isolation, toxins) – rare – Nucleic acid detection • Indirect methods (work with a sample of serum) – Antibody detection (using antigen)

Direct detection of viruses • Culture isolation (virus is often not multiplied, only kept living). Needs cells. More in next part. • Microscopy: electronoptic rather for research than for routine diagnostics; optical for detection of in vivo/in vitro effect of viruses (inclusions, cytopathic effects) • Biochemical identification not here • Animal experiment here = viral isolation • DNA/RNA detection – viruses > bacteria • AG detection in sample – common in viruses

Indirect detection of viruses • Mostly used: CFT, various neutralisations (HIT, VNT) and in recently almost reactions with labelled components (mostly ELISA) • Attention! Not all reaction where serum is used as specimen, are indirect detection methods! In systemic viroses very often the virus itself or its antigen is present in serum and it is possible to find it here by a direct detection

Diagnostics of hepatitis A, C, D, E • HAV. Using ELISA method, we determine anti -HAV Ig. M + Ig. G, or Ig. M and total Ig • HCV. Again ELISA detection of Ig. M and Ig. G, and also PCR method. • HDV. Determining delta antigen (HDAg), antibodies (anti-HD) or viral RNA PCR • HEV. Again Ig. M and Ig. G ELISA detection, PCR is in development

HAV www. wallpaper. cz

Developement of Hepatitis A www-micro. msb. le. ac. uk/3035/HAV. html.

Specific diagnostics of HBV • In the middle of hepatitis B virion there is a nucleocapsid, where DNA is placed. Two core proteins, antigens HBc. Ag and HBe. Ag are present • The envelope is formed by another antigen HBs. Ag • HBs. Ag is overproduced, and so also empty envelopes are present in serum In the empty HBs. Ag, delta agens may invade – hepatitis D causative agent

Delta agens • Delta agens is a viroid, particle with uncertain virology classification • Delta agens may infect humans together with HBV (coinfection), or following after such infection (superinfection) • Presence of delta agens worsens prognosis of viral hepatitis

Complete virions and empty HBs. Ag www. uct. ac. za/depts/mmi/stannard/hepb. html.

HBV diagnostics • HBV has three antigens important for diagnostics. Only two of them can be found in patient‘s serum: HBs. Ag and HBe. Ag. • HBs. Ag is overproduced, present in huge amounts in serum suitable for screening • Antibodies may be assessed against all three of them: anti-HBs, anti-HBe and anti-HBc. • Also PCR is sometimes exploited, hepatic enzymes detection etc. • Interpretation needs combination of results

pathmicro. med. sc. edu/virol/hepatitis-disease 2. htm

HIV diagnostics • Antibodies against envelope glycoproteins using ELISA tests are assessed. When the result looks like positive/borderline, the serum specimen is sent to reference laboratory, that confirms the result using another ELISA and Western blotting. Prior to confirmation result the „positive“ result is referred as „reactive“, not „positive“ • Direct diagnostics using PCR can be performed. Virus isolation is possible, but very difficult and not performed routinely

Practical diagnostic approaches

Practical diagnostics of hepatitis, herpesviral and HIV infection: ELISA • Although some ELISA reactions are to antigen detection and some for antibody detection, practical approaches are similar. • Counting cut off: average of cut off wells, or average of negative controls + constant • Often cut off 10 ± % = borderline values • In some cases, especially in VHA diagnostics, we do not examinate Ig. M and Ig. G antibodies, but Ig. M and total antibodies. It is clear that negative Ig. M and positive total antibodies mean practically presence of Ig. G antibodies.

Practical diagnostics of hepatitis, herpesviral and HIV infection: PCR • PCR is mostly used in HCV diagnostics, eventually HIV diagnostics and diagnostics of some herpesviruses • The procedure is identical to that described in J 09 practical session

Herpetic viruses (including diagnostics)

Herpesviruses • Herpesviruses are DNA enveloped viruses of cubic (icosahedra) symmetry • There exist eight main human herpesviruses. Typical for herpesviruses is latency in either neural or lymphatic tissue. Secondary infection is usually different from the primary infection • They are subdivided into alpha- betagammaherpesvirinae (subfamilies) • Diagnostics is direct (PCR, viral isolation) and indirect

Survey of herpesviruses Abbreviation, subfamily HSV 1 + 2 (alpha) Full name Disease Herpes simplex virus Herpes VZV (alpha) Varicella zoster virus Chickenpox, shingles EBV (gamma) Epstein-Barr virus CMV (beta) Cytomegalovirus Infectious mononucleosis Cytomegalovirosis HHV 6 + 7 Human herpesvirus 6 Exanthema (beta) + 7 subitum HHV 8 (gamma) Human herpesvirus 8 Kaposi sarcoma

A herpesvirus http: //alandroal. weblog. com. pt/arquivo/virus-herpes-simplex. jpg

http: //www. spectrosciences. com/article. php 3? id_article=30 Scheme of a herpesvirus

Viruses of simple herpes (herpes simplex, cold sore) • There exist two types – HSV 1 and HSV 2. The first is supposed to cause herpes labialis, the other rather herpes genitalis – In reality, some sources say that genital herpes is recently caused by HSV 1 more frequently than HSV 2. On the other hand, as for chronicity, the specificity still exist. • The virus survives in neural ganglia. • We have to differentiate a primary infection (from outside, painful blisters) × a secondary infection (without any source)

Viruses of simple herpes (herpes simplex, cold sore) • Clinical symptoms of a primary infection encomprise mostly painful blisters in the mouth, eventualy on genitals • After period of latency and after a trigger (another infection, but also a big psychical stress) reactivations occurs in form of a classic herepes, mostly on the lips. Serious are complications, especially on the eye • Diagnostics: clinical, eventually viral isolation • Treatment by aciclovir, famciclovir and valaciclovir. We cannot get rid of the latency

Herpes labialis http: //missinglink. ucsf. edu/lm/Dermatology. Glossary/herpes_simplex. html

Herpes genitalis http: //www. ecureme. com/atlas/data/herpes_simplex 550_ab. htm http: //www. femail. com. au/genitalherpes. htm

Herpes simplex: live cycle opt. pacificu. edu/ce/catalog/1 4382 -AS/Herpes. html.

Herpes simplex symptoms opt. pacificu. edu/ce/catalog/ 14382 -AS/Herpes. html

Herpes simplex complications opt. pacificu. edu/ce/catalog/ 14382 -AS/Herpes. html.

Ocular forms of infection opt. pacificu. edu/ce/catalog/14382 -AS/Herpes. html.

Herpes simplex: treatments From above: famciclovir, valaciclovir, aciclovir opt. pacificu. edu/ce/catalog/14382 -AS/Herpes. html

Virus of chickenpox and shingles • Both diseases are caused by the same virus • The access of the virus to the organism is through respiratory ways, it multiplies in lymph nodes and spread by blood. Primary infection has the form of chicken pox. Shingles or zoster is a latent infection, activated e. g. by stress. • Diagnostics is difficult. Cultivation on human embryonic cells, PCR, ELISA • Treatment includes aciclovir, valaciklovir and famciclovir.

Varicella zoster virus (VZV) de. wikipedia. org/wiki/Varizella-Zoster-Virus.

Chicken pox www. pediatric. it/varicella. htm http: //www. ecureme. com/atlas/data/Varicella. Zoster_virus 550_ab. htm

Chicken pox http: //blogs. webmd. com/all-ears/2006/01/whyimmunize-against-chicken-pox-when. html

Shingles www. aafp. org/afp/20000415/2437. html. hebra. dermis. net/content/e 404/e 456/index_ger. html

Cytomegalovirus • The name comes from enlarment of infected cells • Acces to the body is by different ways. It spreads by blood. Primary and activated infection in majority of people has no symptoms • Serious is infection of pregrants (it comes through placenta and infects the foetus), in persons with diseased mostly cell mediated immunity (including HIV infection), in persons after transplantation etc. • Diagnostics: serology + viral isolation • Treatment (if needed): ganciclovir, foscarnet

s vir u alo Cy tom eg http: //www. med. unimarburg. de/stpg/ukm/lt/hygiene/vir o/radsak/agradsak. htm

CMV retinitis http: //www. kellogg. umich. edu/theeyeshaveit/congenital/cmv-retinitis. html

Serology answer in CMV http: //membres. lycos. fr/vividal/interpretation-Infection%20 a%20 Cytomegalovirus. htm days weeks months years

Viruses of sixth childhood disease (roseoloviruses, virus of exanthema subitum, HHV 6 A, HHV 6 B a HHV 7*) • These infections are typical for children aged six to nine months. Transmission is by contact • Infections of HHV 6 a HHV 7 are indeterminate diseases with fever, only sometimes also rash. Reactivations are asymptomatic, except transplanted persons and other immunodefficiencies. • Diagnostics ELISA, immunofluorescence • Treatment exceptionally by aciclovir *HHV in all cases means „human herpesvirus“

www. scienceclarified. c om/Ti-Vi/Virus. html HHV 6 www. koori-childrens-clinic. com/2 photo. htm

Exanthema subitum or roseola infantum www. kidspedia. co. il/mamar. asp? id=64

Epstain-Barr virus (EBV, lymphocryptovirus, virus of infectious mononucleosis) • The access to the body is by the mouth. It infects Blymphocytes and damages particular parts of immunity. Also liver may be damaged. There might be no symptoms, of tonsilopharyngitis, liver problems, or other. The virus is oncogene • It is transmitted by kissing, as it is excreted by saliva • Diagnostics: Paul-Bunnell reaction – proof of so called heterophilic antibodies agglutinating sheep RBCs, eventually its „OCH“ and „IM“ modifications, or detection of specific antibodies against different viral antigens (mostly EBNA and VCA) • Treatment: rather symptomatic

Importance of anti-VCA and anti-EBNA • Viral capsid antigen (VCA). Anti-VCA Ig. M appears early in EBV infection and usually disappears within 4 to 6 weeks. Anti-VCA Ig. G appears in the acute phase of EBV infection, peaks at 2 to 4 weeks after onset, declines slightly then persists for the rest of a person’s life. • EBV nuclear antigen (EBNA). Antibody to EBNA slowly appears 2 to 4 months after onset of symptoms and persists for the rest of a person’s life. • There exist also other types of antibodies that are sometimes tested, for example antibodies against the early antigen (EA). Anti-EA Ig. G appears in the acute phase of illness and generally falls to undetectable levels after 3 to 6 months.

EB virus pages 2. inrete. it/mbiomed/photomic. htm http: //www. ordinace. cz/clanek/infekcnimononukleoza/? increase=1

Infections mononucleosis http: //www. healthofchildren. com/I-K/Infectious-Mononucleosis. html

HHV 8 – Rhadinovirus (virus related with Kaposi sarcoma) • It contains unusually big amount of genes, coming from the host cell (molecular piracy). This is related with its oncogenity • Primary infection may resemble infectious mononucleosis, but without heterophilic antibodies • The virus is related with Kaposi sarcoma, a specific kind of tumor in AIDS patients • Diagnostics is still rather experimental

HHV 8 on the cell culture www. ucsf. edu/micro/faculty/ganem_folder/res. html

Kaposi sarcoma http: //www. mf. uni-lj. si/acta-apa-01 -3/poljacki. html

http: //www. dentistry. leeds. ac. uk/oralpath/viruses/viral%20 infections/HHV 8. htm

Kaposi sarcoma worldwide http: //www. elib. hbi. ir/persian/EMERGING_EBOOK/34_HH V 8. htm

The End >>> Lambda phage >>> http: //www. ucm. es/info/genetica/grupod/Cromovibac/cromovibac. htm

More to viruses Viral replication • either in nucleus (mostly in DNA viruses), or in cytoplasm (mostly RNA viruses) of the host cell. • before replication virus has to get rid of its protein envelope • proper course of replication depends on the viral type (RNA/DNA, single stranded – ss/ double stranded – ds).

Individual ways of replication • In majority of DNA viruses DNA-polymerase produces the complementary file • In hepadnaviruses (VHB) DNA is transcribed to RNA, according to that reverse transcriptase makes DNA again • In RNA viruses RNA polymerases are used • In retroviruses (HIV) reverse transcriptase produces DNA along RNA. Cellular RNA polymerase is then used for transcription.

Production of viral proteins • is necessary to enable the virus to exit the cell and to spread further • the proper process is again related with the viral type • in any case, virus uses partially the proteosynthetic apparatus of the host cell.

Individual possibilities of proteosynthesis • • +ss RNA: direct translation possible –ss RNA: „+ file“ has to be completed ds RNA: after – file a + file is completed retroviruses: reverse transcriptase produces DNA and then cellular polymerases work • ds DNA: usually according to – file a + RNA file is formatted • ss DNA: second file is formatted and then is is like in ds DNA

Viruses dependent to other viruses Some strange viruses cannot live without presence of other viruses • Adenoasociated viruses (AAV) belong to parvoviruses. Replication is possible only in presence of helping virus (adenovirus) • Hepatitis D virus – delta agens – is a viroid. It is an incomplete particle, that is only able to survive in envelope of hepatitis B virus (mostly formed by HBs. Ag).

Viruses and outer environment, disinfection • some viruses are very sensitive (e. g. HIV) • on the other hand, some are much more resistant than bacteria (e. g. rhinoviruses) • many disinfectants effective to bacteria have no effect to viruses, or elevated concentrations are necessary; this concerns mostly nonenveloped viruses. Iodine preparations and peroxides use to be effective. • Prions are very resistant towards high concentrations of disinfectants and high temperatures

Viruses as causative agents of diseases A child infected by cytomegalovirus

Ways of transmission • Ways of transmission are various; obviously, they are the same as in bacteria • there are viruses transmitted by droplet infection, faecal oral route, sexual transmission, vector (tick, mosquito) or blood (injection). • in majority of viruses „vertical“ transmission is also possible (mother to foetus)

Pathogenicity factors • Unlike situation in bacteria, in viruses usually individual pathogenic structures are not defined, rather the whole virus is considered to be one pathogen particle • This corresponds also with the fight with infection, where antibodies neutralize the activity of the whole virus. This is also related with elevated importance of neutralisation methods in diagnostics

Course of viral infection • in viruses inflammations are different from bacteria • it is mostly related with intracellular parasitism of viruses • mostly granulocytes are less important, lymphocytes more important, more important cellular immunity • components of non-specific humoral immunity are different, too (mostly interferons).

Latent infection • host cell enables penetration of a virus into a cell • its multiplication and release from the cell is not possible • on the other hand, virus survives in the cell, sometimes it is even integrated into the chromosome • in some cases, later this latent infection may be activated, so the infection appears again • it is typical in some herpesviruses.

Viruses and cancers • Some viruses are related with some types of cancer, mostly EB virus (causative agent of infectious mononucleosis – takes part in formation of Burkitt lymphoma) and HHV 8 together with HIV (Kaposi sarcoma), or human papilomavirus (HPV) – influence to cancer of cervix uteri • The reason is that the viral promotor in such cases may activate expression of human cellular oncogens, that would be otherwise suppressed (it would not be present).

Fighting viruses I – antivirotics • Used in some viruses only • Usually only limited influence in therapy • Enteric, oral or local (sometimes the same chemical, e. g. aciclovir – HERPESIN) • In vitro sensitivity testing still rather experimental • Among the most common: aciclovir, famciclovir, ganciclovir (herpesviruses), amantadin, rimantadin, oseltamivir, zanamivir (influenza), azidotymidin, PMPA (HIV virus)

Fighting viruses II – immunotherapy • We use both passive (antibodies) and active immunisation (vaccination) • Among regular vaccination: – Living attenuated viruses: all components of MMR (mumps, measles, rubella) – Non-living vaccines: viral hepatitis B, poliomyelitis (Salk) • Among other vaccines: lyssa (prophylaxis, not prevention) tick born encephalitis, VHA etc. • Among passive immunisation e. g. antibodies against varicella-zoster virus, HBV etc.

Check-up questions • 1. What is an importance of "total antibodies to HAV"? When could we consider hepatitis A? • 2. What does the expression “confirmation” mean in serology? • 3. What is the way of confirmation in HIV infection? • 4. What are the typical markers of hepatitis B infection? • 5. What group of people is extremely exposed to HCV? • 6. After how long period after a risky situation (e. g. a contact with blood or a sexual intercourse without a condom etc. ) is it reasonable to assess antibodies against HIV? • 7. Where HDV viroid uses to be hidden? • 8. What are the methods used for hepatitis C diagnostics? • 9. What are the ways of cut-off counting in ELISA? • 10. What types of hepatitis are transmitted by faecal-oral route of transmission?