Microbiology

General overview Fungi

1.5 million species, 600-1000 cause human and animal disease Chemo organotrophic Its cell wall contains polysaccharides, often chitin or cellulose and absorbs nutrients It's membrane contain ergosterol as the major sterol (lipid) Classification based on morphology → mushrooms, moulds and yeasts Lichen - symbiotic relationship between fungus and algae/cyanobacteria

Lytic cycle 1

Absorption (attachment) Specific - interaction of tail fibres and host receptor Host range mutation can occur Injection of nucleic acid into host Hershey - Chase experiment (1952) Phage carries genetic information into cells

Predominant Bacterial phyla that make up human microbiome

Firmicutes 62.3% Bacteriodetes 25.6% Verrucromicrobio 3.14% Proteobacteria 2.98% Actinobacteria 1.10%

General Gastronenteritis

Hepatitis A and E virus - not common in Australia, but common in third world countries Hepatitis E related to rubella on left hand side of genome, and right hand side is related to astrovirus → recominbant virus Top 5 foodborne pathogens: norovirus (800 outbreaks of virus in Australia each year, but food only 10% of transmission), salmonella, clostridium perfringens, campylobacter (associated with chickens), Staph aureus Gastroenteritis: 2 billion cases of acute gastroenteritis and 1.9 million deaths each year in children under 5 years (most in developing countries)→ one of leading causes of death by an infectious disease $60 billion in societal costs for the world Vomiting, diarrhoea, fever, malaise 1 episode per person per year in Australia 3 aetiological agents: parasites, bacteria and viruses In developed countries, bacteria is not so much of a problem due to sanitation, but viruses are much sturdier 2004 4.6 billion diarrheal disease → gone down to 1.8 billion

Antibiotic Resistance Mechanisms - target modification

Modification of the bacterial target can prevent antibiotic binding conferring resistance Point mutations at target site Biochemical modification e.g. methylation

Define Antibiotics

Substance produced by one microorganism that selectively inhibits the growth of another Modern interpretation - a compound that kills or controls growth of microorganisms in the host Antibiotics are selectively toxic to microorganisms and not humans - exploit evolutionary divergence of Bacteria and Eukarya

Treatment Enteric Bacteria

Treat symptomatically - antibiotics not recommended except for systemic infections The main defense against these infections are the good bacteria within the gut that the pathogen needs to compete with The antibiotic kills the good bacteria, makes them more sick

Good viruses

Tulip Breaking Virus - helps tulip to colonise regions that are higher in temperature Viruses called bacteriophages can infect and set in motion a genetic takeover of bacteria, such as E coli Viruses lead a "borrowed life" between life forms and chemicals - methylated own DNA and then chop up DNA of life forms Origins of molecular biology lie in early studies of viruses that infect bacteria

Fungal examples - Saccharomyces cerevisiae

Used to make wine, beer and bread Glucophile - ferments than respire glucose Make more ATP from respiration, but yeast ferments to produce alcohol and inhibit the growth of bacteria Primarily found on ripe fruits (e.g grapes) Vector transmission: european hornet, paper wasp → evidence of mating in intestines of social wasps (transmit to progeny) Sporulation occurs in the intestines of the wasp Meiosis creates genetic diverse - more chance of advantages Used as model organism in the lab - single celled, short doubling time (2h), full sexual life cycle (meiosis and mitosis), well defined genetics and easily manipulable Has an a and an alpha haploid → produces an a/alpha diploid Can produce a bud, but can also mate, release pheromones to attract cells, and fuse to form the diploid (conjugation) Meiosis: a diploid producing 4 haploid spores - ascal sac Eukaryotes share same internal complex structure as plants and animals - instrumental in defining key processes

Virions

Virus particles Insert carriers of the genome Assembled inside cells from virus specific components They do not grow and do not form by division The are regarded as the extracellular phase of the virus Take viral genomes from cell to cell They protect the genome in inhospitable conditions

Enteric viral symptoms

Vomiting (more associated with norovirus) Diarrhea Nausea Abdominal cramps Headache, muscle aches Fever (minority) Dehydration in young and elderly victims Some viruses may cause asymptomatic infections

Fungal examples - Cryptococcus spp.

Yeast cells with a polysaccharide capsule Frequently found in soils contaminated with avian excreta → pigeons and eucalyptus trees Capsule prevents desiccation Melanin → protects against UV, protection against heat/cold, reduces susceptibility to degradation by bacteria produced enzymes in soil Enzymes: proteases - nutrient acquisition, urease - nitrogen scavenging

Entero virus classification

7 genogroups Important ones are genogroup 1 and 2 Genogroup 4 infects cats and humans, 5 infects mice, 3 bovine species 40 different norovirus strains Group 2 is virulent pathogenic norovirus that causes pandemics

Antibiotic Resistance Mechanisms - antibiotic inactivation by cleavage

Antibiotic may be enzymatically degraded Beta lactamase (resistance enzymes) confers resistance to Penicillins Bet lactamases breaks lactam ring

Major fungal pathogens of humans

Candida albicans Aspergillus fumigatus Cryptococcus neoformans

Treatment of viral gastroenteritis

Disease is self limiting Rotavirus in young children can lead to life threatening dehydration Aims of intervention Prevention and treatment of dehydration Prevention of severe nutritional compromise Rehydration achieved using balanced glucose-alt oral rehydration solutions

Virus structure - capsid viruses

Environmentally stable → temperature, acid, proteases, detergents and desiccation Consequences Easily spread Retain infectivity after drying Resistant to detergents Survive in gut Resistant to sewage treatment Induce an immune response Usually lyse cell to get out rather than bud out

Discovery of Antibiotics

Fleming discovered the fungi penicillin but didn't know how to isolate the compound - it was left for 16 years Howard Florey and Ernst Chain developed a method for mass producing penicillin (1941) Demonstrated efficacy of penicillin to treat infection There is the emergence of resistance genes shortly after clinical use

Antifungal drugs - Echinocandins

Fungi have two structural sugars in cell wall - chitin and beta 1,3 glucan Only have one class of drugs that target one of the two sugars (beta 1,3 glucan) Non competitively inhibits enzyme that makes beta 1,3 glucan → cell wall is weakened and stressed Only administered IV Resistance is emerging Very low toxicity → targeting structure of fungus that is not like anything in human cells The cell wall of cryptococcus and zygomycetes are very different, so it doesn't work

Bacteriophage applications

Gene cloning and expression Restriction enzymes Phage display systems Phage typing CRISPR/Cas9 Medical application Phage therapy: natural, self replicating system with advantages over antibiotics Biotechnology applications Food production - prevent contamination of food by dangerous bacteria Phage therapy Solution to growing problem of antibiotic resistance Advantages Specific limited off target effects Phage is self reproducing - limited administration required Simple and inexpensive to produce Disadvantages Causative agent must be identified before use

HCV Diagnostics and Treatments

HCV Diagnostics Serology - not useful in acute phase Enzyme Immunoassay (EIA) - looks for antibodies to virus (testing can be done after 3 months - development of antibodies) → if find an antibody, don't know if they have gotten rid of the virus (know they have it but don't know if they still have it) RT-PCR based methods to quantify viral load (testing after 2 weeks) → used to monitor therapy 2012 HCV DAAs N55B-NI → polymerase inhibitors mimic ATP/GTP nucleotides and chain terminate, the replication of the genome is stopped N55B-NNI → bind to the polymerase and inhibit it Protease cofactor 4A → can't chop up the amino acid into the proteins Direct Acting Antivirals New drugs recently approved (2016) EPCLUSA: Sofosbuvir and Velpatasvir for genotypes 1-6 inclusive: SVR 12-94% ZEPATIER: Elbasvir and Grazoprevir for genotypes 1 and 4: SVR 12 - 94-100% Daklinza and Sofosbuvir for genotype 3: SVR 12 - 86-100% (without cirrhosis) TECHNIVIE: Ombitasvir, paritaprevir, ritonavir and ribavirin for genotype 4: SVR 12 >94%

Enteric Virus Transmission

Tolerant to temperature change Survives high levels of chlorine Transmission through fecal-oral route Highly contagious

Discovery of bacteriophage

1896 → volatile agent for limiting cholera 1909 → finds plaques 'transparent holes' and suggest viral agent 1915 → D'Helle father of phage therapy, there was an outbreak of Shigella and used phages to cure, later on treated patients with bubonic plague and cholera in India → pioneered commercial phage production Phage wasn't seen until 1950s Incubated phage with Shigella, plated Shigella and observed plaques, then filtered to get rid of bacteria 1920 → first bacterial strain typing using phages, 1940-1970 → phages become key foundation of molecular biology, 2019 → over 5000 phages examined by EM, phages found in >140 bacterial genera

Mould

A mould develops, a pigment called melanin is produced and incorporated into the cell walls Hypha - mycelium or filament, they branch off Septa - regulates volume of cytoplasm Spore structures - conidiophore, spores called conidia - grow off the hypha

Aminoglycosides

Act by blocking protein synthesis Inhibit function of the small ribosomal subunit (30S) Characterised by amino sugars linked by glycosidic linkage Common aminoglycosides include gentamicin, kanamycin, streptomycin Gentamycin is restricted to serious gram negative infections (toxic)

Patterns of Virus Infections - Persistent or Acute

Acute non persistent infections Rapid recovery - influenza, Hep A virus' Rapid death - rabies Persistence infection with acute onset Symptom free periods - reactivation (herpes) (latent, it is hibernating, reactivation) Long symptom free period followed by illness and death (EBV, HIV, HCB, HBV) (continully replicating, chronic, or CD4 cell count drops low) Chronic disease (HCV, HBV, humanpapilloma virus) HSV Latency Occurs in nerve cells Viral DNA maintained as an episome → not integrated into genome, limited expression of viral genes Reactivation may occur spontaneously or in response to stimuli such as skin trauma, sunburn, UV light or stress Importance of persistent infections May be reactivated → acute disease May cause chronic or progressive disease (Hep B,C and HIV) May lead to development of tumour (HBV, HCV, papillomavirus, Epstein-Barr virus, human T-cell leukemia virus (HTLV) May confuse diagnosis if a persistent infection is detected as an incidental finding

Superficial Fungal infections

Affects 25% of world's population Diseases caused by dermatophytes: Ringworm, Tinea, Athlete's foot Consume keratin - skin, hair and nails Called ringworm or tinea (single spore causes lesions, the tip of growth is inflamed) Diseases caused by Candida spp. (thrush) Thrush: mouth, vagina, penis, nails, skin Vagina - usually when taking antibiotics that usually keep normal fungal numbers in check Chronic mucocutaneous candida infection Can arise in individuals with an unusual combination of endocrine and immune dysfunction APECED autoimmune polyendocrinopathy candidiasis ectodermal dystrophy

Antifungal resistance - Polyene resistant mechanisms

Amphotericin B → gives ergosterol a hug, forms hole in membrane and then lysis Altering target, changes composition of sterol that end in membrane, have lower affinity for amphotericin B, fungal cell is protected Trigger membrane stress response because of altered sterol composition

Antibiotics vs antifungals

Antibiotic - any substance that inhibits the growth and replication of a bacterium or kills it outright Antibiotics work by affecting things that bacterial cells have that human cells do not There are many potential targets for antibiotic cells It is chemo organotroph, cell wall contains chitin and cellulose and membrane consists of ergosterol→ cell wall and ergosterol are main differences Only five classes of antifungal drugs (200 antibacterial classes) Protein synthesis and nucleus (DNA synthesis) do not work well against systemic infections Cholestrol v ergosterol Mammals have cholesterol, gunfi have ergosterol The fungal cell wall Double membrane, inner structural cell wall and outer fibrous layer (mannosylated cell wall proteins chitin and beta(1,3)-glucan Glycosylation, attaching sugars to protein (mannosylation, sugars being attached to proteins are mannans)

Regions that contain microbiota

Any region of human body exposed to outside world has a microbial community Hair Lining of nasal passages Entire surface of skin Vagina Oral cavity Stomach Gastrointestinal tract pH of stomach is 2 → helicobacter pylori Each body area has own characteristics set of bacteria The composition of our microbiota at different body niches is unique Bacterial composition is presented by six most commonly detected phyla: actinobacteria, bacteroidetes, cyanoabcteria, firmicutes, fusobacteria and proteobacteria The fungal composition: aspergillus, candida, malassezia, cladosporium and saccharomyces Viral composition: bacteriophages or eukaryotic viruses

HIV replication cycle

Binding - onto cell surface Fusion - fuse virus membrane with cell membrane and capsid enters cell Reverse transcription - turning RNA into double stranded DNA, which goes into the nucleus of cell Integration - this DNA is then integrated into host DNA Replication Assembly Budding - picks up envelope glycoproteins and cell membrane as it heads out of cell

Gentamycin

Binds irreversibly to 16S rRNA of the ribosomal small subunit Prevents translocation of aminoacyl-tRNA from A site to P site → inhibits protein synthesis Disrupt tRNA anticodon reading → misreading of mRNA Nephrotoxicity - kidney damage Ototoxicity - hearing loss Genetic predisposition for ototoxicity = mitochondrial m.1555A>G mutation 50% of aminoglycoside ototoxicity have this mutation Mitochondria contain bacterial ribosome → mutation increases similarity to bacterial ribosomes → causing cell death, inhibiting mitochondrial function

Phage resistance

Blocking of phage DNA injection Abortive infection systems (Abi) - interfering with phage DNA replication, RNA transcription development and morphogenesis Restriction modification Production of restriction enzymes by the bacteria which cleave at specific DNA motifs Bacteria methylates its own DNA to differentiate phage/host Protect from lytic cycle Can stop phage from coming into the cell Restriction modification system Abortive infection Killing the lysis enzyme, phage will stay in

Factors that lead to change in gut microbiota

Broad spectrum antibiotic (both gram negative and positive) can affect a lot of microbiome Dysiobiosis → causes disease or health (increase in T regulatory cell) Dysbiosis Unnatural shift in composition of our microbiota Results in reduction of numbers of symbionts and/or increase in the numbers of pathobionts Effects of antibiotics on gut microbiota Kill/inhibit infectious bacteria however it disrupts normal gut microbiota → dysbiosis The use of antibiotics has also been associated with intra and extra intestinal diseases Clostridium difficile associated diarrhea Type 1 and 2 diabetes Vancomycin resistant enterococcus Vulvovaginitis Eczema Autism The effect of diet on gut microbiota Food we consume feeds not only us, but also a vast and diverse community of microbiota within our gastrointestinal tract Mounting in vitro and in vivo evidence suggests that diet selects for the gut microbiota composition and that several health promoting and deleterious effects of diet are mediated by gut microbiota

Norovirus

Caliciviridae family Leading cause of acute gastroenteritis globally Positive sense, single stranded RNA virus Non-enveloped, icosahedral capsid Highly infectious and transmissible 699 infections globally → 2.2 million in Australia Economic costs to the world is $60 billion Norovirus detection RT-PCR of stool → sequencing for genotype and strain ID Enzyme immunosorbent assays (EIA) Old: direct and immune EM of stool samples Norovirus can be spread through vomitus Airborne transmission of virus Encodes 9 proteins, on the right hand side forms the icosahedral shell (180 of the proteins) Try and inhibit the production of N57 to prevent illness

Antibiotic Resistance Mechanisms - antibiotic inactivation by modification

Can be inactivated by chemical modification Gentamicin can be inactivated by enzymes (from resistance genes) Addition of proteins means the gentamycin is no longer the right charge or shape to enter through porins and deactivate bacteria

Virus properties

Can pass through 0.22 um filters Viruses discovered in 1900s → use filter and know there's no bacteria, inject and know the disease is caused by a virus Totally dependent on living cells for replication and existence Possess only one species of nucleic acid, either DNA or RNA (can be single or double stranded, linear or circular) Have a component for attaching or docking to cells Able to take over the host cell to propagate themselves

Mechanisms of production of disease

Cell death - cytocidal effect of virus, apoptosis, immune mediated Interference with the function of essential cells - hepatitis or myocarditis Body's response to deal with cell damage - mucus production Local effects of the immune response - rashes Systemic effects of immune response - fever Triggering autoimmune response - postinfectious encephalomyelitis

Different mechanisms of Antibiotics

Cell wall synthesis Folic acid metabolism Cytoplasmic membrane structure and function Lipid biosynthesis Protein synthesis (tRNA) Protein synthesis (30s and 50s protein inhibitors) RNA elongation DNA directed polymerase DNA gyrase

Define Viruses

Cellular organisms whose genomes consist of nucleic acid and which replicate inside host cells using host metabolic machinery to form a pool of components which assemble into particles called virions which serve to protect the genome and transfer it to other cells 2 phases to life cycle: inside cell to make viral proteins hijack machinery of cell, parasite uses the replication machinery of the cell Have to hijack the ribosome to make viral proteins In human cell HIV can make 50 000 copies of itself 25-350nm, pandoraviruses which are as big as bacteria (1 um)

Penicillin

Class of beta lactam Inhibits cell wall synthesis Peptidoglycan synthesis requires transpeptidation of NAM (N-acetylmuramic acid) linked peptide chain Penicillin binding proteins (PBPs) carry out transpeptidation (cross links amino acid chains together and stabilises cell) The beta lactam ring of Penicillin binds and inactivates PBP Unable to make new cell wall - growing cells lyse - cannot cross link amino acids Penicillin works on gram positive bacteria and gram negative bacteria The beta lactam ring can bind to the cell wall without a problem, however, the gram negative inner and outer membrane creates a permeability barrier against many antibiotics

Phage structure

Classic head-tail phage structure Classic T even phage structure -typically dsDNA phages from the family Myoviridae (infects E coli) (odd/even phages) Model organism in molecular biology Tail fibers attach to specific receptors (e.g. surface proteins, LPS, pili, lipoprotein) The sheath serves as a channel for DNA/RNA injection Capsid contains nucleic acid Phage plaque formation Zone of clearance caused by bacterial lysis = 1 plaque forming unit (PFU)

CRISPR-Cas9 System

Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins Originally recognised by Mojica and colleagues and independently by others They recognised these palindromic repeats were derived from extrachromosomal genetic elements (phages) → CRISPR carrier strains were resistant to infection Noticed that the bacteria collected phage sequences in genome, lined them up next to each, used sequences in genome to produce antisense (bind on to incoming phage DNA and stopped phage from infecting bacteria) Works by producing antisense RNA which cleaves the invading elements Acquired defence systems were thought to be present only in vertebrates - landmark discovery Conserved in bacteria and archaea

Enteric bacteria

Common Salmonella non typhoid Campylobacter Shigella Less common E coli Yersinia enterocolitica Aeromonas Plesiomonas shigelloides Nosocomial infections Clostridium difficile Overseas travellers Vibrio cholerae/parahaemolyticus Salmonella typhoid Bacteria - Salmonella Non colonisers - do not invade the gut (pass or imbed), but they do produce toxins Staph aureus, clostridium perfringens (spores), bacillus cereus (spores) Exposure to contaminated foods Short incubation periods (1-6hours) Vomiting Diarrhoea Treatment usually supportive care Gram negative rod with flagella Highly adaptable to different environments Contaminated foods (milk, meat - poultry) Serotyping is important Enteric fever or typhoid often acquired overseas Individual can become carriers Non-typhoid Most common form of salmonellosis Incubation period: 6-48 hours (needs to replicate in gut) Nausea, vomiting diarrhoea Symptoms may last for 2 to 7 days (longer than a virus) Clostridium perfringens Can cause food poisoning when cooked food is then reheated -If food is undercooked some spores may survive the cooking process The spores can germinate into vegetative (live bacteria) and will produce toxin as they undergo sporulation If the food is kept between 4 and 60 degree C for few hours spores can survive Spores can survive much better Colonizers Salmonella Shigella - blood dysentry (take antibiotics) Campylobacter Yersinia E coli Vibrio cholerae/parahaemolyticus Aeromonas and Plesiomonas Clostridium difficile (hospital outbreaks) Invade gut wall, tend to have longer incubation periods (12-24 hours)

Virion architecture - helical

Cylindrical shape (spiral staircase) E.g. ebola, influenza, measles, mumps Nuclear capsid that wind the proteins around the RNA All helical viruses are RNA not DNA Several RNA viruses undergo self assembly as a cylindrical nucleocapsid (hollow tube) The viral RNA forms a spiral within the capsid structure Each capsomer consists of a single protein All animal viruses with helical symmetry have a lipid envelope

Phage transduction

Definition Transfer of DNA to one bacterial cell to another by phage A type of horizontal gene transfer Process During the lysogenic cycle, phage integrates its genome into the host chromosome When phage eventually excises its genome from the host chromosome, it takes a small part of the donor DNA with it The phage genome, along with some host DNA, is packaged into the phage capsid Phage infects another cell and integrates into new host chromosome New recipient host now contains foreign DNA from donor Some have donor bacterium DNA gives to another bacterial cell

Antifungal resistance - Echinocandin resistant mechanisms

FKS1 mutations cause proteins that alter interactions with the non competitive inhibitor of beta 1,3 glucan Hotsports, pick up on where mutations are occurring → lead to altered binding of the drug to the FKS1 protein Another way is through cellular stress response The damage to the wall is sensed by stress response pathways which are activated results in upregulation of chitin synthesis → decreases amount of beta 1,3 glucan in wall and increases amount of chitin in wall → cell robustness restored

Overview of HIV

Fact: Infection rate of HTLV-1 is 45% in Central Australia → highest recorded prevalence rate in the world Acquired immunodeficiency - HIV/AIDS First described in 1981 HIV isolated in 1983 (killed more than 25 million people) First case in Australia in 1981 Otherwise healthy young people started having rare cancer and opportunistic infections only associated with immunocompromised people (esp. Young men) Attacks immune system, enters any cell that is CD4+ cells that constitute the immune system → when CD4+ goes below 250 then get AIDS (8-10 years) History Early 80's homosexual related immune deficiency Blood product recipients e.g. haemophiliacs Some hetersexual trnamission - particualrly in eveloping countries Test for antibody developed in 1984, widespread implementation in 1985 Incidence of HIV infection 10 times higher than previously thought (asymptomatic - 30%) Late 80s epidemic spread to most parts of world with explosiv epidemics in developing countries with few resources to combat speed Throughout 90s numbers increase to become worldwide pandemic Pre exposure prophalaxsis → drug before sexual intercouse to prevent virus → not fully effective (reduces risk by 90%) Death rate has dropped by 80% from 1995 introduction of drug No vaccine - they mutate a lot - need a CTL vaccine (cytotoxic white cell response instead of antibiotic response)

HCV

First identified in 1989 (actually 1987) Enveloped, positive, ssRNA virus Family is Flaviviridae Primarily infections hepatocytes in liver Member of Flaviviridae Family (yellow fever virus, dengue fever virus, west nile virus) Genera: Flavivirus → yellow fever virus, dengue virus, Pestivirus → Bovine viral diarrhoea virus, hepacivirus, HCV Clinical Features Most people are asymptomatic, symptoms 25-30% of people Malaise, legarthy, anorexia, nausea, liver enlargement may cause upper right quadrant pain Jaundice phase (icteric) - bililuria (dark urine, bile pigments in urine), pale stools and jaundice Liver process red blood cells → when damaged, this yellow colour is released into the body Liver Disease Progression Chronic Hep C infection, virus is continually replicating throughout life This leads to fibrosis of liver → then cirrhosis (function goes down, 100 jobs done for body) → leads to Hepatocellular carcinoma (with cirrhosis) Liver cancer is hard to diagnosis, treatment is difficult and often too late (similar to ovarian cancer) History Following the discovery of HAV and HBV, third type of infectious hepatitis was recognised responsible for most cases of post-transfusion hepatitis Virus was finally identified in 1989 by Choo et al. Discovery is probably one of the most elegant uses of molecular virology in recent years

Yeast

Fungi that favour unicellular life cycle Reproduce by budding, start with mother cell that grows and enter cell cycle A bud emerges from mother and grows → when equal in size mitotic division a barrier forms between them (septum), the mother and bud split → results in scars

Why becoming resistant?

Give livestock antibiotics everyday to avoid spreading infection Over-prescription Estimated two thirds of global antibiotics sales occur without any prescription No new classes of antibiotic to market since the 1980s (1984)

HCV Transmission and Epidemiology

HCV Transmission Route of infection - parental is most prevalent, unlike other flaviviruses HCV not transmitted by athopod vectors High rates in IVDU, haemophiliacs, recipients of unscreened blood transfusions Screening of blood products (from 1990) has reduced transmission of HCV by >95% Sexual and mother-to-baby does occur but not common (low prevalence (1.5%) among long term partners) (not sexually transmitted virus, unless blood is involved) HCV Epidemiology 135 million people infected with HCV→ decreasing Injection drug use accounts for the majority of transmission (>50%), 82% in Australia In Australia the peak of epidemic there were 10000 infections per year, around the year 2000 Most common reason for liver transport in Australia and US (50%) → the liver always gets infected again, but get another 20 years Kills 476,000 per year (liver failure/HCC) There are 7 genotypes of virus (35%different at nucleotide level, highly mutated virus more than HIV)→ geographical distribution Dominant is 1, then 3 Australia high rate of genotype 3 infections (45%) In Egypt there was a vaccination program against a parasite in 1970s that resulted in more than 25% of the population becoming infected with HCV (genotype 4)

Molecular biology used to discover HCV

HCV genome were isolated by screening cDNA expression libraries made from RNA (cDNA) from chimpanzees infected with serum from a patient with post-transfusion non-A, non-B hepatitis Lambda GT10 cDNA library made using of infected chimp High level expression, insert RNA fragments in regio, lytic cycle → screen plaque with antibodies To identify portions of the viral genome which are part of the cDNA library that encoded viral proteins, the libraries were screened with antibodies from patients who had non A and no B hepatitis Lambda library Once one of the antibodies attached to the virus, part of the genome could be found, and eventually the rest was figured out Hemophiliac people were given HCV through blood transfusions

Viruses has shaped history

HIV killed 25 million people Viruses killed more people in 1900s than two world wars More than 50% of the population of four indigneous tribes that inhabited the Sydney region died as a direct result of viral infections introduced by the Europeans

Progression to AIDS

High levels of viraemia during acute infection → sharp decline in CD4+ T-cell numbers Emergence of HIV specific immune response → down regulation of viraemia, partial recovery of the CD4+ T-cell count Long period of clinical latency ensues → viraemia relatively low, CD4+ T-cell count declines slowly When CD4+ T-cell count falls below 250 cells/uL → opportunistic infections, increase in viraemia and decline in CD4+ T-cell count Long term nonprogressors → studied Declining immune function and loss of memory T-cells leads to opportunistic infections e.g. Candida Later stages there can be the development of unusual malignancies

Barriers to development of new antibiotics

High rate of re discovery Economic → patient cured (short treatment of chronic diseases), eventual resistance, comparatively small market - narrow approval for specific disease Regulatory → 10-300M from bench to bedside Most are semi synthetic derivatives Small startup companies making antibiotics currently

Human gut microbiota

Home to diverse and complex microbial community of bacteria Essential for human development, immunity and nutrition Bacteria living in and on the human body are not invaders but are beneficial to their host An individual's microbiome may influence their future health Health microbiota Contains balanced composition of many different types of bacteria (immunological equilibrium) The effect of healthy gut microbiota on immune system Peace keeping bacteria release anti-inflammatory products which participate in tuning the host response towards tolerance Helps to prevent the pro-inflammatory effects of any pathobionts present in the microbiota, this maintaining intestinal homeostasis What is function of gut microbiota Gut microbes rely on complex CHO (dietary fibre) to complete their functions in the gut They digest resistant complex polysaccharides that we ingest in plant materials → fruits, vegetables, legumes and whole grains Digestion of these foods by the gut microbiota leads to release of compounds (metabolites) into gut that are taken up into the bloodstream These metabolites help to maintain the balance of immune system and whether we store or burn calories

How pathogens get into food? Foods most at risk?

How do pathogens get into food? The food is grown in contaminated conditions: irrigation water, fertiliser contain human sewage/chicken poo OR sewage enters oyster beds (filter feeders) e.g. Wallace Lake When the food item is prepared for packaging 'dirty' water is used to wash it During the preparation of the food When food is packaged or prepared by a sick worker/caterer who contaminated the food with pathogens The food is left out, gets contaminated and begins to rot (pathogens allowed to replicate) Raw meat containing pathogens is allowed to mix with fresh uncooked foods Foods most at risk for the transmission of gastroenteritis pathogens Shellfish (oysters, clams, mussels) Ready to eat foods that require handling but no subsequent cooking → salads, peeling fruits, deli sandwiches, finger foods, dips, communal foods

Different/new norovirus strains

In 2012, the New Orleans strain was the most dominant strain of norovirus, spread around the world (70% global norovirus infections) Then throughout 2012, a new Sydney strain emerged in March and by Sept/October it was 70% of all cases → new virus = pandemic (in Dec 2012) No population immunity to new strain → went worldwide Numerous new strains emerged since 2016 One of them is a recombinant virus f the 2012 Sydney norovirus → still causing outbreaks Emergence of New Norovirus Strains Every year in Australia, around 2.2 million people become ill from norovirus infections → some asymptomatic but most have gastroenteritis After 3 years 7 million people will be infected and will have some immunity A new strains emerges and herd immunity does not work The virus has become invisible to parts of the immune defence army → antibodies and T cells don't hit the target The new strain outcompetes the 3 year old virus as it has a larger susceptible population to infect, gastro cases for the year increase Viruses of Group 2 lineage have caused all global pandemics since 1996 (occurring around every 3 years): 1996, 2002, 2004, 2006, 2009, 2012, 2016 Norovirus has two primary mechanisms driving its evolution: mutation and recombination (hybrid strains)

Fungal Kingdom

In the past fungal kingdom divided into three phyla → Basidiomycota, ascomycota and zygomycota Separated based on sexual spores - meiosis - spore formation (sporillation) Can also produce asexual spores, used to survive in hard times and to disperse in environment Refer to table

Lysogenic cycle (lambda)

Indefinite persistence of the phage genome in the host cell, without phage production Phage does not kill cell Phage genome replicates at the rate of host DNA No structural proteins produced Restriction enzyme and ligase, cuts and pastes itself into host DNA Then becomes prophage

Cryptococcosis

Infection occurs by inhalation of basidiospores - normally cleared from the lungs by macrophages but people with AIDS or who are immunocompromised, this doesn't happen Most common cause is cryptococcus neoformans → 1 million life threatening infections per year world wide, 500 000 deaths per year in sub-Saharan Africa around 25,8 million people living with HIV in sub-Saharan Africa Diseases caused: pulmonary cryptococcosis, cryptococcal meningitis (AIDS patients), disseminated infection in severely immunocompromised 20-70% mortality, with antifungal intervention Cryptococcus spp. Polysaccharide capsule and melanin are anti phagocitistic prevent recognition of spores Proteases degraged lung tissue, allows for spores to travel to other parts of the body Even when engulfed by macrophage, it doesn't die, ability to scavenge nutrients

Aspergillosis

Infection occurs by inhalation of conidia Usually, macrophages in lungs clears these spores Most common cause is Aspergillus fumigatus → 200000 life threatening infections per year worldwide, stem cell and solid organ transplant patients Diseases caused → asthma, asthma with eosinophilia - allergic asthma, aspergilloma (clump of mould in lungs), invasive bronchopulmonary aspergillosis, disseminated aspergillosis 30-95% mortality, even with antifungals and surgical intervention Aspergillus spp. Secreted enzymes cause damage to lungs Melanin can protect them from being recognized by phagocytes The features developed for a saprophytic lifestyle also enables them to infect humans with weak immune systems

Antifungal drugs - Polyenes

Interacts with ergosterol, surround it to prevent it from forming a proper lipid bilayer, get pores in the membrane It is fungicidal Have administered intravenously, they are nephrotoxic (the differential binding affect 10X more: amphotericin B only hugs ergosterol 10 times more than cholesterol)

Fungal examples - Candida albicans

It is polymorphic Metabolically diverse - grows just as well on other carbon sources as it does on glucose Can grow in many morphologies - yeast, pseudohyphae, hyphae (white cells - propagate stably), chlamydospores (not sexual spores or asexual, growth form in harsh conditions) → depends on the growing conditions It doesn't do meiosis, working with diploid Will grow on any carbon or sugar source Commensal fungal of humans The gastrointestinal (GI) tract of at least 60% of humans is colonised with C.albicans It is also found in the mouth and vagina Poor coloniser of skin No environmental reservoir - only associated with mucous membranes of mostly humans Colonisation occurs at birth New growth morphologies have been discovered- white, opaque Mates inefficiently - two diploid cells that fuse to form tetraploid → randomly releases chromosome until it becomes diploids again Cells can interconvert between white and opaque There is also GUT - gastrointestinally induced transition cells → increased fitness in GI commensalism model, long but no pimples → GUT cells outcompete white cells in the environment, more fit in gut environment, as soon they come out of mouth gut they turn back to white cells (morphological plasticity)

Disease Progression of HCV

Liver usually cannot get rid of the virus, white blood cells will keep on attacking the virus → over a period of time, the virus markers will appear on the cells and will be targeted by immune system cells After time the liver is damaged (10-20 years) 20% of people die from Cirrhosis; HCC, ESLD Drugs can cure people in 8 weeks (98% success rate) Clearance and disease progression of HCV infection Outcome determined by a complex set of virus host interactions Host factors Initial immune response Age Gender Ethnicity Alcohol Host genetics Obesity Pathogen Viral quasispecies (population of viruses/sequences, diverse is worse) Viral load (viral titre: 10^7) Co-infection with HIV

Phage lifecycle (general)

Lytic cycle (virulent phage) Lysis of host and release of progeny particles Viral genes fully expressed Most phages are this type Lysogenic cycle (temperate phage) Phage DNA inserted into host chromosome (prophage) Most phage functions are switched off Prophage becomes active again in response to external stimuli When there are a lack nutrients, the phage can switch to lytic cycle

Phages of interest

MS2 - icosahedron of 180 proteins, used in water industry, filter in aeroplane, test phage, positive control, genome 3600 bp, makes 4 proteins (1 lysis protein) Cystoviridae (Phi 6) → precursor phage to hep c in zika, went from bacteria into animals and created whole families of other viruses Attaches to pillius of bacteria

Antibiotic Resistance Mechanisms - efflux pumps

Membrane transporters that transport molecules out of the cell Resistance to Tetracycline is conferred by the TetA efflux pump Tetracycline inhibits ribosome function and TetA reduces the concentration in the cytoplasm

Define terms (Human Microbiome)

Microbiota: the collection of microorganisms in defined environment Metagenome: the collection of genomes and genes belonging to the members of a microbiota Microbiome: the entire habitat including the microorganisms, their genomes and environment (metabolic/physiological) outputs living inside and on the body Exponential rise in publication investigating the human microbiome and possible links with human disease Evolution of molecula technologies and allows determination of entire microbial communities Prior to technologies, 200 bacterial species were recognised as co-habitants of human body → currently estimated the >10000 different microbial species occupy microbiome

Enteric bacteria Laboratory Analysis

Microscopy White cells - bacteria produce more white blood cells in stool Red cells - colonisers result in more red blood cells in stool as they imbed in gut causing bleeding Food particles Fat globules Cysts, ova, parasites Culture for bacterial pathogens (not done much currently)

Penicillin derivatives

Modification of penicillin side chain allows uptake through gram negative porins Ampicillin and Carbenicillim are semi synthetic Penicillin derivatives that are active against gram negative bacteria Put in an amine or carbonyl group to allow it to enter porins (changing the charge)

Role of phages in ocean

Most abundant and diverse form of DNA replication agent on planet 10million/mL seawater Viruses compromise 94% of nucleic-acid containing particles in the ocean, and 5% of ocean's biomass Essential in biogeochemical cycling (e.g. drive carbon cycling) by microbial lysis Prevent algal blooms

Candidiasis

Most common serious fungal pathogens of humans More than 400000 life threatening infections per year worldwide The GI tract of at least 60% of humans is colonised with it without apparent ill affect because it is kept in check by: Innate immune system Competition with bacterial microbiota Intact gut epithelial layer Groups at risk from invasive infections Cancer patients Organ and bone marrow transplant recipients Immunosuppressive therapy Patients with AIDS Major surgery (abdominal surgery) ICU 46-75% mortality even with antifungal treatment Can cause superficial infections, but systemic infections are serious that can escape the gut

Virion architecture - spherical

Normally in form icosahedron Best way of producing shell of equivalently bonded identical structures Minimum free energy state - can self assemble, no ATP needed Strong structure that can endorse a maximal volume Can be either DNA or RNA Composed of 20 facets, each an equilateral triangle and 12 vertices Because of axes of rotational symmetry is said to have 5:3:2 symmetry The larger ones usually have DNA (not direct correlation) RNA never have genomes over 30 000 base pairs (DNA can be 10X bigger than that)

Gastro/enteric viruses

Norovirus Rotavirus Adenovirus Astrovirus Sapovirus Some picornaviruses - kobuvirus Parvovirus - human bocavirus Naked icosahedral viruses, most have RNA Naked viruses (no envelope) - can survive the gut Common, particularly among infants and young children Occur in colder months in temperature climates (Sept/Oct - late winter, early spring in Australia) Nosocomial and day care and aged care facilities outbreaks reported (immunologically naive), cruise ships, prisons The emergence of new norovirus strains lead to epidemics and pandemics of gastroenteritis Hep A and E share similar properties to enteric viruses (transmission and structure) but cause hepatitis not gastroenteritis

Transduction with PK1C phage

Phage P1 is capable of producing a lytic infection, releasing hundreds of phage particles Ends of P1 change genome have 15kb terminal redundancy - space to incorporate bacterial DNA About 1/10000 of P1 phage particles carry some random donor DNA - occasionally this will be gene of interest P1 lysate from a donor strain is used to infect a recipient strain some recipient cells will have chromosomal DNA from donor cell Recipient cells that have incorporated donor DNA by recombination can be selected for - with nutrient deficient medium Generalised summary All fragments of DNA from any region of the chromosome have a chance to enter the transducing phage Viral enzymes hydrolyse host DNA - no lysogeny required Donor DNA still undergoes homologous recombination into recipient chromosome

Lysogenic cycle 2

Prophage is excised (cut) from genome under certain conditions → phage needs to escape when host is at risk, host DNA damaged or nutrients are low, lytic cycle recommences Lambda phage The life cycle of lambda is controlled by cl and Cro proteins Cl is encoded by the phage and is a lambda transcription repressor When nutrients are low, cl is stable and leads to lysogenic cycle When nutrients are high, Cro protein predominates and leads to lytic cycle

Antibiotic Resistance Mechanisms - reduced permeability

Reduced entry of antibiotic into cell Outer and inner membranes of GNB prevent uptake of many antibiotics Vancomycin tolerant S aureus (VISA) produces thicker cell walls

Antibiotic Resistance Mechanisms

Resistance mechanism are strongly selected in the presence of antibiotic Often encoded on mobile genetic elements like plasmids → efficiently transfer through bacterial population Some mechanisms for antibiotic resistance include Reduced permeability, Efflux pumps, Target modification, Antibiotic inactivation by modification, Antibiotic inactivation by cleavage

Fungal examples - Aspergillus spp.

Saprophytic moulds which are ubiquitous in the environment Dispersal is via conifia (asexual spores) → srivvial in harsh condition A. fumigatis lives in compost → wide fluctuations in temperature (has to have thermotolerance) Produce melanin → protects conidia from UV damage Secret extracellular hydrolases to assimilate nutrients

Combating resistance mechanisms

Semi synthetic derivatives of penicillin designed to be resistant to beta lactamase Compounds that inhibit beta lactamase: clavulanic acid, sulbactam and tazobactam No antibacterial activity Prevent degradation Augmentin = Ampicillin + clavulanic acid

Enteric viral infection -diagnostics

Serology Faecal sample is required to look for viral antigen by EIA or RT-PCR/PCR EIAs detect viral capsid proteins which are present in high numbers (10^8 viruses per g faeces for most enteric viruses) EIA's exist commercially for norovirus, rotavirus, adenovirus, astrovirus but not for sapovirus Molecular Methods Tests detect viral RNA or DNA The test often comes in a multiplex format - one test for all viruses Reverse transcription polymerase chain reaction (RT-PCR) assays for detection of the norovirus, rotavirus, sapovirus and astrovirus RNA is available PCR for adenovirus (a DNA virus, so RT is not needed)

Incubation times of viruses

Short incubation period: Adenovirus conjunctivitis (1-2) Common cold (1-3) Influenza (1-3) Arbovirus infections (3-6) Innate and adaptive immune system will attack the virus Medium incubation Poliomyelitis (7-14) Measles (13-14) Rubella (14-16) Varicella (13-17) Mumps (14-18) Take longer because they are infecting multiple organic (whole body and systemic infections), acute viruses are just in gut or lungs) Long incubation (times in weeks) Hepatitis A (3-5) Hepatitis B (10-12) Infectious mononucleosis (4-6) Rabies (4-7)

HIV antiretroviral use

Since 1993 (high point) incidence of AIDS has been reduced by 4-fold → due to introduction of drugs HIV drugs never get rid of virus → suppress viral load to very low levels If stop taking drugs, the viral load will increase drastically (compared to HCV where it is only a 8 week course) First drug Zidovudine (AZT) -1987 → mimic of base pair nucleotide Treatment of late stages AIDS patients increased their survival for several months In 1994 approved for use in preventing mother to child transmission → reduced chance from 25% to 5% Resistance to AZT quickly emerged - viral loads increased and progression to AIDS resumed HIV antiretrovirals Combination therapy (mid 1990s) Usually 3 or 4 different drugs in combination (>40 combinations available) Reducing viral load to undetectable levels Slows development of resistance Stop CD4+ decline - extending time on AIDS Decreasing transmission of HIV including vertical transmission → 4 fold reduction of HIV because people didn't pass on virus

Source and development of microbiome

Source of microbiome Majority of microbiota comes from other humans Newborn babies encounter microbes for first time during birth when coated within microbes from mother's birth canal Babies born by caesarean section first encounter microbes from the mother's skin and from other individual who touch the baby Development of human microbiome During and immediately following birth, babies are colonised by a range of microorganisms Major sites skin, oro-nasopharynx, gastrointestinal tract, urogenital tract The gut microbiome of babies born by vaginal delivery differs from that of babies born by caesarian section By 6 weeks of age the microbiota in babies born vaginally or by caesarian sections are very similar Meconium is first fecal material that comes from baby Age changes gut microbiota changes Most dramatic changes occur between birth and 2-3 years, when it starts to resemble a typical adult microbiome Over 2-3 years infants gradually acquire a microbiome resembling that in a typical adult Influencing factors Maternal diet, diabetes (gestational) Premature birth (NICU) Breast milk vs. formula Introduction of solid foods Antibiotics Teenage: hormonal changes, diet, obesity Adult: diest, pregnancy, obesity, ethnicity, host genetics, environmental factors

HCV Morphology

Spherical (icosahedral), lipid envelope virus, diameter ~60nm Attaches itself to lipids Has 2 glycoprotein embedded in envelope, coated by viral genome called E1 and E2, highly glycosylated and receptor hunting proteins of the virus to get virus through the cell HCV Genome and protease cleavage Virus genome: positive-sense RNA molecule 9.5kb Translated as a large 3000 reside polypeptide which is then cleaved by both cellular and viral proteases Contains two functionally distinct segments Start codon with untranslated region (to the left) → no protein product (342 bp) Then to the right is the genome and stop codons Get one large polyprotein/gene which is then cleaved by the protease (NS3 and NS2) The protease NS3 cleaves at the triangles → will chop up larger protein into around 10 smaller proteins NS2 cleaces the NS2 and NS3 junction The left hand side creates the icosahedral structure the the E1 and E2 receptors Viruses, particularly small RNA ones split their genome up into the structure of virion and enzymes involved in replicating RNA genome

Virus Structure - enveloped viruses

Still envelope from cell and wrap around capsid During maturation through a process termed budding - from cellular membranes Proteins in the envelope are viral encoded, there are two main types Glycoproteins that form the projections known as spikes Matrix protein - layer on inside envelope added rigidity (helical) No envelope = naked viruses, hydrophilic and are protected from organic solvents (stronger) Properties of enveloped viruses Released by budding and cell lysis Environmentally sensitive: acid, detergent, desiccation and heat Modify host cell membrane during replication Consequences Must remain in moist environments (usually transmitted sexually and through droplets) Spread in large droplets, secretions and transplants Cannot survive in GIT Does not always kill the cell to disseminate Protection from host immune system

Antifungal resistance - Azole resistant mechanisms

Stops ergosterol from being made, instead toxic intermediates accumulate in membrane, cells leak and are fragile So much Erg11 that the drug cannot inhibit all of it Main method of resistance is the upregulation of multidrug transporters Cdr1 and 2 are ABC transporters TAC1 and MRR transcription factors that activate expression of efflux pump → leads to upregulation of them The widespread use of azoles in agriculture → plants and crops → influences amount of resistance

HIV Virion structure and genome

Structure Organisation of a mature HIV-1 virion Envelope glycoproteins project from the outer lipid membrane Two copies of the HIV genome are contained within the core Has a matrix protein A squashed icosahedron into shape of a cone Virus needs RNA converted into DNA Genome Characteristic retroviral elements include the long terminal repeats (LTRs) as well as genes encoding the structural proteins (gag), reverse transcriptase/integrase/protease (pol), and the envelope glycoproteins (env) Also indicated are the genes encoding accessory proteins that are multifunctional and important in the virulence and infectivity of HIV infection

Antifungal drugs - Azoles

Target ergosterol by synthesis First made to clinic in 1980s, less toxic than polyenes No longer see Candida infection in HIV patients due to several therapies, but still problem in sub Saharan Africa This table only represents Fluconazole The azoles inhibits one of the enzymes in the chain → prevents intermediate from forming further, it is toxic sterol which makes the membrane in fungi cell unstable, aters membrane fluidity and growth inhibited Can be given orally Resistance to Azole drugs is a huge problem, serious drug interaction problems

Criteria for Classification of Viruses

The type of nucleic acid Presence of absence of lipid envelope Number of strands of nucleic acid and their physical construction: ss, dd, linear, circular, breaks, segmented Polarity of viral genome - viral genome acts as mRNA are termed 'positive stranded', those where a transcript is made first are termed as 'negative-stranded' Replication strategy The symmetry of nucleocapsid Properties Virion polymerase Virion diameter (nm) Genome size (kb) Representative of family

Antifungal resistance

There are pathogens intrinsically resistant to all classes of drugs Resistance mechanisms Target alteration or overexpression Upregulation of multidrug transporters/efflux pumps Cellular stress responses

Opportunistic invasive fungal infections (fatal)

Top 5 causative agents of opportunistic pathogenic infections Rhizopus oryzae, Cryptococcus neoformans, Pneumocystis jirovecii, Aspergillus spp., Candida albicans Together these create over 2 million life threatening infections per year worldwide Mortality rates are 20-95% despite the availability of antifungal drugs More people die from invasive fungal disease than from tuberculosis or malaria

Gut and Diet (diversity of microbiota)

Traditional populations → have microbiota accessible CHO, these have been shown to increase microbial diversity and metabolic output → can protect from Western diseases In the colon produce metabolites, most important being short chain fatty acids (positive effect → these enter circulation, has effect on gut and other areas of body Industrial populations → decrease in microbiota accessible CHO → microbiota diversity and metabolic output decreases → development of western diseases Over time a number of factors have led to a decrease in diversity of the gut microbiota in industrialized societies Require a MAC diet (high CHO diet) Other factors include diet, sanitation, antibiotics and C-sections and formula Diversity of gut microbiota is largely determined by your diet A low fibre diversity results in reduced microbial diversity Refer to screenshot for more info

Lytic cycle 2

Transcription and translation of phage genes Sequential transcription Immediate/delayed early genes - shut off macromolecule synthesis, replicate phage DNA Middle genes - transcribed throughout infection e.g. DNA replication Late genes - capsid assembly, cell lysis enzymes Utilisation of host ribosomes Individual proteins in different amounts Replication of phage genome Maturation and release Capsid assembly Association of DNA and capsid Lysis of cells and release (100-200 progeny phage)

Phage classification

Type of nucleic acid (DNA, RNA) Host (bacteria, archaea) Morphology (icosahedral, filamentous) → has both helical and icosahedral architecture Life cycle (lytic, lysogenic) Genome conformation (circular, linear) Envelope (rare) / no envelope One phage may infect one or many hosts (most are species specific) The vast majority of phages are unclassified Current phage classification scheme 2 order, 25 families Many family combinations ssDNA, circular, non-enveloped ssRNA, linear, non-enveloped dsRNA, segmented, enveloped dsDNA, linear, enveloped dsDNA, circular, enveloped

Classification of Viruses

Universal system for classifying viruses, and unified taxonomy has been established by the International Committee on Taxonomy of Viruses since 1966 Helps with predictions about replication, pathogenesis, transmission pathway More than 2.475 million virus species belonging to 6 orders, 134 families, 22 subfamilies and 420 genera are currently recognised

foodborne pathogens - faeces

Waste or excrement from digestive tract Water, food residue, bacteria, secretions of intestine and liver Brown colour due to the breakdown of hemoglobin Diarrhoea: frequent passage of watery bowel movements Severe dehydration is killer from diarrhoea

HIV Epidemiology and Transmission

~33 million infected around globe SE Asia and Africa have biggest burden in HIV Increased life expectancy through improved living conditions and health care from the 1950s-80s were eroded as a result of HIV-related mortality Went from 65 to 45 in countries with high HIV prevalence in 1980s Routes of Transmission Sexual transmission (>80%) Blood or blood products e.g. transfusion, intravenous drug use Vertical transmission (including breastfeeding) Occupational exposure to blood (needlesticks)