INFD3012 Infectious Diseases: LT 2 Hepatitis Virus PDF

INFD3012 Infectious Diseases: LT 2 Hepatitis Virus Dr Scott Stimpson Education Innovation | Infection, Immunity and Inflammation Faculty of Medicine and Health [email protected] The University of Sydney Page...

INFD3012 Infectious Diseases: LT 2 Hepatitis Virus Dr Scott Stimpson Education Innovation | Infection, Immunity and Inflammation Faculty of Medicine and Health [email protected] The University of Sydney Page 1  Copyright COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice The University of Sydney Page 2  Learning Objectives – Define the basic routes of transmission of hepatitis B, C and D, and ways to prevent this; – Summarise the clinical features of chronic viral hepatitis, and how to monitor disease progression; – Analyse serology results for chronic viral hepatitis, especially Hepatitis B; – Discuss new direct acting antiviral agents, and their ability to increase SVR rates when added to peginterferon and ribavirin. The University of Sydney Page 3  Types of viral hepatitis www.slideshare.net The University of Sydney Page 4  Hepatitis B and C Both cause acute and chronic infection Both discovered as causes of post-transfusion hepatitis Liver disease in both caused by host response to infection Many asymptomatic infections in all countries The University of Sydney Page 5  Risk of Chronic HBV Depends on age at infection birth – 90% for perinatal infection > mum to - baby giving – 20-50% for children 1 to 5 years old – < 5% for adults The University of Sydney Page 6  Diagnosis of Hepatitis B S-> I mouth post & > - active infection Serology - detection of HBsAg and HBeAg – HBsAg - in blood about 1 month following infection – indicates active HBV infection – HBeAg - indicates active viral replication and increased infectivity – Anti-HBs antibody – protective immunity against infection – confirms immune response to vaccination DNA detection – HBV PCR The University of Sydney Page 7  Hepatitis B Serology The University of Sydney Page 8  Serological markers for HBV infection Serological marker Clinical condition HBsAg Anti-HBs Total anti- anti-HBc HBeAg Anti-HBe Viral DNA HBc IgM Acute hepatitis + - + ++ + - - + ++ Immune tolerance + - + (+) + - ++ or eAg +ve CHB Immune control or + - + - - + + eAg –ve CHB Past infection - + + - - + - - Past immunization - + - - - - - ALT (alanine aminotransferase) helpful to screen for active disease, liver damage ALT usually elevated in chronic hepatitis Usually normal in immune tolerance or immune control The University of Sydney Page 9  Prevention of Hepatitis B AVOID EXPOSURE – screen blood – infection control – safe sex VACCINATION (recombinant HBsAg) – Now universal in childhood – At risk groups too POST EXPOSURE PROPHYLAXIS – babies born to HBsAg +ve (carrier) mothers – needlestick in non-immunes The University of Sydney Page 10  Why Treat Hepatitis B? Prevent cirrhosis > - liver tissue replaced with scar tissue Prevent liver failure Prevent hepatocellular carcinoma (HCC) > - liver cancer The University of Sydney Page 11  R.E.V.E.A.L: high baseline viral load is associated with increased incidence of cirrhosis Cumulative incidence of liver cirrhosis All subjects (N=3,582) Cumulative incidence of liver cirrhosis (% 40 Baseline HBV DNA level, copies/mL 36.2% 6 (n=602) 105–95% Single dose better compliance Age group 0–14: 359 20–24: 30018 30–39: 10865 15–19: 14639 25–29: 19745 40+: 6281 The University of Sydney Page 13  Genital Lesions obvious visually Papules Ulcers: Warts (HPV) HSV Syphilis Syphilis Chancroid (Haemophilus ducreyi) Vesicles Donovanosis (Calmmatobacterium granulomatis) HSV Candidiasis Scabies Gonorrhea Trichomonas Crusted HSV Scabies The University of Sydney Page 14  Genital Warts - Human papillomavirus (HPV), genotypes 6 and 11 are associated with 90% of genital warts harder to before treat if no vacive - - Recurrent but complications rare dry scal edges , - Genital Wart Lesions: Morphology varies from flat to filiform. - HPV 16, 18 ( oncogenic) main risk factor for cervical and anogenital cancer: PAP smear for screening vaccine for prevention of acquisition (6, 11, 16, 18) The University of Sydney Page 15  Genital Warts Clinic based: Cryotherapy, laser ablation, podophyllin 25% solution, trichloroacetic acid Patient administered: podophyllotoxin paint (0.5%) Imiquimod cream (immune modulator) immune reaction for treatment www.ecotarget.com/health The University of Sydney Page 16  Genital lesions: HSV-1, HSV-2 Genital herpes (ulcers) may be caused by Herpes simplex viruses: HSV-1 or HSV-2 (mostly). cold fores -more females F>M. Commonly asymptomatic. usually multiple, painful lesions + dysuria/discharge + viral symptoms fluid inside - Blister, ulcer heals within 2-4 weeks Recurrence common (generally same site) Genital herpes from HSV-1 infection: - Oral sex, milder than HSV-2, recurs less frequently The University of Sydney Page 17  Genital lesions: HSV-1, HSV-2 HSV in pregnancy: risk of disseminated infection and spontaneous abortion high risk of transmission if primary lesions during pregnancy Treatment: Primary Infection Aciclovir 400mg Famciclovir 125mg Valaciclovir 500mg Recurrence: Shortened by 1-2 days if treatment started within 72 hours The University of Sydney Page 18  Mpox Mpox (formerly known as monkeypox) caused by infection with a virus, the Monkeypox virus. from monkey L originated This virus is part of the same family as the virus that causes smallpox. Monkeypox virus was discovered in 1958, when two outbreaks of a pox-like disease occurred in colonies of monkeys kept for research. First human recorded case was in 1970. In 2022, mpox spread around the world. Examples of Mpox Rashes Photo credit: UK Health Security Agency  Mpox There are two types of Monkeypox virus: subtype I and subtype II. Subtype I causes more severe illness and deaths. Some outbreaks have killed up to 10% of the people who get sick, although more recent outbreaks have had lower death rates. Subtype I is endemic to Central Africa. Subtype II is the type that caused the global outbreak that began in 2022. Infections from subtype II mpox are less severe. More than 99.9% of people survive. Subtype II is endemic to West Africa.  Mpox Both types of the virus can spread through: Direct contact with infected animals Close contact (including intimate contact) with a person with mpox Direct contact with contaminated materials Preventing Mpox: Avoid close skin-to-skin contact with people who have a rash that looks like mpox and animals that carry the mpox virus. Get Vaccinated: Smallpox vaccine (JYNNEOS) ~80% efficacy A same vacum for mpox  Syphilis bacterial infection Treponema pallidum in human have to live Cannot be cultured > - dormant until Most detected disease is latent ~ active Clinically evident disease rare in Australia more > - fetus Primary mode of transmission is STI, second is transplacental Transmission occurs during early disease stages (primary and secondary) Requires exposure to open lesions with organisms present The University of Sydney Page 22  Syphilis Age group 0–14: 16 20–24: 604 30–39: 1706 15–19: 284 25–29: 927 40+: 1708 The University of Sydney Page 23  Treponema pallidum Slow growing spirochete (doubles every 30 hours) Only known host is human- obligate parasite Small genome (1041 proteins) Hard to work out why it is such a good pathogen: molecule -energy - lack of metabolic capacity (hard to make ATP) - Lack of obvious virulence factors - No toxins, doesn’t lyse cells in vitro: predicted hemolysins in genome show no hemolytic activity stable - Can't manipulate genetically doesn't mutate Very The University of Sydney Page 24  Primary rukcer Chancre at site of inoculation Usually painless Heal spontaneously Syphilis quickly becomes systemic – Spread to local lymph nodes The University of Sydney Page 25  Secondary Weeks to months after initial infection 25% with untreated initial infection Symptoms vary – Rash (most common) – Fever – Malaise – Anorexia The University of Sydney Page 26 Early Latent Infection demonstrable by serologic testing No signs/symptoms Duration of 1 year or less Potentially infectious Late Latent Asymptomatic infection beyond one year Slower metabolism and prolonged dividing time Requires longer treatment duration Thought too not be infectious ↑ have cases of transmit The University of Sydney Page 27  Tertiary -into organ and brain & From 1 to 30 years after initial exposure May never have clinically apparent primary or secondary lesions Untreated, 25-40% develop tertiary The University of Sydney Page 28  Neurosyphilis (4-25 years p.i.) – General paresis depends on which region was affected Personality change; hyperactive reflexes – Opthalmic involvement; Auditory symptoms – Cranial nerve palsies; Meningitis symptoms Tertiary Manifestations Gummas (1-46 years p.i.) Cardiovascular heart - nodular lesions of skins (10-30 years p.i.) -attack and bones - Aortitis - Tumor-like growths - Aortic aneurysm The University of Sydney Page 29  Congenital Syphilis – Transmission to fetus at any stage of disease in mother – Most likely primary or secondary – 40% result in stillborn – 40-70% of survivors infected at birth – 12% of infected die of complications Saber shin Hutchinson’s Teeth The University of Sydney Page 30  Diagnosis – Darkfield microscopy – Quickest and most direct method Primary and secondary syphilis Direct visualization of spirochete from moist lesions Requires experienced lab tech and proper equipment Negative results do not exclude disease. ↓ Rarely used in practice the bacteria may from have moved location that The University of Sydney Page 31  Diagnosis: Serological Tests – Nontrepomal test Non-specific VDRL - Venereal Disease Research Lab RPR - Rapid Plasma Reagin – Tests for reactivity (IgM, IgG Abs) to cardiolipin-cholesterol- lecithin antigen unless syphicis - – Used as screening tests Cheap, Sensitive – Reported as titers -useful to assess success of treatment or reinfection be false positive – False positives to autoimmune diseases, viral infections may women HIV , leprosy , malaria 2 in pregnant The University of Sydney Page 32  Diagnosis: Serological Tests – Treponemal test FTA-ABS - fluorescent treponemal antibody absorption TPHA -hemaglutination test for antibody to T. pallidum TPPA - T. pallidum particle agglutination assay patient's antibody bind to the bacteria – Typically used as confirmatory tests FTA-ABS – Detect antibodies directed against Treponemal cellular components – Qualitative – reactive or non-reactive Syphillis is a – False positive to other Treponemal bacteria > sub-type - https://www.memorangapp.com/flashcards/58561/Treponema,+Chlamydia,+Neisseria The University of Sydney Page 33  Diagnosis – Neurosyphilis – Assess positive patients for signs symptoms Opthalmic signs or symptoms fluid Evidence of tertiary syphilis cerebal sinus - – Lumbar puncture with CSF studies needed if any evidence of neuro involvement Cell count (>20 WBC per ml) Protein concentration - elevated CSF-VDRL: diagnostic in absence of blood contamination Treponemal tests not always recommended – FTA highly sensitive, many false positives The University of Sydney Page 34  Treatment For most stages of disease Neurosyphilis – First line First line – Penicillin G (i.m) – Penicillin G (i.v) Alternatives Alternatives G – Doxycycline – Ceftriaxone – Azithromycin in case allergy The University of Sydney Page 35  Treatment: Follow Up Early disease: Reassess response at 6 months and 12 months with titers - 4 fold increase in titer indicative of failure or reinfection Latent disease: Reassess response at 6, 12 and 24 months with titers - 4 fold increase in titer indicative of failure/ re-infection Study CSF, re- treat if signs or symptoms of syphilis Neurosyphilis: Repeat CSF every 6 months until cell count normal - Consider retreatment if not decreased after 6 months or not normal at 2 years The University of Sydney Page 36  INFD3012 Infectious Diseases: LT 5 Parasites in the Clinic Dr Scott Stimpson Education Innovation | Infection, Immunity and Inflammation Faculty of Medicine and Health [email protected] The University of Sydney Page 1  Copyright COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice The University of Sydney Page 2  Learning Objectives Describe the clinical presentation of different parasites and the settings these may be acquired. Recall the major pathogens responsible for many common symptoms. Outline how different assays can be used to identify and diagnose parasites. Outline treatment methods used for a variety of different parasites. The University of Sydney Page 3  Parasites in the clinic…. – Most patients present to a regular doctor's office. – Parasites can come from anywhere in the world, due to international travel. – This means diseases that are not native to the country could be the issue! – Crucial to understand the process taken to identify the exact parasite(s). The University of Sydney Page 4  Case study 1 – A 38-year-old asplenic man residing in California was hospitalised with a 40ºC (104º F) fever, hematuria, myalgia, fatigue and thrombocytopenia. – The patient had recently traveled to Arizona, Kansas and Oklahoma in the USA. – A blood specimen collected in EDTA tube was sent to the hematology lab for routine work-up. Images were captured and sent to the DPDx Team for diagnostic assistance. The University of Sydney Page 5  A thin smear for differential WBC count was used A. B. C. Smith,blue In some infections with intraerythrocytic parasites, the morphologic characteristics observed on microscopic examination of blood smears do not allow an unambiguous differentiation. Moreover, potential blood donors may have subclinical symptoms and very low parasitemia, undetectable in blood smears. In such cases, the diagnosis can be derived from molecular techniques, such as PCR. The University of Sydney Page 6  So, what was it? – This was a case of babesiosis caused by Babesia sp. – Morphologic features included: Pleomorphic ring forms in normal-sized red blood cells Figures A and B (blue arrows). Characteristic tetrad (Maltese cross) forms Figures B and C (green arrows). Extracellular form Figure C (red arrow). Presence of only rings and ring-like forms. - > SPCR diagnosis The University of Sydney Page 7  Babesiosis Babesiosis is caused by apicomplexan parasites of the genus, Babesia. Humans enter the cycle when bitten by infected ticks. During a blood meal, a Babesia- infected tick introduces sporozoites into the human host. cred blood cells Sporozoites enter erythrocytes and undergo asexual replication (budding). Multiplication of the blood stage parasites is responsible for the clinical manifestations of the disease. fick > - human mouse > - The University of Sydney Page 8  Treatment – The disease is more severe in patients who are immunosuppressed, splenectomised, and/or elderly. – For ill patients, babesiosis usually is treated for at least 7-10 days with a combination of two prescription medications — typically either: Atovaquone PLUS azithromycin; OR Clindamycin PLUS quinine (this combination is the standard of care for severely ill patients). The University of Sydney Page 9  Case study 2 – A 27-year-old female sought emergency medical attention in San Pedro Hospital, Logroño, Spain due to a foreign object in her eye. – Medical personnel removed the object, captured images and submitted to the DPDx Team for diagnostic assistance The University of Sydney Page 10  Under the microscope… A. B. C. Identification to the genus or species level involves comparing certain morphological structures on the larvae, including the anterior and posterior spiracles, mouthparts and cephalopharyngeal skeleton, and cuticular spines. Travel history can also be helpful for genus or species-level identification. The University of Sydney Page 11  So, what was it? maggots , sheep fly / – This was a case of ocular myiasis caused by Oestrus ovis, the sheep botfly. – Diagnostic features shown in the images were: Cylindrical, swollen or heavy-looking anterior end Backward projecting thick spines in widely spaced bands around the body Location in patient/host determined by morphology The University of Sydney Page 12  Oestrus ovis, the sheep botfly. – Adult females of O. ovis are larviparous and deposit their larvae directly into the nasal cavities of the normal host (sheep). – Larvae then move to the frontal sinuses and attach to the mucous membrane. When mature, they leave the host to form puparia in the soil. In humans, eggs are often laid directly into the conjunctiva by female flies. into mucosa first layer Close-up of the anterior end of a larva, showing the mandibles and one of the anterior The University of Sydney spiracles. Page 13  Treatment – Fly larvae need to be surgically removed. – No medications approved for treatment. – Preventing possible exposure is key advice for patients traveling in tropical areas of Africa and South America. Those with untreated and open wounds are more at risk. The University of Sydney Page 14  Case study 3 – A 22-year-old male noticed painless, ulcerating lesions with crusting and mild swelling on his back and face. – He had travelled to the rain forest of Peru five months earlier, where he experienced multiple insect bites all over his body. The University of Sydney Page 15  Under the microscope… A. B. A biopsy specimen from a lesion on his back was sectioned and stained with hematoxylin and eosin (H&E) distinguish between genetic material (Figure A). foreign bodies and A smear of a scraping from the biopsy site was also made and stained with Giemsa stain (Figure B). The University of Sydney Page 16  So, what was it? parasite – This was a case of cutaneous leishmaniasis caused by Leishmania species. The diagnosis was based on: Characteristic amastigotes in both figures. Amastigotes are small (1-2 mm x 1-5 mm), spherical to ovoid structures in which a nucleus and a kinetoplast are seen Presence of amastigotes (a protist cell that does not have visible external flagella or cilia) Travel history to an endemic region and reported exposure to the insect vector (sand flies) The University of Sydney Page 17  Cutaneous Leishmaniasis – Leishmaniasis is a vector-borne disease that is transmitted by sandflies and caused by obligate intracellular protozoa of the genus Leishmania. – Human infection is caused by about 21 of 30 species that infect mammals. – The different species are morphologically indistinguishable, but they can be differentiated by isoenzyme analysis, molecular methods, or monoclonal antibodies. start with macrophage and distribute via cymphatic system Ulcerative skin lesion, with a raised outer border, on a Guatemalan patient who has cutaneous leishmaniasis. The University of Sydney Page 18  Treatment – Decisions about whether and how to treat should be individualised. – The treatment approach depends in part on the Leishmania species/strain and the geographic area in which infection was acquired; the natural history of infection, the risk for mucosal dissemination/disease, the parasite’s drug susceptibilities in the pertinent setting; and the number, size, location, evolution, and other clinical characteristics of the patient’s skin lesions. – Conventional amphotericin B deoxycholate traditionally has been used as rescue therapy for cutaneous (and mucosal) leishmaniasis. – Oral agent miltefosine for treatment of cutaneous leishmaniasis in adults and adolescents who are not pregnant or breastfeeding The University of Sydney Page 19  Case study 4 – A 60-year-old non-smoking male patient presented to his primary care physician with a chronic cough and shortness of breath. – He reported no recent travel outside of the Southern United States. – A CT scan revealed focal consolidation and cavitation. Later, a lung biopsy was performed on a lesion that revealed possible parasitic forms on histopathological examination. The University of Sydney Page 20  Under the microscope… A. B. C. Diagnosed by the finding of the distinctive coin lesions on chest X-rays. The species that produce subcutaneous nodules are diagnosed by the finding of adult worms in biopsy specimens of these nodules. The University of Sydney Page 21  So, what was it? – This is a case of pulmonary dirofilariasis caused by Dirofilaria immitis, the canine heartworm. 4 can infect rumans from dog – Diagnostic features included: Smooth, thick cuticle with internal lateral ridges (arrows Figure C). Tall polymyarian and coelomyarian musculature. The presence of two reproductive tubules. The University of Sydney Page 22  Dirofilaria immitis, the canine heartworm. The genus Dirofilaria includes vector-borne filarial nematodes, which are usually associated with carnivore hosts. Some Dirofilaria spp. are zoonotic; the most commonly seen species in human patients are D. repens, D. tenuis, and D. immitis (the dog heartworm). Human dirofilariasis is generally divided into pulmonary dirofilariasis (D. immitis) and subcutaneous dirofilariasis The University of Sydney Page 23  Treatment – The definitive treatment of Dirofilaria infection in humans is surgical removal of lung granulomas and nodules under the skin; this treatment is also curative. In many cases, no treatment with medicines is necessary. – Dirofilaria immitis most often causes pulmonary disease in the human host but can also uncommonly cause nodules in other tissues. Humans are suboptimal hosts and larvae that migrate to the heart usually die.thence lung – Dead worms produce infarcts when they lodge in pulmonary vessels; these infarcts are usually referred to as “coin lesions” on chest radiography, which may be mistaken for malignancy. Following embolisation, patients may present with vague systemic symptoms (e.g., malaise, fever, chills) and respiratory distress. There is often a mild eosinophilia. – Ensure dogs receive heartworm treatment. The University of Sydney Page 24  Case study 5 – A 63-year-old man returned from visiting with family in Nigeria. – He developed fever, chills and a mild headache three days before presenting to the clinic. – A medical evaluation revealed that he also had not taken any drug prophylaxis prior to or during his visit. – A blood smear was ordered and stained with Giemsa The University of Sydney Page 25  Under the microscope… A. B. C. Plasmodium Malaria 4 showed infection = with Giemsa is a preferred stain, as it allows for detection of certain morphologic features (e.g. Schüffner’s dots, Maurer’s clefts, etc.) that may not be seen with other stains. Ideally, the thick smears are used to detect the presence of parasites while the thin smears are used for species-level identification. Quantification may be done on both thick and thin smears. The University of Sydney Page 26  So, what was it? – This was a case of malaria caused by Plasmodium falciparum. Morphologic features shown included: Numerous ring stage parasites in the thick smears. Schizont (rarely seen in peripheral blood) with 9-10 merozoites and compact pigment in one thick smear (Figure B). Infected red blood cells (RBCs) normal size in the thin smears (Figures C). Applique forms and ring stage parasites with delicate cytoplasm in the thin smears. – In addition to the morphologic features, PCR testing results also revealed that only Plasmodium falciparum was present.~ strain's treatment is different for different The University of Sydney Page 27  Malaria caused by Plasmodium falciparum. Blood parasites of the genus Plasmodium. There are approximately 156 named species of Plasmodium which infect various species of vertebrates. Four species are considered true parasites of humans, as they utilize humans almost exclusively as a natural intermediate host: P. falciparum, P. vivax, P. ovale and P. malariae. The University of Sydney Page 28  Treatment – Treatment of malaria depends on many factors including disease severity, the species of malaria parasite causing the infection, and the part of the world in which the infection was acquired. – These two characteristics help determine the probability that the organism is resistant to certain antimalarial drugs. – Additional factors such as age, weight, and pregnancy status may limit the available options for malaria treatment. – Extensive document available for treatment of different types of malaria infections.. The University of Sydney Page 29  So, who still wants to travel? – International travelers can be at risk for a variety of infectious and non-infectious diseases. Travelers may acquire parasitic illnesses: through ingestion of contaminated food or water, by vector-borne transmission, or through person-to-person contact. Many great diagnostics and treatments available today for the very wide range and diverse parasites we know of. The University of Sydney Page 30  INFD3012 Infectious Diseases: LT 6 Malaria and the host Dr Scott Stimpson Education Innovation | Infection, Immunity and Inflammation Faculty of Medicine and Health [email protected] The University of Sydney Page 1  Copyright COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice The University of Sydney Page 2  Learning Objectives – Identify the steps of the malaria life cycle – Compare and contrast the major types of human Plasmodia – Describe the epidemiology and features of malaria infection – Explain the influence of genetics on the development of malaria – Recognise the clinical features of both uncomplicated and complicated malaria – Recall the current drugs used for anti-malaria therapy, as well as other methods of prevention (e.g., vector control, vaccines) The University of Sydney Page 3  Malaria Parasitic disease causes by protozoa of genus Plasmodium, transmitted by Anopheles mosquito Major cause of mortality & morbidity with huge socio-economic impact 1. Plasmodium falciparum malaria - More severe, with greatest mortality (0.5 million pa) from cerebral & other complications, 60-75% total malaria - Sub-saharan Africa & South-east Asia (99.7% of estimated malaria cases in the WHO African Region, 2018) 2. Plasmodium vivax malaria - Can cause anaemia & pulmonary oedema (excess fluids in lungs) - SE Asia & Western Pacific, parts Africa & South America (3/4 cases) The University of Sydney Page 4  Life cycle of Plasmodium infection 2 stages www.thelancet.com Vol 391 April 21, 2018 The University of Sydney Page 5  Major types of human Plasmodia P. falciparum Single liver phase 5-7 days, then release 105-106 merozoites Infect RBC  mature from small ring form to trophozoite nucleus divides by asexual replication  mature shizont that ruptures after 48 hr, releasing merozoites 2.days after infected RBCs & , areexploded from all the merozoites Adherence “sticky” RBC to endothelium causes cerebral malaria and other blood cluts ↓ blood clots in brain complications causes immure react to system Massive expansion infection: after 12-13 days parasites increase from 10 to 108-10, patient starts to have fever from from parasitised ~ cytokine & interlenkin production RBIs non-immune host, rapidly progress severe disease with huge parasite numbers. (1012-13) The University of Sydney Page 6  Major types of human Plasmodia P. vivax > - need constant malaria pills Longer liver phase (12 d to months), some persist in liver & cause relapsing malaria Invades young RBC preferentially via Duffy RBC A chemokine receptor on RBC (can stop HIV but preferential to help malaria) antigen Similar erythrocytic cycle 42-48 hr, with fewer merozoites per shizont P. ovale (low prevalence, mainly West Africa & Asia) Also has prolonged infection liver  relapsing malaria 48 hr erythrocytic cycle P. malariae (low prevalence, West Africa) 72 hr erythrocytic cycle The University of Sydney Page 7  RBCs don't expand > - look larger RBCs do expand older RBCs ruffled RBCs do expand membrane www.thelancet.com Vol 391 April 21, 2018 The University of Sydney Page 8 Vector: Female Anopheles mosquito ~460 species Anopheles, >100 transmit malaria Development of sporozoites takes 6-12 days, period varies between vectors (shorter if warmer) Climate dependent: 16-33oC, altitude 7 day) - behaviour & blood-feeding pattern (evening, inside) - breeding characteristics (larvae breed in water) Anopheles gambia: highly efficient vector: feeds mainly humans, bites evening, bites indoors and rests indoors The University of Sydney Page 9 Epidemiology Complex interplay vector, host & parasite factors: Climate & climate change Seasonal rainfall increases breeding mosquitoes - larvae on surface fresh/salt water, marshes, swamps, rice fields Genetic factors Strong correlation malaria & poverty - impact malaria on fertility, population growth, premature mortality, productivity, economic development The University of Sydney Page 10  Global malaria prevalence https://ourworldindata.org/ The University of Sydney Page 11 Pathogenesis: Anemia & fever Anemia destroying RBCs Destruction of parasitised RBC, shortened RBC survival Fever Cytokine-mediated: raised plasma levels of TNF, IL-1, IL-6 GPI (glycosyl-phosphatidyl-inositol) anchor of P. falciparum is potent inducer of TNF; increase ICAM-1 & VCAM (tissue migration), promote sequestration of RBC in brain & coma The University of Sydney Page 12  Pathogenesis: Cytoadherence of P. falciparum-infected RBC & cerebral malaria Parasitised RBC: Erythrocyte Membrane Protein 1 (PfEMP1): express in RBC membrane, highly variable; help escape immune control Cytoadhesion starts after 12 h, highly effective, few mature Pf-RBC in blood Endothelium receptors: CD36: constitutively expressed in most tissues except brain ICAM-1: receptor in brain Chondroitin suphate A (CSA): main receptor in placenta Aird, William C. Blood 123 2 (2014): 163-7 The University of Sydney Page 13  Different genes influence development of malaria: High selection pressure of malaria for protective SNPs RBC genes 1. Entry: Duffy Blood Group antigen (chemokine receptor); Receptor for P. vivax: absent from Central Africa 2. RBC variants: limit growth of parasites; haemoglobinopathies (structural defects of hemoglobin) HbS: Sickle cell trait, heterozygote is protective, but confers risk sickle cell disease in homozygotes Thalassaemia: small RBCs; protects against Malaria Mediterranean & SE Asia Melanesian (South-East Asian) ovalocytosis: haemolytic anaemia in which the red blood cell is oval-shaped, confers resistance cerebral falciparum malaria. The University of Sydney Page 14 Mechanisms of Anti-Plasmodium Immunity: innate response The University of Sydney Page 15 Mechanisms of Anti-Plasmodium Immunity: adaptive response The University of Sydney Page 16  Clinical features Critically dependent immune status host Stable high transmission - severe malaria age 6 m to 3 yr, mild older children & adults have partial immunity & are asymptomatic Moderate transmission - symptomatic disease shifts to older age Low transmission & travellers - severe & symptomatic disease in all ages Pregnant women have risk of symptomatic & severe malaria at all ages The University of Sydney Page 17  Clinical features of uncomplicated Malaria Symptoms start when parasite start to infect RBCs Incubation period: ~ 2 wk but vary widely (travellers with P. falciparum, 90% within 8 wks; Vivax up to 9-12 m) Symptoms: Headache, myalgia (muscle pain), lethargy Fever, shivering & chills, followed high temp (40) & then fall As infection continues: enlarged liver & spleen, anaemia The University of Sydney Page 18  Clinical features of complicated Malaria Multisystem disease; in Africa mainly paediatric, but Asia & in travellers may be adult Cerebral malaria, seizures & coma: - if survive, generally recover well Severe anaemia, hypoglycaemia, renal impairment/failure Respiratory distress, liver failure The University of Sydney Page 19  Diagnosis of malaria Biosensors and Bioelectronics Volume 105, 15 May 2018, Pages 188-210 The University of Sydney Page 20  Treatment: types of drugs Aims Causation Therapy Drugs To alleviate Symptoms are Blood Chloroquine, symptoms caused by blood schizonticidal quinine, artemisinin forms of the drugs combinations parasite To prevent Relapses are due Tissue Primaquine relapses to hypnozoites schizonticidal of P. vivax/ P. drugs ovale To prevent spread Spread is through Gametocytocidal Primaquine for P. the gametocytes drugs falciparum, Chloroquine for all other The University of Sydney Page 21  Treatment: types of drugs Treatment of Malaria – Summary Type of infection Suppressive Treatment Radical Treatment P. vivax and P. ovale Chloroquine over 36-48 hours Primaquine for 14 days. P. malariae and P. knowlesi Chloroquine over 36-48 hours None P. falciparum Treatment depends on Primaquine in single dose as severity and sensitivity gametocytocidal Artesunate+Pyrimethamine- sulphadoxine or other artemisinin-based combination therapy (ACT), OR Quinine plus tetracycline The University of Sydney Page 22  Global distribution of drug-resistant Plasmodium falciparum www.thelancet.com Vol 391 April 21, 2018 The University of Sydney Page 23  Prevention & Malaria Control Minimise contact vector & human host - impregnated bed nets (highly effective while in bed & application associated with improved control) - affluent traveller (screens, protective clothing, insect repellants) Vector control - indoor residual insect spraying (IRS): highly effective with DDT in past & pyrethroids now, but issue of insecticide resistance & hard to sustain - larval stage (control ground water, insecticide spray, but environmental effects) The University of Sydney Page 24  Prevention & Malaria Control Prophylactic drugs to human host - travellers (see specific country guidelines) - Intermittent presumptive therapy: targeted to infants & pregnant women - Children aged 3–59 months in areas of seasonal transmission Anti-malarial vaccines - Two malaria vaccines are currently recommended for use in children living in moderate to high malaria transmission areas. - Current malaria vaccines reduce uncomplicated malaria by ~40%, severe malaria by ~30%, and all-cause mortality by 13%. - Malaria vaccines should be delivered in conjunction with other control interventions The University of Sydney Page 25  INFD3012 Infectious Diseases: LT 7 Central Nervous System Infections Dr Megan Steain Infection, Immunity and Inflammation School of Medical Sciences Faculty of Medicine and Health [email protected] The University of Sydney Page 1  Copyright COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice The University of Sydney Page 2  Learning Objectives – Describe how pathogens access the CNS – Compare and contrast the features of meningitis, encephalitis and brain abscesses and give examples of the pathogens responsible for each – Describe a prion and how it can be transmitted and lead to disease – Explain how different infectious agents can cause damage to the CNS – Recall methods to diagnose and treat different types of CNS infections The University of Sydney Page 3  Divisions of the nervous system Nervous System Central Peripheral Nervous System Nervous System (CNS) (PNS) Brain Spinal Cord Somatic Autonomic http://visual.merriam-webster.com/human-being/anatomy/nervous-system/central-nervous-system_1.php The University of Sydney Page 4  Protection against CNS infection – Bones of skull and Durible , fibroblast vertebral column – Meninges – Dura mater – Arachnoid mater Subarachnoid space: CSF and major arteries – Pia mater – The blood-brain barrier Kristensson, 2011, Nature reviews Neuroscience The University of Sydney Page 5  gating system The blood-brain-barrier selective – Specialised endothelial cells that line blood vessels in brain tight-controllingI limiting movements - – Form tight junctions with adjacent cells – Wrapped by astrocytes and pericytes vascular > - controls tone ↳ glialcells ↳ modulate functions & signalling Dahm et al., 2016, Mediators of Inflammation The University of Sydney Page 6  Pathogen entry into the CNS – Direct invasion via bloodstream – Cross endothelial cells infect the cells Viremia/ Bacterioria , directly Direct infection e.g. Poliovirus > - barrier Transit between tight junctions e.g. Neisseria meningitidis > tricking - the Disrupt BBB e.g. Nipah virus > break down the parrier - – Trafficking via infected blood cells & E.g. HIV, L.monocytogenes immure cell Trojan Horse , wide with the The University of Sydney Page 7  as > - peripheral Pathogen entry into the CNS - or (NS from infect sending neurons ↓ Rabies & Measles Herpes Spinal cord – Retrograde axonal transport from peripheral nerves The University of Sydney Page 8  Pathogen entry into the CNS – Olfactory route ~ smelling Olfactory neurons are exposed to external environment Allows for axonal transport of pathogens 9 Kristensson 2011. Nature reviews: Neuroscience, Vol 12. The University of Sydney Page 9  brain-eating The olfactory system and Naegleria fowleri - amoeba · freshwater s hish velocity The University of Sydney Page 10 https://onlinelibrary.wiley.com/doi/full/10.1111/j.1574-695X.2007.00332.x?sid=nlm%3Apubmed Pathogen entry into the CNS – Direct spread Contiguous infection e.g. sinuses, mastoid Following surgery or trauma Charlton et al.m BMJ Case Rep 2019; 12 The University of Sydney Page 11  Patterns of CNS infection Can have combination es : 1 +2 2+ 3 – 1 Meningitis Inflammation of the mengial layer Infection of arachnoid mater and cerebrospinal fluid (CSF) in subarachnoid space and cerebral ventricles Inflammation of meninges – 2 Encephalitis Widespread infection within brain or spinal cord Characterised by abnormalities of brain function – 3 Myelitis Infection of spinal cord polio – & Abscess Localised collection of pus The University of Sydney Page 12  Meningitis – Bacterial (pyogenic) or Viral (aseptic) – Symptoms include: Fever Headache Neck stiffness stretching meningial layer Photophobia optic nerve Others: focal neurological deficits, seizures, skin rash (meningococcal) 4) Skin rash – Absence of both parenchymal brain involvement and spinal cord inflammation The University of Sydney Page 13  Bacterial meningitis – Bacterial pathogens responsible vary with age of patient – High morbidity and mortality – Neonates ( - pus in urine > - The University of Sydney Page 12  Uropathogenic E. coli Cause up to 90% of all diagnosed UTIs 10 % undiagnosed UPEC use distinct virulence mechanism to cause disease in the bladder (cystitis) and kidney (pyelonephritis) UPECs use discrete virulence mechanisms compared to other pathogenic E. coli e.g. no type III secretion, invade host cells Frontiers in Microbiology | 1 August 2017 | Volume 8 | Article 1566 The University of Sydney Page 13  Attachment- Bladder Bladder surface lined by epithelial cells (urothelium) which is 3 to 4 cell layers deep tensile stretching against pressure ~ (umbrella cells) , Large, highly differentiated multinucleate ‘facet’ cells are the first cells exposed to pathogens Facet cells express four integral membrane proteins on their surface termed uroplakins Uroplakin embedded membrane stabilizes facet cells and prevents bladder rupture The University of Sydney Page 14  Attachment- Bladder Type 1 pili- binds mannosylated uroplakin proteins on luminal surface of bladder FimH-adhesion protein FimA, FimF and FimG- structural proteins The University of Sydney Page 15 Type 1 pilus-mediated bacterial attachment to bladder epithelium filarial bacteria in - epithelial cells The University of Sydney Page 16  Attachment-Kidney P pili bind glycosphingolipids on kidney epithelium Expressed by 80% strains causing pyelonephritis Adhesion protein PapG at distal end of P pili mediates adhesion to host cells not ~ binding to kidney Asymptomatic bacteria generally don’t express P pili P-pili trigger host response via Toll like receptor 4 The University of Sydney Page 17  Attachment-Kidney Frontiers in Microbiology | 1 August 2017 | Volume 8 | Article 1566 ascendin into Kidney ~ The University of Sydney Page 18  Invasion Type 1 pilus mediated invasion- via uroplakin proteins and integrans on bladder cells entry activ Fir H birding to ural picton > changing voles - & The University of Sydne y Page 19  Host cell invasion- FimH mediated internalisation Ladhesion & effector molecule Type 1 pilus mediated invasion- via uroplakin proteins and integrans on bladder cells The University of Sydney Page 20  Host cell invasion CNF1-mediated invasion: single chain toxin, secreted by 1/3 of UPEC strains - apoptosis bladder cells, aids bladder cell exfoliation & access underlying tissue Afa/Dr adhesions: can bind type V collagen which improves UPEC persistence in kidney Afa/Dr & Type 1-pili invasion directs UPEC enter cells via vacuoles, may help avoid fusion with lysosomes June 2016 Volume 80 Number 2 mmbr.asm.org 351 The University of Sydney Page 21  UPEC-associated toxins alpha-haemolysin: type-I secreted toxin, ~50% UPEC strains, correlates with clinical severity - high conc. inserts into membrane and lyses host cells travel into kidney , out into blood - Lower conc. modulates host signalling pathways, limit stimulation of inflammatory pathways June 2016 Volume 80 Number 2 mmbr.asm.org 351 The University of Sydney Page 22  UPEC-associated toxins & a L aid bacterial attachment The University of Sydney in bladder & Kidney Page 23  H antigen: flagella Appears to aid ascension of UPEC from the bladder to kidney http://www.ecl-lab.com/ The University of Sydney Page 24  for extracelinor infection Host defence- neutrophil recruitment neutrophil recruitment critical for bacterial clearance recruitment mediated by interleukin 8: high level of IL-8 in patients with UTI from tola receptor 4 IL-8 secretion induced by TLR4, mice lacking TLR4 highly susceptible to UTIs In humans low expression of IL-8 receptors (CXCR1) correlates with UTI incidence ninher 4k 8 - = duT The University of Sydney Page 25  recognise not alertin immure system 11-8 key cutlive = cannot recognise IL-8 The University of Sydney Page 26  Exfoliation of bladder cells facilitates bacterial clearance from the host but leaves smaller underlying immature cells more susceptible to infection 6 hours post-infection The University of Sydney Page 26 The University of Sydney Page 28 The University of Sydney Page 29 UPEC Type I filli interetre attachment & on create bistims G replication ascension into 2 interfe attachment Kidney I from epithelial cells recruitmentof S neutrophils I produced by epithelial cell - extracellular space so unsue their 1 response D make it no antibodies to Unable bind The University of Sydney Page 30  UPEC Kidney Bladder The University of Sydney Page 31  INFD3012 Infectious Diseases: Pathogenesis of Shigella Professor Jamie Triccas Infectious Diseases and Immunology Faculty of Medicine and Health [email protected] The University of Sydney Page 1  Copyright COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice The University of Sydney Page 2  Learning Objectives – Describe the features of Shigellosis – Compare the pathogenic mechanisms used by Shigella, with particular emphasis on: type III secretion systems and host cell invasion actin-based motility O-antigen glucosylation The University of Sydney Page 3  Shigellosis – a global public health problem Infection gastrointestinal tract, acute and severe inflammation: rapid & destructive to epithelial lining of the GI tract loss fluid diarrhea of , highly contagious, faecal-oral route >150 million cases worldwide/year; ~0.6 million deaths poor canitation young children unclean water sources , , 99% of all disease occur in developing countries. 70% children under 5 yo. The University of Sydney Page 4  Symptoms – acute inflammation of the mucosal tissues in the gut – Infection characterised by: – fever, severe abdominal cramps – watery diarrhoea – mucoid and bloody stools, characteristic of bacillary dysentery. – Secondary complications may occur: – septicaemia in the blood – pneumonia blood cells – Haemolytic Uremic Syndrome destruction of red The University of Sydney Page 5  Shigella spp. – Shigella are gram-negative bacteria from the family Enterobacteriaceae. E coli ete. , – Four species, S. flexneri causes the greatest level of mortality leprosy in human – Non motile, non-spore forming, rod-shaped I genome facultative anaerobic bacteria – Restricted host range: humans (and some non-human primates) are the only host – major hurdle for research no good animal model to study The University of Sydney Page 6  Host adaptation – Shigella are expertly adapted to human host Stomach Gastric acidity Duodenum Proteases Bile salts Jejunum and Ileum Antimicrobial peptides Colon High osmolarity Anaerobic Mucous barrier Organic acids Epithelial cell barrier Cellular response to infection The University of Sydney Page 7  Shigella pathogenesis The University of Sydney Can J Infect Dis Med Microbiol, 2006 Vol 17 Page 8  Shigella virulence A 215 kb virulence plasmid encodes ‘invasive’ phenotype 30 kb central pathogenicity island necessary for entry into epithelial cells & macrophage apoptotic death. This region plasmid encodes the structural components of a Type Three Secretion System. The University of Sydney Page 9  Secrete rivulence factors involves energy only 2 proteins that al very important The University of Sydney Page 10  Type VI protein secretion in bacteria Role of T6SS effector proteins in pathogenicity and inter-bacterial competition. direct well lysis The University of Sydney Page 11  Type VII protein secretion in (myco)bacteria McLaughlin et al Plos Pathogen, 2007 The University of Sydney Page 12  Type Three Secretion System – allows for delivery of a range of effector proteins directly from bacterial cytoplasm into host cell cytoplasm – Referred to as a molecular syringe Sekiya et al., PNAS 2001 Enninga et al., Cellular Micro 2009 The University of Sydney Page 13  The Shigella T3SS meadle Stabilise the Basal body Barison et al, Cellular Micro, 2013 The University of Sydney Page 14  Model of the secretion process through the T3SS the IpaD-guided membrane insertion of the IpaB-IpaC translocon at the needle tip > tells the bacteria Spa32 when it's long enough length regulation IpaD IpaC/IpaB stabilisation form) needle complex The University of Sydney Barison et al, Cellular Micro, 2013 Page 15  Ogawa et al, Nature Reviews Microbiology The University of Sydney Page 16  Shigella cell invasion – effector molecules ‘injection’ cause rearrangement of the host cell cytoskeleton and allows uptake of the bacterium because of IpaB/C – Shigella cause non-phagocytic cell to become phagocytic and internalise the bacteria Ipa proteins Sansonetti, FEMS Microbial Rev 2001 The University of Sydney Page 17  Actin based motility – Shigella utilise host cell actin as basis for motility Forms actin tail – Localisation of IcsA to one bacterial pole elicits accumulation of F-actin IscA N-wasp and Arp2/3 complex The University of Sydney Cossart, 2000 Cell Micro Page 18  Intracellular movement of S. flexneri by directed actin polymerization induce IscA- actin accumulation - cell deeth IscB- block autophagy site on IscA IscP- IscA localisation to one pole VirA- microtubule degradation 4 clear path to move to spread a propagate The University of Sydney Page 19  Actin-based motility Gouin, 1999 Cell Sci Cossart, 2000 Cell Micro The University of Sydney Page 20  S. flexneri-induced macrophage death Type III translocators/effectors: IpaB,IpaC Secretion of IpaB and IpaC aids escape from the phagosome Secreted IpaB triggers the proteolytic activation of procaspase-1 Active caspase-1 executes cell death Relase of proinflammatory cytokines IL-1 and IL-18- inflammation characteristic of shigellosis The University of Sydney Page 21  Shigella pathogenesis S TLR4 Can J Infect Dis Med Microbiol, 2006 Vol 17 The University of Sydney Page 22  Lipopolysaccharide of S. flexneri 5a N-acetylglucosamine O-antigen Rhamnose O-antigen O-antigen Core repeating sugars unit Glucose Lipid A Outer Phospholipids membrane Outer core The University of Sydney Page 23  O-antigen glucosylation – The serotype of S. flexneri is designated by the position of the glucose and/or O-acetyl residues on the O-antigen backbone ~ at different positio rhamnose Glucosyl group Il N-acetylglucosamine O-acetyl group different serotypes 1) Y 3a different virulence 1a 3b 2a 5a The University of Sydney Page 24  The “Sword and Shield” Theory resistant to killing > – a balance between the length/conformation of the LPS O-antigen to protect the bacterium against innate immune effectors (shield) and the function/accessibility of the TTSS needle (sword). type 3 into host cell Long LPS Balanceof needle & LPS Long needle /small UPS The University of Sydney West et al, Science. 2005 Page 25  INFD3012 Infectious Diseases: LT10 COVID-19 The Virus Dr Megan Steain Infection, Immunity and Inflammation School of Medical Sciences Faculty of Medicine and Health [email protected] The University of Sydney Page 1  COMMONWEALTH OF AUSTRALIA Copyright Regulation WARNING! This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice The University of Sydney Page 2  Learning objectives – Outline the major components of a SARS-CoV-2 virion and give an overview of its replication cycle – Describe how antivirals inhibit SARS-CoV-2 replication – Discuss the immune response to SARS-CoV-2 and immune evasion tactics employed by the virus The University of Sydney Page 3  SARS-CoV-2 – SARS-CoV-2 is a newly identified member of the Coronaviridae family – Virus responsible for COVID-19 (Coronavirus disease 2019) – Characteristics of Coronaviruses: – Single-stranded, positive sense, RNA genome, non-segmented – Enveloped – 80-90 nm diameter Image sources: Biorender and Almeida and Tyrrell, 1967, Journal of General Virology 1 The University of Sydney Page 4  Human Coronaviruses Virus Genus Year of Disease discovery HCoV-229E Alphacoronavirus 1966 Common cold HCoV-OC43 Betacoronavirus 1967 Common cold SARS-CoV Betacoronavirus 2002 Severe acute respiratory syndrome HCoV-NL63 Alphacoronavirus 2004 Mild respiratory illness/common cold HcoV-HKU1 Betacoronavirus 2005 Mild respiratory illness/common cold MERS-CoV Betacoronavirus 2012 Middle east respiratory syndrome SARS-CoV-2 Betacoronavirus 2019 Coronavirus disease 2019 (COVID-19) The University of Sydney Page 5  SARS-CoV-2 virion Spike protein Trimer, glycosylated + single-stranded (ss) RNA Binds to host cell ACE-2 receptor 30 kb N protein Bound to viral RNA M protein Forms the viral ribonucleoprotein Transmembrane protein (vRNPs) complex Organises virion assembly E protein Transmembrane protein Involved in assembly and viral The University of Sydney budding Page 6 Image sources: Biorender  Initiating SARS-CoV-2 infection – Exposure of mucosal surfaces to infectious virus can initiate infection – The spike protein of SARS-CoV-2 attaches to cells expressing the angiotensin-converting enzyme 2 (ACE-2) receptor- epithelial cells Image sources: Biorender and Leung 2021, The University of Sydney Nature reviews microbiology, 19 Page 7  SARS-CoV-2 replication cycle 1. Attachment and entry 2. Translation of the replicase gene (ORF1a and ORF1b) 3. Replication of the RNA genome and subgenomic RNAs 4. Virion assembly 5. Virion release The University of Sydney Page 8 Image sources: https://www.nature.com/articles/d41586-021-02039-y  Entry ACE-2 + – Spike protein interacts with ACE-2 – Facilitates attachment to target cells – Requires cleavage by host cell proteases (TMPRSS2) to mediate fusion of viral and cellular membranes – Or is cleaved by Cathepsin B/L if endosomal entry pathway is utilized Hoffman et al., 2020 Cell 181 Shang et al., 2020 Nature 581 The University of Sydney Page 9  2. Translation of the replicase gene (ORF1a and ORF1b) Viral RNA acts as messenger RNA to allow for expression of non-structural proteins (NSPs) NSP1 induces the cleavage of host mRNAs Blocks nuclear export of host mRNA Directs translation toward viral mRNAs NSPs remodels the host cell, creating ‘virus factories’ The University of Sydney Page 10  Virus Factories – Common strategy used by RNA viruses – Can provide: – High local concentration of proteins required for virus RNA replication – Shield from the innate cytoplasmic receptors (PRRs) – Double-membrane vesicles (DVMs), formed from cellular membranes (ER-golgi) – Form within 2 hr post-infection – 7 hr post-infection 200-300 observed in a single cell (Vero E6) – Merge later in infection The University of Sydney Page 11 Klein et al., https://www.biorxiv.org/content/10.1101/2020.06.23.167064v1  SARS-CoV-2 genome Replication of the RNA genome +ve RNA → -ve RNA → +ve RNA Replicase directly translated upon infection Structural Accessory proteins proteins 5 nsps 11 nsps Including RdRp The University of Sydney Page 12  4. Virion assembly Wolff et al., 2020 Science, 369 RNP: RNA genome + N protein https://science.sciencemag.org/content/369/6509/1395 The University of Sydney RTC: Replication-transcription complex Page 13  4. Virion assembly Accumulation of SPIKE protein The University of Sydney Page 14 Klein et al., https://www.biorxiv.org/content/10.1101/2020.06.23.167064v1  5. Virion