Viral Infection of Gastrointestinal Tract

Pharanai Sukhumungoon

Department of Microbiology, Faculty of Science Prince of Songkla University Overview Enteroviruses Hepatitis viruses Poliovirus Hepatitis A Enterovirus 71 Hepatitis B Coxsackieviruses Hepatitis C Gastroenteritis viruses Hepatitis D Hepatitis E Rotavirus Norovirus Astrovirus Picornaviridae

P = Polio i = insensitivity to ether c = Coxsackie o = Orphan r = Rhino rna = RNA

Pico = 10-12 (very very very small) • Poliovirus, insensitivity to ether, Coxsackie, orphan virus, rhinovirus, RNA • 35 genera • Enteroviruses • Poliovirus types 1, 2, and 3 • Coxsackie A virus types 1 to 22 and 24 • Coxsackie B virus types 1 to 6 • Echovirus types 1 to 9, 11 to 27, and 29 to 34 • Enterovirus 68 to 71+ • Rhinovirus types 1 to 100+ • Cardiovirus • Aphthovirus • Hepatovirus • Hepatitis A virus • Virion is a naked, small (25 to 30 nm) icosahedral capsid enclosing a single-stranded positive RNA genome. • Smallest virion in size and complexity • Contains 60 subunits, • RNA (30%), protein (70%) • Genome: single stranded, positive sense RNA, infectious, contains VPg (viral encoded protein) • Virus replicates in cytoplasm. • Viral RNA is translated into polyprotein, which is then cleaved into enzymatic and structural proteins. • Protein: VP1 and VP3: antibody binding sites, VP4 internal protein Icosahedral structure • Enteroviruses are resistant to pH 3 to pH 9, detergents, mild sewage treatment, and heat. • Tolerant to: 70% ethanol, other disinfectants in the lab, all antibiotics. • Susceptible to: 0.3% formaldehyde, 0.1 N HCl • Enteroviruses are thermo-labile (just 50ºC is enough to deactivate them) but it must be without magnesium chloride. • They can survive in frozen state for years and for weeks in refrigerator. -Portal of entry is alimentary tract -Initial multiplication of virus in lymphoid tissue of pharynx and gut  viremia  reticuloendothelial system

-Spread to target organs, eg. Spinal cords, brain, meninges, myocardium, skin

-Incubation period (time from exposure to onset of disease) about 7-14 days

-Virus is shed in stool for weeks and can be found in pharynx 1-2 wks after infection. Historical Highlight

CDC/Public domain

♣ First found enterovirus JollyRoger/Public domain ♣ Acid resistant ability at pH 3 or less ♣ First infect and multiply in the tonsil, lympnode of the neck, Peyer’s patch, small intestine  then, CNS is invaded by way of circulating blood Clinical features

♣ Asymptomatic infections ♣ Abortive poliomyelitis (the most frequent form of this disease) ♣ Viral meningitis (Aseptic meningitis or nonparalytic poliomyelitis) : can recover within 2-10 days ♣ Paralytic poliomyelitis: flaccid paralysis ♣ Progressive post-poliomyelitis muscle atrophy (PPMA) Progressive post-poliomyelitis muscle atrophy (PPMA)

-Not from a consequence of persistent infection but rather a result of physiological and aging change in paralytic poliomyelitis patients.

-PPMA may be caused by the loss of anterior horn cells or peripheral disintegration of individual nerve terminals in motor units that were reinnervated during recovery after acute disease. These 2 factors can be occurred with age.

-Seldom occurs before 60 years old. Paralytic poliomyelitis

CDC/Public domain Prevention and Control

Vaccine is composed of 3 types of polioviruses (type 1, 2, and 3)

♣ Salk vaccine (killed virus) ♣ Sabin vaccine (attenuated virus) better than salk in the aspect of epidemiological prevention

-Disadvantage of sabin vaccine is the back mutation. Thus, in the region that almost lack polio cases, salk should be used.

USAID/Public domain Reference:

Tobus/CC BY-SA 4.0 Virology Clinical Syndrome 1. Properties are common to • Coxsackie A virus poliovirus • Herpangina • Aseptic meningitis 2. Antigenically heterogenous • Paralytic disease 3. Two groups, A and B • Infantile diarrhea 4. Group A: serotype 1-24 • Minor illness (cold-like) • Coxsackie B virus 5. Group B: serotype 1-6 • Myocarditis 6. Pathogenic of suckling • Aseptic meningitis mice • Respiratory disease • Paralytic disease • Pericardial effusion • Pancreatitis  insulin- dependent diabetes

Pleurodynia (Bornholm disease)

• Chiefly caused by Coxsackievirus B virus (but also reported to be associated with Coxsackie A and Echoviruses).

• Distinguishing characteristic of this disease is the severe pain in the lower chest, often on one side, believed to arise from inflammation of fibrous tissue.

• The slightest movement of the rib cage causes a sharp increase of pain, which makes it very difficult to breathe.

• The illness lasts about a week and is rarely fatal. • Chiefly caused by Coxsackievirus A (A1-A6, A8, A10, and A22) • Illness is characterized by an abrupt onset of fever and sorethroat (there may be dysphagia, vomiting, abdominal pain) © James Heilman / CC BY-SA 4.0 • Pharynx is usually hyperemic and a few characteristic tiny discrete vesicles with a red areola occur on the posterior pharynx and over 24 hours these become shallow ulcers, rarely larger than 5 mm diameter. • Usually occur in small children in summer • Self limited (ulcer healed within 7 days)

•It can be differentiated from herpetic gingivostomatitis by the positioning of vesicles. In herpangina, they are typically found on the posterior pharynx, as compared to gingivostomatitis where they are typically found on the anterior pharynx and the mouth.

*The term herpangina refers to Coxsackie A virus even if it can cause by Coxsackie B or Echovirus. Hand, Foot, and mouth disease

• Most cases are due to Coxsackievirus A16 or Enterovirus 71 • Almost Universal sign is enanthema (eruption of mucous membrane), most commonly on the buccal mucosa • Exanthema soon follows, most frequently on the hands and feet. • The intraoral lesion are ulcerative but on feet and hands are usually vesicular.

Virology Clinical findings 1. Properties are • Aseptic meningitis common to Poliovirus • Encephalitis 2. Numerous type • Respiratory diseases 3. Cause no disease • Diarrhea in suckling mice • Minor illness 4. Found in alimentary tract

-Almost all Coxsackie and most of echoviruses are associated to some degrees of meningitis and (very rare instances) with paralytic CNS disease. • Most enterovirus infections are mild and resolve spontaneously • Intensive supportive care may be needed for cardiac, hepatic, or CNS diseases • IV, intrathecal (through spinal cord reaching CSF), or intravenous immunoglobulin halt the progression of disease • chronic enterovirus meningoencephalitis and dermatomyositis in patients with hypogammaglobulinemia or agammaglobulinemia • Good hand-washing practices and the use of gowns and gloves are important in limiting nosocomial transmission • Inflammation of the stomach and small and large intestines caused by a variety of viruses that results in vomiting or diarrhea. • Often called the “stomach flu” • Acute infectious gastroenteritis is a common illness that affects persons of all ages worldwide • It is a leading cause of mortality among children in developing countries • Elderly persons, are also at risk of severe complications and death from acute gastroenteritis • Most viral gastroenteritis is caused by RNA viruses

Characteristics of Reoviridae

♣ Size : ~ 70-85 nm. ♣ Segmented dsRNA ♣ 10-12 segments, grouped in 3 sizes, Large, Medium, small ♣ Icosahedral capsid, non-enveloped virus

♣ Double-protein capsid shells

♣ Replication occurs fully in cytoplasm ♣ Lack of complete uncoating of virion ♣ Possession of all enzymes required for dsRNA transcription Electron micrograph showing virion characteristic of Rotavirus Proteins encoded by 11 segments of Rotaviral genome VP4 protein (P-serotype) (acts as hemagglutination) ♣ Protein spike 60 pieces with a knob-like structure ♣ Encoded by segment 4 (1.5% of virions) ♣ induce neutralizing antibody

VP7 protein (G-serotype) (acts as outer capsid) ♣ Glycoprotein encoded by segment 9 ♣ Induce major neutralizing antibody

♣ Form smooth external surface of the outer shell (30% of virion proteins) VP6 protein (inner capsid) ♣ Inner capsid of the virus, encoded by segment 6 (51% of virion proteins) ♣ Determine the group of virus Rotavirus infection

VP4, VP7 VP6

Particle double-shelled single-shelled core Structure Triple-layered double-layered single-layered proteins VP1, 2, 3, 4, 6, 7 VP1, 2, 3, 6 VP1, 2, 3 Biological properties Infectious Non-infectious Non-infectious

Low infectious dose Rotavirus mortality rates by country, per 100,000 children <5 years of age Rotavirus: Pathogenesis • Rotaviruses infect and ultimately destroy mature enterocytes in the villous epithelium of the proximal small intestine. • The loss of absorptive villous epithelium, coupled with the proliferation of secretory crypt cells, results in secretory diarrhea. • NSP4, functions as an enterotoxin and contributes to secretory diarrhea. • Rotavirus may evoke fluid secretion through activation of the enteric nervous system in the intestinal wall. • Ranges from sub-clinical infection to severe gastroenteritis leading to life-threatening dehydration • The stools are characteristically loose and watery and only infrequently contain red or white cells • In severely immunodeficient children, rotavirus can cause protracted diarrhea with prolonged viral excretion Risk factors affecting the increase of Rotavirus infection in children

1. Lack of breast-feeding 2. Low birthweight 3. Daycare attendance 4. Maternal factors (young age, smoking)

♣ Prolonged gastroenteritis in immunocompromised host but the severity is comparable • Diagnosis can usually be confirmed by a wide variety of commercially available EIAs or by techniques for detecting viral RNA, such as Polyacrylamide gel electrophoresis (PAGE), hybridization, or RT-PCR • Treatment: • Standard oral rehydration therapy or IV • Antibiotics and antimotility agents should be avoided • Prevention • Rotavirus vaccines Prevention and control

Vaccine recommended by WHO ♣RotaTeq® (RV5), licensed in 2006, is given in 3 doses at ages 2 months, 4 months, and 6 months. ♣Rotarix® (RV1), licensed in 2008, is given in 2 doses at ages 2 months and 4 months.

Both are given orally Norovirus (Norwalk virus)

• Gastroenteritis that occurred in a school in Norwalk, Ohio, in 1968 • Norwalk virus is the prototype strain of a group of (+) ssRNA genome, nonenveloped, small (27–40 nm), round, icosahedral viruses • Not been adapted to cell culture, they often are shed in low titers for only a few days, and no animal models are available • Classified in Caliciviridae • Two genera belonging to the family Caliciviridae: the noroviruses and the sapoviruses (previously called Norwalk-like viruses and Sapporo-like viruses, respectively • Major cause of epidemic nonbacterial gastroenteritis • Incubation period 24-48 h, recover in 60 h • Broadening and blunting of the villi • Shortening of the microvilli • Vacuolization of the lining epithelium • Crypt hyperplasia • Infiltration of the lamina propria by polymorphonuclear neutrophils and lymphocytes

Lack of author detail / public domain • Symptoms include diarrhea, nausea, vomiting, low fever, abdominal cramp, headache, malaise • Vomiting is more prevalent among children • The stools are characteristically loose and watery, without blood, mucus, or leukocytes • Diagnosis assays based on polymerase chain reaction (PCR) for detection of virus in stool and vomitus • Worldwide distribution in all age range • Supportive treatment, practice good hygiene, no vaccine • Immunity to Norovirus is lasting for 6-14 weeks (protecting the same serotype)

• Classified in Astroviridae • 6,800 nt genome (+)ss RNA linear • 8 serotypes, cause diarrhea in infant, small children, immunocompromised patients, nursing home for elderly person • Primarily pediatric pathogens, causing 2–10% of cases of mild to moderate gastroenteritis in children • Incubation period 1-4 days, recover in 1-4 days • Symptoms resemble that of Rotavirus infection • Epidemiology: eating, drinking contaminated food-water, close contact with patients -Can be cultured in human embryonic kidney (HEK) cells (differred from Norovirus) -Affects young children throughout the world -Astrovirus serotype 1 is the most prevalent strain -Fatal rate is low

© Jer cas / CC BY-SA 4.0 Astrovirus “Star-like appearance” Roles of Astrovirus to immunocompromised host ♣ Diarrhea ♣ can be found in 12% of fecal specimens from HIV patients but just 2% from normal people ♣ Organ transplantation patient (4.7%) ♣ Chronic Astroviral diarrhea in children with Bone marrow transplantation until death Diagnosis of Astrovirus ♣ Electron microscopy ♣ Cell culture (HEK, LLCMK2; rhesus monkey kidney epithelial; cells) ♣ Reverse Transcription-PCR (recommended) ♣ ELISA (recommended) . Viral hepatitis is a systemic disease primarily involving the liver . Hepatitis viruses produce acute inflammation of the liver . Clinical illness characterized by fever, gastrointestinal symptoms such as nausea and vomiting, and jaundice • Acute viral hepatitis are caused by one of five viral agents • Hepatitis A virus (HAV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), the HBV-associated delta agent or Hepatitis D virus (HDV), and Hepatitis E virus (HEV) • Transfusion-transmitted agents (e.g., "hepatitis G" virus and "TT" virus, have been identified but do not cause hepatitis) • All types of viral hepatitis produce clinically similar illnesses Typical course of acute viral hepatitis • Former name, enterovirus 72 • Picornaviridae • Genus Hepatovirus • 27 nm Naked virion, (+) ssRNA • Stable at low pH • 1 serotype • No CPE in cell culture

CDC/Betty Partin/Public domain Inactivated by: Chlorine treatment of drinking water Formalin (0.35%, 37° C, 72 hours) Peracetic acid (2%, 4 hours) β-Propiolactone (0.25%, 1 hour) Ultraviolet radiation (2 µW/cm 2/min) Multiplication of virus

Infection Penetration Uncoating in cytoplasm

Translation to polyprotein

Polyprotein cleaved into New progeny many smaller protein viruses types (structural & non- structural proteins) Pathology

Largely virus Infection by multiplication in fecal-oral route Viremia intestine

Hepatocyte destruction

Resolved without chronic phase and Icteric phase immuned (ALT ) Symptoms eg. nausea, malaise, loss of appetite, diarrhea, flu-like symptom Pathology Diagnosis 1. Primary replication site: intestinal lining 1. Test for IgM by RIA or ELISA 2. Children aged 5-15 years 2. Direct demonstration of virus 3. Symptomless in younger 3. PCR patient 4. LFT (liver function test) 4. Incubation period: long (4 weeks) 5. Preicteric: Nausea, Malaise 6. Jaundice: increased bilirubin, dark bile 7. Fatality rate: 0.3% 8. Risk groups: waste-associated person, drug abuse (injection), gay men, traveler to endemic area Scheme of typical clinical and laboratory features of hepatitis A • Spread: fecal-oral route and contamination of food and water • Seasonal: autumn and winter • Treatments: symptomatic • Passive immunization: immune globulin for travelers to endemic area, this IgG cannot prevent infection but just relieve clinical symptoms and then immuned. • Vaccination: Killed virus prepared from African green munkey kidney or human diploid cell cultures (kill by formalin) • Vaccine Trade name  Havrix, Biovac A etc.  2 doses, second dose should be given 6-12 months later for boosting. • Virology • Hepadnaviridae • dsDNA virus 3.2 kb genome • Dane particle (infectious particle, about 42 nm diameter) • Spherical and tubular particles are incomplete particles (free HBsAg) • Ag: HBs, HBc, HBe CDC/Public domain • 4 subtypes: adw, adr, ayw, ayr DNA virus replicates via REVERSE TRANSCRIPTASE

TimVickers/Public domain

HBsAg = Glycoprotein on Indication envelop of virus of infection

© T4taylor/CC BY-SA 3.0 HBcAg = Core protein of capsid HBeAg = Protein inside Indication of viral replication core. In addition, this in hepatocyte protein can be found when (patient in transcription occurs. danger) Scheme of typical clinical and laboratory features of acute hepatitis B

• Incubation period: 2-5 months • Predominate in males • Clinical course is more severe than HAV • Preicteric (prodromal), jaundice, and fulminant • Destruction of hepatocytes -Hepatocellular carcinoma -Cirrhosis • Now, about 300 million world population are chronically infected, among these, at least 1 million are dying. Fates of HBV Infection

Symptomatic Acute 25% Symptomatic and Asymptomatic 65% chronic 10% (HCV 50-70%) Immuned

Fulminant Recoverd hepatitis 1% and immuned Healthy Active viral carrier replication Death

Primary hepatocellular Cirrhosis carcinoma

Death Death Disease HBsAg Anti-HBs Anti-HBc Anti-HBe HBeAg stage

Pos Neg Pos Neg Neg/Pos Acute

Neg Pos Pos Pos/Neg Neg Resolved

Pos Neg Pos Neg Pos Chronic

Later stage Pos Neg Pos Pos Neg in chronic infection

Neg Pos Neg Neg Neg Vaccinated Disease HBsAg Anti-HBs Anti-HBc Anti-HBe HBeAg stage

Neg Pos Pos Pos/Neg Neg Resolved

Disease HBsAg Anti-HBs Anti-HBc Anti-HBe HBeAg stage

Neg Pos Neg Neg Neg Vaccinated

Disease HBsAg Anti-HBs Anti-HBc Anti-HBe HBeAg stage

Pos Neg Pos Neg Neg Acute Disease HBsAg Anti-HBs Anti-HBc Anti-HBe HBeAg stage

Neg

Can be resolved or vaccinated or no immunity

-The younger a person is when infected with Hepatitis B virus, the greater his or her chance of developing chronic Hepatitis B. Approximately 90% of infected infants will develop chronic infection (CDC). Approximately 25%–50% of children infected between the ages of 1 and 5 years will develop chronic hepatitis. The risk drops to 6%–10% when a person is infected over 5 years of age.

-Can Hepatitis B be spread through food? Unlike Hepatitis A, it is not spread routinely through food or water. However, there have been instances in which Hepatitis B has been spread to babies when they have received food pre-chewed by an infected person. How is Hepatitis B spread?

Hepatitis B is spread when blood, semen, or other body fluid infected with the Hepatitis B virus enters the body of a person who is not infected. People can become infected with the virus during activities such as:

-Birth (spread from an infected mother to her baby during birth) -Sex with an infected partner -Sharing needles, syringes, or other drug-injection equipment -Sharing items such as razors or toothbrushes with an infected person -Direct contact with the blood or open sores of an infected person -Exposure to blood from needle sticks or other sharp instruments

CDC • Treatments Prevention • Antiviral: Ganciclovir and • Recombinant vaccine foscarnet, famciclovir, lamivudine derived from yeast or • Immune modulator: IFN- mamalian cells • Passive immunization • Thai infants receive vaccine at 0, 2, and 6 months • Virology • Pathology and Epidemiology • Flaviviridae • Incubation period in post • 40-50 nm, 9.4 kb (+)ssRNA transfusion: 6-8 weeks enveloped icosahedral • Less severe than hepatitis B virion • Fluctuation of ALT • High mutation • Chronic v.s. alcoholic cirrhosis • No cell culture for isolation • Fulminant hepatitis rate is • 23 genotypes about 1% • Infects only in man and • About 75% are asymptomatic chimpanzee (narrow host range) • Route of transmission  Blood, Sex (not by fecal- oral route) Chronic Hepatitis C

Modified from Rose et al, 1992 • Hepatitis delta virus firstly found by detecting delta antigen in hepatocyte of chronic HBV patient. • Unclassified family • Virion: 35-41 nm virion envelop virus (envelop derived from HBV). So, it is surrounded with HBsAg (look like a non-infectious one) • No clear capsid but inside, HD Ag is bound with genome as ribonucleoprotein complex. • Circular (-) ssRNA genome 1.7 kb in size. • Require HBV (HBV is a helper) : Coinfection or Superinfection • Clinical manifestation: • Diagnosis: Detection of antibody by RIA, HD Ag in liver • Epidemiology: Central Africa, Middle east; Drug abuser, Blood product • Control and Prevention:IFN-, HBV vaccine • Caliciviridae • 7.5 kb (+) ssRNA naked icosahedral virion • Infected by fecal-oral route  intestine  viremia  Liver  shedding into feces. • Incubation period: 2-9 weeks

• Different from HAV by slow raising Ab titer but Ab titer is down CDC/Public domain rapidly. • Clinical features are like other hepatitis viruses. Self-limiting within 2-6 weeks • Low fatality rate  about 0.5-3% • Epidemiology: through food, principally through drinking water in undeveloped countries. • Control: Sanitization, Vaccine is in develoment. • HEV causes acute infection but may cause chronic disease in immunocompromised patient, particularly in organ transplant patients. -Every year, there are an estimated 20 million HEV infections worldwide, leading to an estimated 3.3 million symptomatic cases of hepatitis E

-WHO estimates that hepatitis E caused approximately 44,000 deaths in 2015 (accounting for 3.3% of the mortality due to viral hepatitis)

• Only 1 serotype but many genotype. At least 4 different types : genotypes 1, 2, 3 and 4. Genotypes 1 and 2 in humans. Genotype 3 and 4 circulate in several animals (including pigs, wild boars, and deer) without causing any disease, and occasionally infect humans.