RESPIRATORY VIRUSES

Viruses that initiate infection via respiratory tract Site of infection Family Viruses Local respiratory infection Orthomyxoviridae Influenza А and В viruses Paramyxoviridae Parainfluenza viruses (4 types), respiratory-syncytial virus RSV (3 types) Picornaviridae Rinoviruses (113 types) Reoviridae Reoviruses (3 types) Coronaviridae Types 1-4 Adenoviridae Types 1-7, 14, 21 Generalized diseases, usually with initial respiratory symptoms Herpesviridae Varicella virus, Epstain-Barr virus (EBV), cytomegalovirus Mumps and measles viruses Togaviridae Rubella virus Some enteroviruses Bunyaviridae Hantaviruses Arenaviridae Lassa fever virus

Family Orthomyxoviridae. Influenza virus (-) RNA enveloped virus with haemagglutinin and neuraminidase spikes Pleomorphic Helical symmetry Diameter 80-120 nm 3 types: A, B and C

Structure of influenza virus The internal antigens (matrix and RNP) are the type-specific proteins and are used to determine whether a particular virus is A, B, or C. The external antigens (hemagglutinin and neuraminidase) are subtype and strain- specific antigens of influenza A virus: H1N1, H2N2, H3N2

Comparison of influenza A, B and C viruses Severity of illness ++++ ++ + Animal reservoir yes no Human pandemics Human epidemics Surface antigens 2 1 Antigenic changes shift, drift drift Amantadine, rimantadine sensitive no effect Zanamivir

Influenza virus replication

Hemagglutinin (HA) protein is involved in attachment and membrane fusion in the endosome of the infected cell.

The neuraminidase protein digests sialic acid (neuraminic acid) on the surface of cells, when the virus binds to the cell, and it is internalized (endocytosed). Promotes the release of virions from infected cells.

Types of hemagglutinin and neuraminidase

Antigenic variations Antigenic drift is due to mutation. Both HA and NA after a few years may accumulate changes in genome that an individual immune to the original strain is not immune to the drifted one. Antigenic drift results in sporadic outbreaks and limited epidemics. Antigenic shift is due to reassortment. It is the process by which at least two different strains of a virus combine to form a new subtype having a mixture of the surface antigens of the two original strains. As a result, the population has no immunity against the new strain. Antigenic shift results in an epidemic or pandemic. Antigenic drift creates influenza viruses with slightly modified antigens, while antigenic shift generates viruses with entirely novel antigens.

Past antigenic shift 1918 H1N1 “Spanish Influenza” 20-40 million deaths 1957 H2N2 “Asian Flu” 1-2 million deaths 1968 H3N2 “Hong Kong Flu” 700,000 deaths H1N1 Re-emergence No pandemic 2009 H1N1 “Swine Flu Mild Pandemic At least 15 HA subtypes and 9 NA subtypes occur in nature. Up until 1997, only viruses of H1, H2, and H3 are known to infect and cause disease in humans.

Theories behind antigenic shift 1. Reassortment of the H and N genes between human and avian influenza viruses trough a third host. 2. Recycling of pre-existing strains (this probably occurred in 1977 when H1N1 re-surfaced). 3. Gradual adaptation of avian influenza viruses to human transmission. Coinfection with human and animal strains of virus can generate very different virus strains by genetic reassortment

Reassortment of H and N genes between human and avian influenza viruses Sometimes pigs can be infected with more than one virus type at a time, which can allow the genes from these viruses to mix. This can result in an influenza virus containing genes from a number of sources, called a “reassortant” virus.

Bird or avian flu (2006) The outbreaks affecting some Asian countries was caused by influenza A/H5N1 virus. It caused severe infection in humans. While avian influenza spreads rapidly among birds, it does not infect humans easily, and there is no confirmed evidence of human-to-human transmission.

2009 Influenza A (H1N1) Virus The 2009 Swine flu outbreak in humans was due to a new strain of influenza A virus subtype H1N1 that derived in part from human influenza, avian influenza, and two separate strains of swine influenza (American swine and Eurasian swine viruses). Swine influenza viruses are most commonly of the H1N1 subtype, but other subtypes are also circulating in pigs (e.g., H1N2, H3N1, H3N2).

Epidemiology and pathogenesis Spread: via small particle aerosols. The incubation period is about 18 to 96 hours. Site of infection: the epithelial cells of the respiratory tract: trachea and bronchi

Pathogenesis Infection of mucosal cells results in cellular destruction and desquamation of the superficial mucosa. The resulting edema and mononuclear cell infiltration are accompanied by local symptoms: nonproductive cough, sore throat, and nasal discharge. Systemic symptoms: fever, muscle aches, headache, and general prostration.

Symptoms

Pathogenesis and immunity Immunity to an influenza virus is type-specific and lasts for many years. Recurrent cases of influenza are caused primarily by antigenically different strains.

LABORATORY DIAGNOSIS OF INFLUENZA Specimen: the nose and the throat swab. 1. Detection of antigen by immunofluorescence (IF) and ELISA. 2. The virus isolation in cell culture or chicken eggs. Identification of influenza strain and type: hemagglutination inhibition (HAI) and hemadsorption inhibition (HadsI). 3. Serology: HAI, neutralization test (NtT), ELISA, CFT in pared sera.

Immunofluorescence (IF) to detect virus into host cells direct IF indirect IF

Hemadsorption (Hads) Virus growth in cell cultures is detected by testing for hemadsorption: red cells are added to the culture and adhere to virus budding from infected cells. If the culture tests positive, hemadsorption inhibition test with specific antisera is used to identify the virus. cell culture positive Hads

Chicken embryo structure

Chick embryo culture method Inoculation of chick embryo Removing allantoic fluid Fluid from the amniotic or allantoic cavity of chick embryos is tested for the presence of newly formed viruses by haemagglutination test; the virus in positive fluids is then identified by haemagglutination inhibition test with specific antisera.

Haemagglutination inhibition test (HAI) Influenza viruses bind to red blood cells using the haemagglutinin causing the formation of a lattice. HA: two-fold serial dilutions of a virus are prepared, mixed with red blood cells, and added to the wells of a plastic plate. The red blood cells that are attached to virus particles form a lattice that coats the well. The red blood cells that are not bound by virus sink to the bottom of a well and form a button. The basis of the HAI assay is that antibodies to influenza virus will prevent attachment of the virus to red blood cells. By adding specific antibodies to the virus it is possible to block this interaction and detect the virus. If antibodies to the virus are specific, hemagglutination will not be observed. negative HA positive HA negative HAI positive HAI

TREATMENT Class Effective against Drug name M2 inhibitors (adamantane derivatives) Influenza A Amantadine, rimantadine Neuraminidase inhibitors Influenza A & B Zanamivir, oseltamivir

Mechanism of Action of Neuraminidase Inhibitors

Interferon The innate immune system forms the first line of defence against viruses - Interferons (IFNs), glycoproteins known as cytokines. Interferons are produced by: the cells of the immune system (leukocytes, fibroblasts, or lymphocytes) in response to challenges by foreign agents such as viruses, parasites and tumor cells; cells infected with a virus. Interferons assist the immune response by: inhibiting viral replication within host cells, activating natural killer cells and macrophages, inducing the resistance of host cells to viral infection.

VACCINES The vaccine is multivalent and the current one is to two strains of influenza A and one of influenza B. Inactivated preparation of egg-grown virus. Live, attenuated vaccine. Split-vaccine consists of all viral antigenes. Sub-united vaccine consists of hemagglutinines and neuraminidases (H1N1, H3N2 and B).

Unique features of Adenoviruses Double-stranded linear(+)DNA. Naked icosahedral capsid has fibers (viral attachment proteins) and vertices. 47 human serotypes Viruses cause: lytic, persistent, latent infections in humans, some strain can immortalize certain animal cells.

Life cycle of adenoviruses

Pathogenesis of adenoviruses infections Virus is spread: by aerosol, direct contact, fecal-oral. Virus infects: epithelial cells in respiratory and gastrointestinal tract, conjunctiva and cornea. Virus persists in lymphoid tissue (tonsils, adenoids, Peyer’s patches).

Mechanism of adenovirus spread within the body

Time course of adenovirus respiratory infection

ILLNESS ASSOCIATED WITH ADENOVIRUSES Incubation period is 2-14 days. Clinical syndromes: Eye Epidemic keratoconjunctivitis, acute follicular conjunctivitis, pharyngoconjunctival fever. Respiratory system Common cold (rhinitis), pharyngitis, tonsillitis, bronchitis, pneumonia. Genitourinary Acute hemorrhagic cystitis, orchitis, nephritis. Gastrointestinal Gastroenteritis, mesenteric adenitis, appendicitis. Rare results of adenovirus infections: Meningitis, encephalitis, arthritis, myocarditis, hepatitis. Fatal disease may occur in immunocompromised patients, as a result of a new infection or reactivation of latent virus

Respiratory consequences of adenovirus infections

IMMUNITY PREVENTION Strong, type-specific A vaccine is available against Adult Respiratory Distress Syndrome. Oral, live-attenuated vaccines against serotypes 4 and 7 of adenovirus is administered in tablet form. It is given to new recruits into various arm forces around the world.

LABORATORY DIAGNOSIS OF ADENOVIRUSES INFECTION 1. Detection of antigen from pharyngeal and eye secretions and feces by: IF, ELISA and polymerase chain reaction. 2. The virus isolation in cell culture. Cytopathic effects (CPE) include swelling and rounding of cells. Identification: HAI, CFT; type virus – by NtT. 3. Serology (rise in antibody titer): HAI, NtT, CFT.

Family Paramyxoviridae Genus Respirovirus (parainfluenza viruses types1 and 3) Genus Rubulavirus (parainfluenza viruses types 2 and 4, mumps virus) Genus Morbillivirus (measles virus) Genus Pneumovirus (respiratory syncytial virus)

Parainfluenza viruses ss (-) RNA viruses Enveloped viruses with hemagglutinin and neuraminidase spikes and fusion (F) protein Helical symmetry 4 types: 1, 2, 3, 4a, 4b

Clinical features of parainfluenza (PIV) Incubation period is 2 to 6 days. Clinical symptoms: Rhinitis, pharyngitis, cough, fever, croup (laryngotracheobronchitis), bronchiolitis, and pneumonia. Croup - the subglottic region becomes narrower and results in difficulty with breathing, a seal bark-like cough and hoarseness. There is clinical variation between the different PIV types. PIV-1 and 2: croup in children ages 2-6 years in autumn/early winter. PIV-3: bronchiolitis and pneumonia, and croup sporadically, without a particular seasonal occurrence. PIV-4: mild upper respiratory infections.

LABORATORY DIAGNOSIS OF PARAINFLUENZA 1. Detection of antigen from nasopharyngeal aspirates and throat swab by IF and PCR. 2. The virus isolation in cell culture. Indication: Haemadsorption of erythrocytes on the surface of cells infected with virus. Identification: HadsI, HAI, NtT, CFT. 3. Serology – detection of rise in titer of IgG in pared sera: NtT, ELISA, CFT, HAI.

Respiratory Syncytial Virus (RSV) ss RNA enveloped virus Considerable strain variation exists, may be classified into subgroup A and B. CPE - the formation of multi-nucleate syncytium Clinical features:

MUMPS Transmission by: Mumps is a viral infection that primarily affects the salivary glands causing them to become inflamed, resulting in the characteristic “chipmuck cheeks” Transmission by: salivary gland secretions, mainly just before and shortly after clinical onset direct and close person-to-person contact and airborne route. The virus enters the body through the pharynx or the conjunctiva.

Clinical symptoms of mumps The incubation period usually is 18 to 21 days, but may extend from 12 to 35 days. Prodromal phase: fever, anorexia, malaise, myalgia. The symptoms: Fever. Parotitis. Pain from parotitis swelling persists for 7 - 10 days. It may be unilateral or bilateral. Up to 20% infections result in no symptoms.

Complications of mumps Orchitis 20-50 % Meningitis and meningoencephalitis 15 % Ovaritis 5 % Pancreatitis 2-5 % Rare complications: polyarthritis, diabetes, nephritis, thyroiditis, deafness, myocarditis.

LABORATORY DIAGNOSIS OF MUMPS The virus isolation from the saliva, liquor or urine in cell culture (or chicken eggs). CPE: giant multinucleated cells formation. Identification: HAI, NtT, IF, CFT. 2. Serology: HAI, NtT, ELISA, CFT (demonstrating IgM in the first serum and detecting IgG rise in paired sera).

Measles virus Family Paramyxoviridae Genus Morbillivirus enveloped RNA virus which is composed of a helical nucleocapsid, and matrix protein. The envelope surface is covered with hemagglutinin and fusion proteins which are embedded in a lipid bilayer. Electron Micrograph

Age distribution of measles cases Measles is one of the most contagious of all human viruses, with about 40 million infections world wide each year, and one to two million deaths.

Measles a systemic infection, disseminated by viremia, with acute disease manifestations involving the lymphatic and respiratory systems, the skin, and sometimes the brain. Measles virus enters through the oropharynx and the conjunctiva. Incubation period is 10 days until the onset of fever and 14 days to rash. The clinical symptoms: Fever, cough, laryngitis, coryza, conjunctivitis, Koplick’s spots, and maculopapular rash. Complications: Pneumonia, otitis media, optic neuritis Encephalitis, subacute sclerosing panencephalitis (SSPE) Hemorrhagic measles

Measles pathogenesis

Koplick’s spots

Conjunctivitis. Maculopapular rash

Subacute Sclerosing Panencephalitis

LABORATORY DIAGNOSIS OF MEASLES Clinically Koplick’s spots are pathognomonic. 1.Detection of antigen from nasopharyngeal aspirates and throat swab by IF. 2. The virus isolation in cell culture. CPE: giant multinucleated cells formation. Identification: HAI, IF, NtT. 3. Serology: HAI, NtT, CFT.

Family Togaviridae. Rubella virus It is an enveloped (toga=cloak), RNA virus with icosahedral symmetry. There is only one major antigenic type.

Pathogenesis and clinical features of rubella Spread via an aerosol route through infected droplets. Replication: in the epithelium and lymph nodes of the upper respiratory tract leading to viremia and  spread to other tissues. Incubation period is 12 to 23 days (the average 18 days). Enlarged tender lymph nodes (post-auricular, post-cervical, and suboccipital) usually develop 4-6 days before rash. Maculopapular rash begins on the face and lasts from 12 hours to 5 days.

Rubella rash

Complications of rubella Encephalitis Arthritis Thrombocytopenic purpura Congenital syndromes:

Laboratory diagnosis of rubella Serology: Rising Ig G antibody titres - 4-fold or greater: HAI, CFT, NtT, IF. Presence of rubella-specific IgM: ELISA. Isolation the virus in cell cultures from respiratory tract secretions and, in infants with congenital infection, from urine, cerebrospinal fluid, and blood. Identification: viral interference, HAI, IF, ELISA.

Vaccination Combined measles-mumps-rubella live-virus vaccine (MMR) is recommended for all healthy 12 to 18-month-old children. The second dose of: rubella vaccine is given either at school entry or at entry to high school. mumps vaccine is recommended either at 6 or 12 to 13 years of age. Live measles vaccine should be given to anyone who does not have a history of measles or has not received vaccine after the age of 15 months.