Myxoviridae

            The name Myxovirus came from their ability to adsorb onto mucoprotein receptors on erythrocytes causing haemagglutination. It is classified into two families – orthomyxoviridae consisting of the influenza virus and paramyxoviridae consisting of the Newcastle disease virus, mumps virus, parainfluenza viruses, measles virus and respiratory syncytial virus.

Orthomyxovirus

            Orthomyxoviridae family has three genera : Influenza virus A, B, C. They infect humans, pigs, horses, seals, whales and birds. Influenza virus A and B are morphologically similar, but C differs in some respects, especially in having only a single type of glycoprotein spike.

The influenza virus is typically spherical, with a diameter of 80-120nm diameter, pleomorphism is common. Filamentous forms are frequent in freshly isolated strains. The nucleocapsid has helical symmetry with a core of eight segments of single stranded minus sense RNA. RNA segments are associated with nucleoprotein (RNP). Three polymerase proteins, an acidic and two basic, associated with RNP at its end. They are responsible for RNA replication and transcription. Influenza C virus contains seven segments of RNA lacking a neuraminidase gene. The nucleocapsid is surrounded by an envelope, which has an inner membrane protein layer and an outer lipid bilayer. The protein layer or matrix protein or ‘M’ protein, is composed of two components, M1 and M2. The M2 protein projects through the envelope to form ion channels which allow pH changes in the endosome. The protein part of the envelope is virus coded but the lipid layer is derived from the modified host cell membrane. Projecting from the envelope are two types of spikes or peplomers : haemagglutinin(H) spikes, about 500 tapered projections(triangular cross section), of 10nm in length and neuraminidase (N) spikes, about 100 mushroom-shaped projections, 9nm in length.

            The viral particles bind to the host cell receptors(especially RBC), sialic acid, by H spikes and the effect is known as haemagglutination. It is important in laboratory work because it provides a simple and rapid method for detection of viruses in egg or tissue culture fluid. The highest dilution of the virus suspension that produces agglutination of a fixed quantity of cells is known as its haemagglutination titre. Haemagglutination is followed after a time by the detachment of the virus from the cell surface, reversing haemagglutination. This is known as elution and is caused by the N spikes. Plasma membrane of infected cells contains haemagglutinins. Therefore, RBCs get adsorbed to the surface of such cells, this is known as haemadsorption, a technique by which the growth of the influenza virus can be identified in cell culture.

            Virus particles which have eluted from red cells are still capable of agglutinating fresh red cells, but red cells once acted on  are not susceptible to agglutination by the same strain of the virus, because of the destruction of the specific cell receptors by the initial treatment with the virus.

Antigenic structure – The antigens of the influenza virus can be classified as the internal antigens and the surface antigens.

• The internal antigen is the ribonucleoprotein and is hence called the RNP antigen. It is found free in infected tissues and occurs in the supernatant when the virus containing fluid is centrifuged. It is genus specific, stable and does not exhibit any antigenic variation.
• M protein is also genus specific.
• H spike is a glycoprotein composed of two polypeptides, HA 1 and HA 2. It is strain specific and is capable of great variation.
• N spike is a glycoprotein enzyme which destroys cell receptors by hydrolytic cleavage. It is strain specific and exhibits variation.

Antigenic variation – A unique feature of the influenza virus is its ability to undergo antigenic variation, which is of great importance in the epidemiology of  the disease. Antigenic variability is highest in influenza virus type A and less in type B, not demonstrated in type C. The internal RNP antigen and M protein antigen are stable but both surface antigens, H and N peplomers undergo independent antigenic variations, which may be of two types – antigenic drift or antigenic shift.

Antigenic drift : results from mutations in genes that code for haemagglutinin and neuraminidase. Such mutations change the configuration of the part of the antigen molecule that stimulates the production of specific antibodies. The antibodies formed against the parental haemagglutinin or neuraminidase are less effective in inhibiting the mutated forms of these viral components. This drift accounts for the periodical epidemics of influenza.

Antigenic shift : results from gene reassortment, possibly after two different viruses infect the same cell, for example, bird and human influenza viruses infect a pig cell and exchange large segments of their genomes. It represents more dramatic changes, the viral strains that emerge are significantly different antigenically from previously known strains. Antigenic shift accounts for the major pandemic.

Susceptibility – The virus is inactivated by heating at 50⁰C for 30 minutes. It remains viable at 0-4⁰ C for about a week. It can be preserved for years at –70⁰C or by freeze drying. They remain viable on fomites for two weeks. Ether, formaldehyde, phenol, salts of heavy metals and many other chemical disinfectants destroy infectivity. Iodine is particularly effective. Haemagglutinating, enzymic and complement – fixing activities of the virus are more stable than infectivity.

Pathogenicity – The route of entry is the respiratory tract. Very small doses can initiate the infection in experimental infections, larger doses if by intranasal inoculation. The viral neuraminidase facilitates infection by hydrolyzing the mucus film lining the respiratory tract and exposing the cell surface receptors for virus adsorption. Ciliated epithelial cells of respirarory tract are the main sites of virus infection. These cells are damaged and shed,  exposing the basal cells in the trachea and bronchi. This renders the respiratory tract highly vulnerable to bacterial invasion. Very rarely the virus isolated from spleen, liver, kidneys and other organs (1957 pandemic).

            The incubation period is 1-3 days. The disease varies in severity from a mild coryza to fulminating and rapidly fatal pneumonia. The illness is characterized by a sudden onset of systemic symptoms such as chills, fever, sore throat, headache, cough, myalgia and malaise. Respiratory symptoms are prominent and severe prostration is common. Abdominal pain and vomiting may occur, especially in type B infection in children. The uncomplicated illness resolves within about 7 days.

            The most important complication is pneumonia, mainly due to bacterial secondary infection. Rarely influenza produce cardiac complications and neurological involvement, such as myocarditis and encephalitis respectively. It is also associated with Reye’s syndrome which is characterized by a severe encephalomyelitis accompanied by liver degeneration. Type B infections may sometimes cause gastrointestinal symptoms (gastric flu).

Laboratory diagnosis –

1. Demonstration of viral antigen : Rapid diagnosis of influenza made by demonstration of  the antigen on the surface of the nasopharyngeal cells by immunofluorescence. The samples used for this are nasopharyngeal secretions and nasal swabs or centrifuged depositions of throat garglings.
2. Virus isolation : Influenza viruses can be isolated from patients during the first 2 or 3 days. Throat garglings are collected using suitable buffered salt solution. If short delay is expected, store at 4⁰C, and if in case of  long delay, store at –70⁰C. The specimen is treated with antibiotics to destroy bacteria. Isolation can be made in eggs or monkey kidney cell culture.

The material is inoculated into the amniotic cavity of 11-13 day old eggs. After incubation at 35⁰C for 3 days, the eggs are chilled and the amniotic and allantoic fluids harvested separately. The fluids are tested for haemagglutination using guineapig and fowl erythrocytes at room temperature and 4⁰C, in parallel. The isolate is identified based on the haemagglutination and typed by complement fixation test with antisera to types A, B and C.

Inoculation into monkey kidney or other suitable continuous cell cultures is the preferred method where the facility is available. Virus growth can be identified by haemadsorption. Rapid results can be obtained by demonstrating virus antigen in infected cell cultures by immunofluorescence.

3. Serological tests : Complement fixation and haemagglutination inhibition tests are employed for the serological diagnosis of influenza. Rise in the antibody titre can be demonstrated  by examining the paired sera in parallel. Complement fixation tests with the RNP antigens of 3 types, V antigens can be done. Because of its complexity, CF tests are used rarely. HI is a sensitive test for the serological diagnosis of influenza. A disadvantage of this serological technique is the presence of nonspecific inhibitors(glycoproteins–alpha, beta, gamma) in the sera, which causes nonspecific inhibition of haemagglutination. They are inactivated by treatment with RDE, trypsin, CO₂ without affecting the antibody content of sera. The highest dilution of serum that inhibits haemagglutination is its HI titre.

Immunity – An attack of influenza confers protection effective for about one or two years. The apparent short duration of immunity is due to the antigenic variation that the virus undergoes frequently. Following infection and immunisation, circulating antibodies are formed against the various antigens of the virus. This viral infection induces cell mediated immunity.

Prophylaxis – Vaccination is the main method of preventing influenza, but major difficulty in immunoprophylaxis is the frequent change in the antigenic structure of influenza virus. Vaccines cannot be made in bulk, as the appearance of a new variant will make them obsolete. The most important indication for immunoprophylaxis is when a pandemic is threatened by a new virus. In such cases the time taken for the manufacture of the vaccine with the new variant is crucial, as the virus is likely to spread fast and infect whole populations before the vaccine becomes available. Besides, the fresh isolates do not grow well in eggs till they are passaged serially. To overcome this recombinant vaccine has been introduced, which possesses the growth characters of old established strains and carries the surface antigens of the new variant. The recombinant will grow well in eggs, facilitating vaccine manufacture.

            Killled vaccines induce the formation of circulating antibodies. The influenza virus is grown in allantoic cavity of chick embryos, inactivated by formalin or BPL, purified by centrifugation and disrupted with antigens. Temperature sensitive mutants can be used as live vaccines, administered by aerosol spray, stimulates the production of local IgG antibodies. Recombinant live vaccines may be obtained by hybridisation between the ts mutants of established strains and a new antigenic variant.

            Amantidine and rimantidine used as anti influenza drugs, they block the viral M2 protein which functions as ion channel, act only with type A. They reduce the average duration of the disease. Zanamivir, a new drug designed to block viral neuraminidase has been found effective in the treatment and prevention of influenza, administered as nasal spray.

Paramyxovirus

            The family Paramyxoviridae contains important pathogens of infants and children, responsible for acute respiratory infections, measles and mumps. The viruses resemble orthomyxoviruses in morphology, but are larger and more pleomorphic. They are enveloped viruses with 100-300nm diameter, the helical nucleocapsid with a diameter of 18nm. The paramyxoviruses are genetically stable, because they are composed of a single strand of unsegmented, negative sense RNA that does not undergo recombination normally. The nucleocapsid is surrounded by a lipid envelope which has the matrix protein, M at its base and two types of glycoprotein spikes. The longer spike is the haemagglutinin, H, which may also possess neuraminidase, N activity, hence known as HN protein. It is responsible for adsorption of the virus to the host cell surface. The second spike is the F or fusion protein, responsible for the fusion of the viral envelope with the plasma membrane of the host cells. It brings about cell to cell fusion, causing large giant cells or syncytia which are characteristic of paramyxovirus infections. The F protein also mediates the haemolytic activity of paramyxoviruses.

Mumps – is an acute infectious disease that occurs primarily in school-aged children. It is characterised by nonsuppurative enlargement of the parotid glands. The virus is transmitted by way of respiratory and oral secretions and respiratory tract is the portal of entry, sometimes through conjunctiva also. It multiplies in the upper respiratory tract and in local lymph nodes. The virus then enters the blood stream and the infection spreads to many organs of the body. The incubation period is about 12-25 days. Parotid swelling is usually the first sign of illness, is accompanied by fever, local pain and tenderness. Parotitis resolves within 1-2 weeks. But the involvement of extraparotid sites can be serious. Epididymo-orchitis is a complication seen in postpubertal male patients. The testis becomes swollen and acutely painful, with accompanying fever and chills, sterility or low sperm counts may result. It may also cause meningoencephalitis, pancreatitis, arthritis, myocarditis, renal dysfunction.

            Mumps is endemic worldwide, household spread is common. Humans are the only natural hosts. The source of infection is a patient in the late incubation or early clinical stage of illness. Infection is transmitted by direct contact, air borne droplets or fomites contaminated with saliva, and also possibly urine. One attack of mumps confers lasting immunity.

            The diagnosis may be established by virus isolation and serological tests. The virus may be isolated from the saliva, urine or CSF. The specimens have to be inoculated soon after the collection. The prepared specimen is inoculated into monkey kidney cell cultures or human cells or HeLa cells. Virus growth can be detected by haemadsorption and immunofluorescence, the CPE will be little. Isolation can also be made by inoculation into 6-8 day old chick embryos, the virus can be identified by HI using specific antisera.

            The Jeryl-Lynn strain of mumps virus, attenuated by passage in eggs and grown in chick embryo fibroblast culture is used as the vaccine. It is recommended for use only after one year of age as maternal antibodies may interfere with the multiplication of the vaccine virus if given earlier. The vaccine is given as a single subcutaneous injection, either alone or in combination with measles and rubella vaccines (MMR vaccine).

Respiratory Syncytial Virus – RSV is recognized as the most important cause of lower respiratory tract infection in infants. It is pleomorphic and ranges in size from 150-300nm. The viral envelope has two glycoproteins- the G protein by which the virus attaches to cell surfaces, and the fusion protein, F.it causes cells in cultures to fuse their plasma membranes and become multinucleate masses or syncytia. It does not possess haemagglutinating activity, neuraminidase and haemolytic properties. It does not grow in eggs, but in human cell cultures. It is highly labile and inactivated rapidly at room temperature, can be preserved by lyophilisation.

            The virus is shed in respiratory secretions, infection is transmitted by contact with contaminated hands and surfaces. Nosocomial infections are frequent in nurseries and paediatrics wards. Incubation period is about 4-5 days. The virus multiplies in the mucus membranes of  the nose and throat. In infants the disease may begin as mild diarrhoea, cough, wheezing and leads to viral pneumonia. RSV is an important cause of otitis media in young children.

            RSV can be isolated from nasopharyngeal swabs or nasal washings. Samples should be inoculated in cell cultures immediately after collection. The virus is detected by CPE and immunofluorescence tests. Serological diagnosis is by demonstration of rising antibody titres by ELISA, CF, neutralisation or immunofluorescence tests. No effective vaccine.

Measles – the virus is roughly spherical, but pleomorphic. The tightly coiled helical nucleocapsid is surrounded by the lipoprotein envelope carrying on its surface H spikes, F proteins etc. they grow well on human or monkey kidney cultures. The virus is readily inactivated by heat, ultraviolet, ether, formaldehyde.

            Virus gains access to the human body via the respiratory tract, multiplies locally. The infection then spreads to the regional lymphoid tissue and multiplies further. It seeds the epithelial surfaces of the body, such as skin, respiratory tract, conjunctiva. They also replicate in lymphocytes which aids in dissemination throughout the body. Multinucleated giant cells with intranuclear inclusions are seen in lymphoid tissues throughout the body. After an incubation period of 10-12 days , patient develops upper respiratory tract infections with high fever, rhinitis, cough and conjunctivitis. Koplik’s spots of 13mm diameter, bluish white spots surrounded by erythema can be seen on the buccal mucosa. After 1-2 days, the acute symptoms decline with the appearance of characteristic maculopapular rash which appears first on the neck and then spreads to the rest of the body. In next 10-14 days, rash fades. The infection confers lifelong immunity.

            The diagnosis can be done by the demonstration of multinucleated giant cells in Giemsa stained smears of nasal secretions. Demonstration of measles specific IgM is confirmatory.

            People are the only natural hosts. Children of the age of 15 months are given MMR vaccine, followed by a booster at the age of 4-6 years.