Picorna Virus: Classification and Laboratory Diagnosis

In this article we will discuss about Picorna (pico, small, rna, RNA) virus or Poliomyelitis which contains single stranded RNA:- 1. Classification of Picorna Virus 2. Resistance of Poliomyelitis Virus 3. Pathogenesis and Disease in Man 4. Laboratory Diagnosis 5. Antigenic Structure 6. Epidemiology 7. Coxsackie Viruses 8. Clinical Findings 9. Echo Virus 10. Entero Virus 11. Use and Effectiveness of Salk Vaccine.

Contents:

1. Classification of Picorna Virus
2. Resistance of Poliomyelitis Virus
3. Pathogenesis and Disease in Man
4. Laboratory Diagnosis of Poliomyelitis
5. Antigenic Structure of Polio Virus
6. Epidemiology of Poliomyelitis
7. Coxsackie Viruses
8. Clinical Findings of Poliomyelitis
9. Echo Virus
10. Entero Virus
11. Use and Effectiveness of Salk Vaccine

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1. Classification of Picorna Virus:

Picorna viruses of medical importance comprise two genera:

Entero-viruses of enteric tract and rhinoviruses of upper respiratory tract.

Though poliomyelitis is known as one of the most ancient contagious diseases, its infec­tious nature was ascertained only in 1905 by Wickman; later, in 1908-09, it was proved to be of viral aetiology.

Polio virus ranges from 8-27 nanometer (nm) (1 nm = 10^-9 metre) or equal to millimicron (mp) and forms intra-nuclear inclusions. It is cultivated in vitro in tissue culture of monkey kidney or HeLa cells.

There are three types of viruses which do not possess common immunogenic properties:

Type I viruses include Brunhilde strains which are pathogenic to man and monkey;

Type II viruses include the Lansing strains which are responsible for disease in man, monkey and rodents (rats, mice, hamsters etc.);

Type III viruses (the Leon strain) are pathogenic only to man and monkeys and differ immunologically from the first two types.

2. Resistance of Poliomyelitis Virus:

The poliomyelitis virus survives in sterile water at room temperature for more than 100 days; in milk for 90 days; in faeces in cold for more than 6 months and in sewage for several months. It withstands exposure to 0.5-1 per cent phenol solution and remains viable for several weeks at pH 3.8-8.5. It is very sensitive to calcium chloride, formalin, potassium permanganate and hydrogen peroxide. It is rapidly killed on boiling.

3. Pathogenesis and Disease in Man:

The virus is extremely neurotropic. It causes the degenerative inflammatory process in the anterior horns of spinal cord and in the grey sub­stance of the brain. However, the virus circulates in the blood. It may be discharged in the faeces of patients and convalescents for 2-7 weeks and is discharged through the nasopharyn­geal mucous membrane secretion and sometimes up to 4 months and during the incubation period.

Healthy carriers also excrete the virus with the faeces. This virus may be found in the nasopharyngeal mucus and tonsils approximately 3 days before the temperature rises and for 3 days after the onset of the disease. According to the clinical symptoms, three forms of poliomyelitis are distinguished—abortive, non-paralytic, and paralytic.

(a) Abortive or In-Apparent Infection without Symptom:

It is the most common form (90%) of the disease (abortive poliomyelitis). Patient recovers in a few days.

(b) Non-Paralytic Poliomyelitis (Aseptic Meningitis):

In this category, the patient shows clinical symptoms of stiffness, pain in back and neck for 2-10 days. There is rapid and complete recovery.

(c) Paralytic Poliomyelitis:

Due to damage of lower nerve motor neurons, flaccid paralysis develops and it is classified into spinal, bulbar, and bulbospinal on the basis of nerve involvement. Muscle involvement is most frequent. Within six months, the recovery is maximum, but the residual paralysis may last longer.

4. Laboratory Diagnosis of Poliomyelitis:

1. Isolation of Virus:

Virus can be recovered from the pharyngeal aspiration during first 1-5 days and from the faeces up to 5 weeks and it cannot be usually recovered from CSF. It is cultivated in human or similar cell culture and its cytopathic effect is observed in 2-3 days. Its identification is performed by neutralisation test with standard era of 3 types.

2. Direct demonstration of virus in stool is done by dried electron microscopy and by immune electron microscopy.

3. Serology:

Paired serum samples can be tested for neutralizing complement fixing antibodies.

Immunity:

There are three immunologically distinct types of polio viruses: I, II and III. Immunity to one does not confer immunity to another. A very stable immunity is produced following the disease. No case of reinfection has been described. Immunity is associated with the presence of virus neutralizing antibodies in the blood.

5. Antigenic Structure of Polio Virus:

Each type of polio virus contains 2 types of specific antigens-—C and D. C antigen of all types are cross-reactive but D antigen is not. One attack of poliomyelitis gives permanent immunity only against the type causing the infection.

Both live and killed vaccines are available and offer excellent protection. Killed vaccine induces only humoral antibodies (Ig M and Ig G) but does not produce intestinal immunity. Live vaccine induces humoral antibodies (Ig M and Ig G) and local secretory and circulating Ig A antibodies and makes intestine resistant to reinfection.

This live oral attenuated polio vaccine (OPV-Sabin vaccine) is administrated orally and is indicated for active immunization of all infants of 2 months of age simultaneously with the first DPT injection. The second and third doses are administrated at 2 month intervals. The fourth dose is administrated at 1 years of age.

6. Epidemiology of Poliomyelitis:

Poliomyelitis caused by three types of polio virus is distributed worldwide mainly affecting children in tropical countries with poor sanitation.

The most important source of dissemination of polio virus in the community is usually due to sewage contamination of drinking water with faeces of poliomyelitis patient, sometimes pharyngeal (tract) secretions may spread the virus in the early stage of disease; about 90% of children of tropical countries with poor hygiene have antibodies to all three types of polio virus by the age of 5 years and hence epidemics do not occur; 80% paralysis occurs before the age of 3 years in developing countries.

Immunization has brought down the incidence to 20-40/100,000 population. Vaccination of whole popula­tion may eradicate the disease.

7. Coxsackie Viruses:

These viruses was first isolated in 1949 in Coxsackie of New York State. They resemble polio viruses in properties and epidemiology.

They are divided into two groups:

A (23 types) and B (6 types).

They can be isolated from throat secretions or faeces in suckling mouse brains.

Coxsackie B and A9 grow in monkey kidney tissue culture. Due to existence of several types in each group, sero diagnosis is difficult. Similarly, vaccination is not susceptible to physical, chemical agents; is similar to those of entero viruses.

8. Clinical Findings of Poliomyelitis:

Incubation period is 2-9 days. Infection is transmitted by faecal-oral routes. Clinical mani­festations of coxsackie virus infection are:

1. Herpangina (Vesicular Pharyngitis):

The syndrome caused by certain group A viruses (2, 4, 5, 6, 10) is characterised by abrupt onset of fever, pharyngitis, headache and pain in abdomen. Pharynx becomes hyperaemic with discrete vesicles. It is a self-limiting disease.

2. Aseptic Meningitis:

Both group A and B viruses are responsible for this condition. Sometimes clinical picture may resemble paralytic meningitis.

3. Pleurodynia (Epidemic Myalgia, Bornholm Disease):

This disease is caused by group B virus and is characterised by fever and sub-sternal chest pain intensified by .movement. Its duration is 2 to 15 days.

4. Myocarditis:

In children and adults, coxsackie B virus may result in myocarditis.

5. Trans-Placental Passage:

Trans-placental passage of Coxsackie B virus may result in disseminated lesions (hepatitis, myocarditis, meningoencephalitis and adrenal-cortical disease).

6. Juvenile Diabetes:

Coxsackie is associated with this condition.

9. Echo Virus:

Echo viruses (enteric cytopathic human orphan viruses) infect human enteric tract. Only man is infected. They were called “Orphan viruses” as they are not related to any particular clinical disease.

There are 34 serotypes which cause asymptomatic infection; only few are responsible for overt disease (outbreak of fever and aseptic meningitis (echo 4, 6, 9, 16), infantile diarrhoea (echo 18, 20), and mild respiratory disease (echo 28).

Echo viruses resemble other picorna viruses in their properties. Diagnosis is by isolation of virus from throat secretion, stool, CSF in monkey kidney CPF, and neutralisation test. Serology is difficult due to existence of many types of echo viruses. When echo viruses were first identified they were not pathogenic to laboratory animals.

10. Entero Virus:

Out of five types of viruses of this group, four (68, 70, 71, 72) are associated with human diseases and type 72 is now designated as hepatitis A virus.

Pathogenesis:

Coxsackie virus A24 and entero virus 70 caused two massive epidemics of acute haemorrhagic conjunctivitis. Its incubation period is about 24 hours.

Rhino Virus:

Rhino virus cause common cold and are isolated usually from nose and throat and rarely from faeces. Because of their adaptation to grow in the nose (Rhine, nose), they are called Rhino virus.

Properties:

These viruses resemble other picorna viruses except that they are destroyed at pH 3-5 and are more heat stable. There are about 113 serotypes of Rhino virus.

Host range and Cultivation:

Only man harbours rhino viruses, chimpanzees can be infected experimentally. Based on growth in human or monkey kidney cell cultures with cytopathic effect, rhino viruses are divided into three groups: M (Monkey) strains and H (Human) strains. H strains can grow in both human and monkey cells. O (Organ) strains grow in organ cultures of nasal or tracheal ciliated epithelium.

Pathogenesis:

Human rhino viruses cause common cold which is an acute respiratory tract infection. Its transmission is mainly in droplet inhalation and the incubation period is 2-3 days. During this period the virus is present in the nasopharyngeal secretions.

Laboratory Diagnosis:

Isolation of virus is performed by inoculation of nasopharyngeal secretion in human or monkey kidney cell cultures and incubated at 33°C. Due to multiplicity of serotypes, it is not possible to isolate serologically.

Epidemiology:

Rhino virus infection is widespread and is transmitted by inhalation. Rhino virus 13 inactivated vaccine gave in volunteers protection against some strains for a very short period depending on Ig A antibodies. It is difficult to prepare an effective vaccine due to multiplicity of antigenic types.

Polio Vaccine:

In 1949, Enders, Weller and Robbins (Nobel Prize winners) were able to cultivate poliomyelitis virus in tissue cultures, made from human, monkey kidneys, human intestinal tissues, human embryonic tissues and human cancer (HeLa cells). This virus multiplies and kills the tissue cells.

The Salk vaccine was prepared by Jones Salk by cultivating polio virus Types I, II, III in live monkey kidney tissue cells and, after an incubation, the dead tissue cells and other debris are removed and the remaining fluid containing polio virus is collected and formaldehyde (1:4,000) is added to kill the virus and allowed to act for one week, and excess of formaldehyde is removed.

Safety tests were then carried out to determine whether the virus is really killed and the fluid is not contaminated with bacteria. If satisfactory, the preservative is added and the vaccine is ampouled and used.

11. Use and Effectiveness of Salk Vaccine:

Three doses are recommended: first two doses at an interval of about four to six weeks apart and the third at an interval of 1-3 years or more.

This vaccine does not prevent poliomyelitis, but the incidence is reduced to a very, low level, when it is used consistently, because of the antibodies in the blood and the tissues of vaccinated persons which interpose a barrier between the gastrointestinal source of virus and the central nervous system.

Thus, the most important effect of the vaccine is to prevent severe paralytic polio (e.g. crippling and deadly “spinal paralysis”). This vaccine is 90 per cent effective in preventing the paralytic polio.

Vaccination in Pregnancy:

Immunization of the mother with the vaccine probably gives some passive protection to the infants for a few months.

The Sabin or Oral Polio vaccine:

On cultivation, the polio virus would probably lose its virulence for man and become available as living, attenuated virus vaccine.

Sabin and World Health Organisation conducted a large scale experiment with this living attenuated virus vaccine and the vaccine was found to be excellent and approved in 1962 for general use. The vaccine is given routinely by oral route to millions of people by incorporating it in candy, food, beverage.

Following the oral administration of the Sabin vaccine, the virus multiplies in the alimen­tary canal and the virus is discharged in the faeces. No symptoms are observed. In addition to the excellent antibody response, the intestinal infection apparently results in an immunization of the intestinal tract as well, thus preventing subsequent reinfection.