Rotavirus was first discovered in animals. In humans, the virus was first detected by electron microscopy in duodenal biopsy of a child with acute gastroenteritis. The shape of the virus appeared like a wheel under electron microscopy which led to the naming of virus as “Rotavirus” (“Rota” means wheel).
VIRUS
Rotavirus belongs to the family Reoviridae.
Structure of virus (Fig. 1): Rotavirus (RV) is a non-enveloped virus of 100 nm particle having icosahedral symmetry. The fully infectious virus particle consists of three concentric layers of proteins also termed triple layered particle (TLP).
Fig1. Structure of rotavirus
Three layers from outside inwards are outer capsid, inner capsid and internal core which are made up of VP7, VP6 and VP2, respectively. These layers surround the inner genome which is a double-stranded RNA having 11 segments. Each gene segment encodes for single protein.
Outer capsid proteins: VP4 and VP7 are structural proteins, present in the outer capsid. VP7 forms the smooth layer of the outer shell. VP4 forms the spikes on it. They are the outer most components of the virus and act as the neutralization antigens and target of neutralizing antibody. That makes them important candidate for vaccine preparation.
• VP7: It is a glycosylated protein. Defines the G serotypes. There are currently 27 G serotypes, of which 12 are human strains.
• VP4: It is a protease cleaved protein. It defines the P serotypes. P types have been divided into serotypes and genotypes.
Inner capsid protein: VP6 contains the group specific antigen. Based on VP6, RV has been divided into 8 groups A to H. Group A rotaviruses are most common human pathogen responsible for diarrhea in children.
• Inner core: This layer is made up of VP2 protein.
• VP1 (RNA dependent RNA polymerase) and VP3 (a methyl transferase), together makes a complex and situated on the inner aspect of VP2. VP1 and VP3 are required for viral replication.
• NSP4 is a non-structural protein. It is also called viral enterotoxin. It acts a virulence factor for the virus.
• Replication of the virus in cell culture requires prior treatment with proteases which cleaves the outer capsid spike protein VP4 and increases the infectivity of the virus.
PATHOGENESIS
Pathogenesis of rotavirus diarrhea is multifactorial.
Malabsorption Mediated Diarrhea
• Destruction of epithelial cells: Rotavirus (RV) infects the differentiated enterocytes at the top of the villi, leads to vacuolization and destruction of epithelial cells which in turn leads to disruption of function of enterocytes.
• Action of rotavirus enterotoxin NSP4: This viral enterotoxin is released from the infected cells and exerts the paracrine effect on its neighboring cells. It activates the cell signaling pathway by which there occurs intracellular mobilization of calcium and chloride secretion.
• Villus ischemia and activation of enteric nervous system also plays role in causing malabsorption.
Pathogenesis of vomiting: Enterochromaffin cells of the gut when gets infected with RV, stimulates the secretion of serotonin. This in turn activates the vagal afferent nerves and stimulates the part of the brainstem that controls the vomiting.
Role of Viral Proteins in Pathogenesis
• VP4 and VP7: Mediate viral entry to host cells.
• VP3, VP6, NS2 and NS3: Efficient viral replication
• NSP1 and NSP3: Extraintestinal spread
• NSP4: Enterotoxin
In neonates and immunosuppressive individuals, RV can infect and replicate in various extraintestinal sites like liver, biliary tract and pancreas leading to biliary atresia and pancreatitis.
CLINICAL FEATURES
Rotavirus in children: Symptomatic infection of rotavirus usually occurs in children. Rotavirus causes moderate to severe degree of diarrhea associated with vomiting and fever. Diarrhea is usually preceded by vomiting. Electrolyte disturbance due to diarrhea and vomiting leads to dehydration. Fever generally is of mild grade. Severe dehydration and cardiovascular failure are mainly responsible for rotavirus diarrheal death in developing countries.
RV usually causes severe diarrhea in certain group of immunosuppressed individuals like bone marrow or stem cell transplant recipients. Rarely it has been associated with chronic symptoms in immunocompromised patients.
The role of rotavirus in causation of intussusceptions is still not clear. However, it is presently accepted that wild RV can cause intussusception but with extreme rarity.
Temporal association of rotavirus has been reported with necrotizing enterocolitis and hemorrhagic gastroenteritis in neonates.
Rotavirus in adults: In adults, repeated infection can occur due to rotavirus. However, most of the infections are asymptomatic in nature. Mild symptoms can occur rarely. In contrast to other parts of the world, RV is the second cause of acute gastroenteritis in China, next to norovirus.
EPIDEMIOLOGY
Rotavirus is one of the important causes of acute gastroenteritis amongst children below 5 years age leading to near 5% deaths worldwide. This accounts for approximately 453,000 deaths worldwide annually, of which more than one-fifth (22%) is contributed by India. It is estimated to account for near 2.5 millions hospitalizations globally and 10 lakhs in India. Majority of rotavirus deaths (80%) occurs in Southeast Asian countries and Africa. Annual rotavirus deaths per year in India, China and Pakistan are 1,22,000; 27,000; and 20,000, respectively.
Rotavirus is seen both in developed and developing countries including North America, Europe, Australia, Japan amongst the developed world, and Asia, Africa and South America amongst the tropical developing countries. In both developed and developing countries, the age and symptom pattern is same but due to early and effective management the death rate in developed world is much less as compared to that of developing countries.
Molecular Epidemiology
Group A rotavirus: Group A rotaviruses are genetically divergent. It consists of 27G types and 35 P types. Of these, 12 G (1 to 6, 8 to 12, and 20) and 15 P types (1 to 6, 8 to 11, 13, 14, 19, 25, and 28) have been isolated from human beings. G1 is most frequently detected serotype from each continent.
G1–G4 constitute 97% of all G types isolated from Asia, North America and Australia. G9 occasionally emerges as the predominant strain in several continents.
Amongst the combination of G and P types, five combinations are predominant throughout the world—G2P[4], G1P[8], G3P[8], and G4P[8] and G9P[8].
Group B rotaviruses: Group B rotaviruses otherwise known as adult diarrheal rotavirus (ADRV) are responsible for causing several large outbreaks of severe gastroenteritis in adults in China and other south Asian countries like India and Bangladesh. Large outbreak affecting near 20,000 people has been reported in China. Cholera-like symptoms with severe watery diarrhea is the main symptom. Contaminated water has been implicated as the source of infection.
Rotavirus diarrhea also occurs quite often in children in day care centers and health care settings.
Infectiousness of RV: Transmission of rotavirus occurs through feco-oral route. Rotaviruses are highly infectious in nature as compared to other diarrheal pathogens. The high infectivity occurs due to:
• Low infective dose: One tissue culture infective dose can cause infection in susceptible host.
• A large number of viruses shedding in diarrheic stool (up to 1011/mL).
• Survival of rotavirus in adverse environ mental conditions and resistance to physical inactivation helps in efficient transmission.
• Short incubation period: 1–2 days.
• Shedding of virus in stool starts before the onset of diarrhea and continues after cessation of diarrhea.
Transmission through respiratory route also has been proposed because:
• Occurrence of rotavirus outbreak without evidence of feco-oral transmission.
• High seroprevalence of rotavirus during first year of life even in developed countries.
• Occasional occurrence of respiratory symptoms in rotavirus infected patients. Isolation of RV from upper respiratory tract.
However, there is no definite proof for respiratory mode of transmission.
Role of animal rotavirus strains: Rotaviruses are host-specific but genetic recombinations can occur between RVs of same as well as different hosts. Various reassortant strains, human-bovine, human-pig and human simian strains, have been isolated from humans particularly from high endemic countries like India and Brazil.
Seasonal pattern: In topical developing countries, rotavirus infection is seen throughout the year. In southern India, rotavirus infection is seen throughout the year, but in north India it is mostly observed during the cold dry months of the year.
In temperate climate countries, peak rotavirus infection occurs during winter months of January to April.
IMMUNE RESPONSE
Presence of maternal antibody in the neonate protects from severe rotavirus infection. In neonate, severe rotavirus diarrhea is thus seen between 3 and 24 months of age, 7 and 15 months being the peak. This possibly reflects the decreasing titer of maternal antibody.
Repeated infection in neonates also protects them from subsequent severe diarrhea.
Humoral immunity plays an important role in protection of severe rotavirus infection. Serum antibody is one of the major indicators of protection after natural and vaccine-induced infection.
• VP6 is the main immunodominant anti gen. IgG or IgA antibody against this acts as the measure of protective immunity.
• Antibodies against the surface antigens VP4 and VP7 also play important role in protection against infection. Their role in vaccine-induced immunity is not clear. Though it is believed that antibody against the surface protein is essential for vaccine-induced protection, vaccine producing low titer of serum antibody also has been found to be effective.
• Local gut immunity also considered as important for protection.
DIAGNOSIS
Detection of virus or its components like viral antigen or genome from diarrheic stool is the mainstay of diagnosis.
Detection of antibody against RV is done to check the immunity status of the individual after vaccination or infection.
Stool sample during early phase (1–4 days) is the best sample because of high number of virus.
Detection of Virus
• Direct detection of virus in the stool sample was previously done by electron microscopy (EM). Because of very typical wheel-like shape of rotavirus, it can be diagnosed by EM. However, as the method involves expensive equipment and tedious procedure, it is no more used for routine diagnosis purpose.
• Isolation of rotavirus can be done by cell culture method which further can be used for serotyping of the virus. However, this is time consuming and technically demanding, so not a preferred method for patient diagnosis. However, cultivation of virus is important to study the virus.
Antigen Detection
Detection of viral antigen by enzyme immune assay is routinely used for its high sensitivity, specificity as well as for its convenience. Commercial ELISA systems are available for group A and also for group B and C. It requires at least 104 to 107 virus particle/mL of specimen to be positive.
Viral RNA Detection
• Viral RNA can be detected by reverse transcriptase polymerase chain reaction (RT-PCR). This test is 1000 times more sensitive than ELISA and also can be used for genotype detection.
• Dot-blot hybridization was used as a good sensitive method. This was based on the principle of hybridization between the labeled rotavirus single strand RNA and heat denatured double strand rotavirus RNA immobilized to nitrocellulose mem brane. The technique was more sensitive than ELISA but no more preferred after the wide use of RT-PCR.
• Older methods: Gel electrophoresis of RV RNA, countercurrent immunoelectrophoresis, latex agglutination were previously used and not used presently because of availability of better tests.
Detection of Antibody
• IgA, IgG and IgM against rotavirus can be detected to check the immune status and generally not required for diagnosis. Rotavirus antibody detection is done commonly by ELISA or neutralization assay.
• Complement fixation, immune adherence hemagglutination, hemagglutination inhibition, immunofluorescence, etc. were used previously.
TREATMENT
The primary goal of treatment is fluid replacement and electrolyte maintenance. Oral rehydration salt therapy is recommended by World Health Organization (WHO) and widely used for treatment of acute gastroenteritis. Intravenous administration is applicable whenever oral therapy is not possible.
PREVENTION
Isolation of infected patient, hand hygiene and environmental disinfection are the primary key to prevent the spread of virus.
• Surface decontamination with hypochlorite with free chlorine content of minimum 20,000 parts per million should be used instead of phenolic disinfectants.
• Ethyl alcohol hand sanitizer is able to destroy the virus.
VACCINE
Rotavirus vaccine has come a long way and presently several licensed vaccines are available. Many countries have already adapted the vaccination in their national program.
The need of vaccine to prevent rotavirus is self-explanatory from the high number of mortality and morbidity associated with it in children below 5 years age.
Important Factors in Rotavirus Vaccine
Protective Immunity by Natural Infection
• Natural infection provides immunity from severe infection.
• Decrease in severity with repeated infection.
• Severity of infection is almost nil after second infection.
Circulating rotavirus serotypes: Multiple G and P serotypes and genotypes circulate worldwide. This was thought to be important for vaccine preparation, so that vaccine can be made based on the locally prevailing serotypes. Presently it is evident that both monovalent and polyvalent vaccines are able to provide effective immunity.
Jennerian approach: Edward Jenner had used animal virus, cowpox virus, for preparation of vaccine against the smallpox that is caused by the variola virus which is antigenically similar another poxvirus. This approach is based on the principle that animal viruses that share antigenic relatedness and non-pathogenic for human host, when infect the human host do not produce disease but able to mount protective immunity against the pathogenic human strains because of cross-protection. The concept of using an antigenically related but non-pathogenic virus for production of protective antibody is called Jennerian approach. This approach has been tried in rotavirus vaccine preparation with animal rotaviruses as they share a major common antigen.
Presently Licensed Vaccine
Monovalent Vaccine (RV1)
• This is a live attenuated vaccine prepared from a virulent human rotavirus strain 89 12, of serotype G1 P[8].
• Attenuation has been done by several passages in tissue culture.
• It is given orally, 2 doses at 6 weeks and 10 weeks.
• It provides protection against G homotypic and heterotypic strains as well as P homotypic strains.
• Protective efficacy against severe disease in Asia and Africa is »60% and in developed countries »95%.
• Manufactured by GlaxoSmithKline, with brand name Rotarix (RV1).
Polyvalent Bovine-human Reassortant Vaccine (RV5)
• This is a pentavalent reassortant vaccine which consists of
– VP7 of G1, G2, G3 and G4 in 4 strains and VP4 of P1A[8] in 1 strain.
– All five strains are reassortant with WC3 strain which provides the genetic back bone.
• It is given orally, in 3 doses at 2, 4, and 6 months age.
• Protective efficacy against severe disease in Asia and Africa is »50% and in developed countries »85%.
• Manufactured by Merck with the brand name RotaTeq (RV5).
Both the vaccines are licensed in more than 100 countries worldwide.
Both the vaccines show rare temporal association with intussusceptions, < 1:50,000.
Indian Nursery Strain 116E
• This is a live attenuated monovalent vaccine which is now licensed in India.
• The vaccine is prepared from the strain 116E G9 P8[11], a naturally occurring human-bovine reassortant strain. This strain was first found in nursery of All India Institute of Medical Sciences, New Delhi. It contains 10 genes from human rotavirus and VP4 from bovine rotavirus.
• The strain was found to cause subclinical infection and protects from severe rotavirus for up to 3 years.
• The vaccine is given in 3 doses along with DPT with a vaccine efficacy against severe gastroenteritis of 56.4%.
• The vaccine has been launched in several states across India as first phase with a plan to expand the program to all over the country.
• It is manufactured by Bharat Biotech with the brand name of Rotavac.
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