Rotavirus

Rotavirus is the most common cause of severe diarrhoea among infants and young children, and is one of several viruses that cause infections often called stomach flu, despite having no relation to influenza. It is a genus of double-stranded RNA virus in the family Reoviridae. By the age of five, nearly every child in the world has been infected with rotavirus at least once. However, with each infection, immunity develops, and subsequent infections are less severe; adults are rarely affected. There are five species of this virus, referred to as A, B, C, D, and E. Rotavirus A, the most common, causes more than 90% of infections in humans.

The virus is transmitted by the faecal-oral route. It infects and damages the cells that line the small intestine and causes gastroenteritis. Although rotavirus was discovered in 1973 and accounts for up to 50% of hospitalisations for severe diarrhoea in infants and children, its importance is still not widely known within the public health community, particularly in developing countries. In addition to its impact on human health, rotavirus also infects animals, and is a pathogen of livestock.

Rotavirus is usually an easily managed disease of childhood, but worldwide more than 500,000 children under five years of age still die from rotavirus infection each year and almost two million more become severely ill. In the United States, before initiation of the rotavirus vaccination programme, rotavirus caused about 2.7 million cases of severe gastroenteritis in children, almost 60,000 hospitalisations, and around 37 deaths each year. Public health campaigns to combat rotavirus focus on providing oral rehydration therapy for infected children and vaccination to prevent the disease.

History
In 1943, Jacob Light and Horace Hodes proved that a filterable agent in the faeces of children with infectious diarrhoea also caused scours (livestock diarrhoea) in cattle. Three decades later, preserved samples of the agent were shown to be rotavirus. In the intervening years, a virus in mice was shown to be related to the virus causing scours. In 1973, Ruth Bishop and colleagues described related viruses found in children with gastroenteritis.

In 1974, Thomas Henry Flewett suggested the name rotavirus after observing that, when viewed through an electron microscope, a rotavirus particle looks like a wheel (rota in Latin); the name was officially recognised by the International Committee on Taxonomy of Viruses four years later. In 1976, related viruses were described in several other species of animals. These viruses, all causing acute gastroenteritis, were recognised as a collective pathogen affecting humans and animals worldwide. Rotavirus serotypes were first described in 1980, and in the following year, rotavirus from humans was first grown in cell cultures derived from monkey kidneys, by adding trypsin (an enzyme found in the duodenum of mammals and now known to be essential for rotavirus to replicate) to the culture medium. The ability to grow rotavirus in culture accelerated the pace of research, and by the mid-1980s the first candidate vaccines were being evaluated.

In 1998, a rotavirus vaccine was licensed for use in the United States. Clinical trials in the United States, Finland, and Venezuela had found it to be 80 to 100% effective at preventing severe diarrhoea caused by rotavirus A, and researchers had detected no statistically significant serious adverse effects. The manufacturer, however, withdrew it from the market in 1999, after it was discovered that the vaccine may have contributed to an increased risk for intussusception, a type of bowel obstruction, in one of every 12,000 vaccinated infants. The experience provoked intense debate about the relative risks and benefits of a rotavirus vaccine. In 2006, two new vaccines against rotavirus A infection were shown to be safe and effective in children, and in June 2009 the World Health Organization recommended that rotavirus vaccination be included in all national immunisation programmes to provide protection against this virus.

Signs and symptoms
Rotavirus gastroenteritis is a mild to severe disease characterised by vomiting, watery diarrhoea, and low-grade fever. Once a child is infected by the virus, there is an incubation period of about two days before symptoms appear. Symptoms often start with vomiting followed by four to eight days of profuse diarrhoea. Dehydration is more common in rotavirus infection than in most of those caused by bacterial pathogens, and is the most common cause of death related to rotavirus infection.

Rotavirus A infections can occur throughout life: the first usually produces symptoms, but subsequent infections are typically asymptomatic, as the immune system provides some protection. Consequently, symptomatic infection rates are highest in children under two years of age and decrease progressively towards 45 years of age. Infection in newborn children, although common, is often associated with mild or asymptomatic disease; the most severe symptoms tend to occur in children six months to two years of age, the elderly, and those with  compromised or absent immune system functions. Due to immunity acquired in childhood, most adults are not susceptible to rotavirus; gastroenteritis in adults usually has a cause other than rotavirus, but asymptomatic infections in adults may maintain the transmission of infection in the community. Symptomatic reinfections are often due to a different rotavirus A serotype.

Transmission
Rotavirus is transmitted by the faecal-oral route, via contact with contaminated hands, surfaces and objects, and possibly by the respiratory route. The faeces of an infected person can contain more than 10 trillion infectious particles per gram; only 10–100 of these are required to transmit infection to another person.

Rotaviruses are stable in the environment and have been found in estuary samples at levels as high as 1–5 infectious particles per US gallon. Sanitary measures adequate for eliminating bacteria and parasites seem to be ineffective in control of rotavirus, as the incidence of rotavirus infection in countries with high and low health standards is similar.

Disease mechanisms
The diarrhoea is caused by multiple activities of the virus. Malabsorption occurs because of the destruction of gut cells called enterocytes. The toxic rotavirus protein NSP4 induces age- and calcium ion-dependent chloride secretion, disrupts SGLT1 transporter-mediated reabsorption of water, apparently reduces activity of brush-border membrane disaccharidases, and possibly activates the calcium ion-dependent secretory reflexes of the enteric nervous system. Healthy enterocytes secrete lactase into the small intestine; milk intolerance due to lactase deficiency is a particular symptom of rotavirus infection, which can persist for weeks. A recurrence of mild diarrhoea often follows the reintroduction of milk into the child's diet, due to bacterial fermentation of the disaccharide lactose in the gut.

Diagnosis and detection
Diagnosis of infection with rotavirus normally follows diagnosis of gastroenteritis as the cause of severe diarrhoea. Most children admitted to hospital with gastroenteritis are tested for rotavirus A. Specific diagnosis of infection with rotavirus A is made by finding the virus in the child's stool by enzyme immunoassay. There are several licensed test kits on the market which are sensitive, specific and detect all serotypes of rotavirus A. Other methods, electron microscopy and polyacrylamide gel electrophoresis, are used in research laboratories. Reverse transcription-polymerase chain reaction (RT-PCR) can detect and identify all species and serotypes of human rotavirus.

Treatment and prognosis
Treatment of acute rotavirus infection is nonspecific and involves management of symptoms and, most importantly, maintenance of hydration. If untreated, children can die from the resulting severe dehydration. Depending on the severity of diarrhoea, treatment consists of oral rehydration, during which the child is given extra water to drink that contains small amounts of salt and sugar. Some infections are serious enough to warrant hospitalisation where fluids are given by intravenous drip or nasogastric tube, and the child's electrolytes and blood sugar are monitored.

Rotavirus infections rarely cause other complications and for a well managed child the prognosis is excellent. There are rare reports of complications involving the central nervous system (CNS) where rotavirus was detected in the fluid of the CNS in cases of encephalitis and meningitis,  and recent studies have confirmed that rotavirus infection is not always confined to the gut, but can cause viremia.

Epidemiology


Rotavirus A, which accounts for more than 90% of rotavirus gastroenteritis in humans, is endemic worldwide. Each year rotavirus causes millions of cases of diarrhoea in developing countries, almost 2 million resulting in hospitalisation and an estimated 611,000 resulting in death. In the United States alone—before initiation of the rotavirus vaccination programme —over 2.7 million cases of rotavirus gastroenteritis occurred annually, 60,000 children were hospitalised and around 37 died from the results of the infection. The major role of rotavirus in causing diarrhoea is not widely recognised within the public health community, particularly in developing countries. Almost every child has been infected with rotavirus by age five. It is the leading single cause of severe diarrhoea among infants and children, being responsible for about 20% of cases, and accounts for 50% of the cases requiring hospitalisation. Boys are twice as likely to be admitted to hospital as girls. In temperate areas, rotavirus infections occur primarily in the winter, but in the tropics they occur throughout the year; the difference is partly explained by seasonal changes in temperature and humidity. The number attributable to food contamination is unknown.

Outbreaks of rotavirus A diarrhoea are common among hospitalised infants, young children attending day care centres, and elderly people in nursing homes. An outbreak caused by contaminated municipal water occurred in Colorado in 1981. During 2005, the largest recorded epidemic of diarrhoea occurred in Nicaragua. This unusually large and severe outbreak was associated with mutations in the rotavirus A genome, possibly helping the virus escape the prevalent immunity in the population. A similar large outbreak occurred in Brazil in 1977.

Rotavirus B, also called adult diarrhoea rotavirus or ADRV, has caused major epidemics of severe diarrhoea affecting thousands of people of all ages in China. These epidemics occurred as a result of sewage contamination of drinking water. Rotavirus B infections also occurred in India in 1998; the causative strain was named CAL. Unlike ADRV, the CAL strain is endemic. To date, epidemics caused by rotavirus B have been confined to mainland China, and surveys indicate a lack of immunity to this species in the United States.

Rotavirus C has been associated with rare and sporadic cases of diarrhoea in children in many countries, and outbreaks have occurred in Japan and England.

Prevention
Because improved sanitation does not decrease the prevalence of rotaviral disease, and the rate of hospitalisations remains high, despite the use of oral rehydrating medicines, the primary public health intervention is vaccination. In 2006, two vaccines against Rotavirus A infection were shown to be safe and effective in children: Rotarix by GlaxoSmithKline and RotaTeq by Merck. Both are taken orally and contain attenuated live virus. Rotavirus vaccines are licensed in more than 100 countries, but only 17 countries have introduced routine rotavirus vaccination.

The Rotavirus Vaccine Program is a collaboration between PATH, the World Health Organization, and the U.S. Centers for Disease Control and Prevention, and is funded by the GAVI Alliance. The Program aims to reduce child morbidity and mortality from diarrhoeal disease by making a vaccine against rotavirus available for use in developing countries.

On June 5, 2009, WHO announced that clinical trials of Rotarix vaccine “in high-mortality, low-socioeconomic settings of South Africa and Malawi, found that the vaccine significantly reduced severe diarrhoea episodes due to rotavirus.” WHO now recommends that rotavirus vaccine be included in all national immunisation programmes and the incidence of rotavirus disease in the United States has declined since introduction of rotavirus vaccination for children. Additional rotavirus vaccines are under development.

Infections of animals
Rotaviruses infect and cause diarrhoea in the young of many species of animals. They infect mammals (for example, apes, cattle, pigs, sheep, rats, cats and dogs, mice, horses, rabbits) and birds (chickens and turkeys). These rotaviruses are a potential reservoir for genetic exchange with human rotaviruses. There is evidence that animal rotaviruses can infect humans, either by direct transmission of the virus or by contributing one or several RNA segments to reassortants with human strains. Rotavirus are a pathogen of livestock and cause economic loss to farmers because of costs of treatment associated with high morbidity and mortality rates.

Types of rotavirus
There are five species of rotavirus, referred to as A, B, C, D and E. Humans are primarily infected by species A, B and C, most commonly by species A. All five species cause disease in other animals. Within rotavirus A there are different strains, called serotypes. As with influenza virus, a dual classification system is used, which is based on two structural proteins on the surface of the virion. The glycoprotein VP7 defines G-types and the protease-sensitive protein VP4 defines P-types. Strains are generally designated by their G serotype specificities (e.g., serotypes G1 to G4 and G9), and the P-type is indicated by a number and a letter for the P-serotype and by a number in square brackets for the corresponding P-genotype. (P-serotypes are difficult to characterize; therefore, molecular methods based on sequence analysis are often used to define the corresponding P-genotype instead. These genotypes correlate well with known P-serotypes). Because the two genes that determine G-types and P-types can be passed on separately to offspring, various combinations occur in any one strain. The Wa strain is classified in full as G1P1A[8].

Structure
The genome of rotavirus consists of 11 unique double helix molecules of RNA which are 18,555 nucleotides in total. Each helix, or segment, is a gene, numbered 1 to 11 by decreasing size. Each gene codes for one protein, except genes 9 and 11, which each code for two. The RNA is surrounded by a three-layered icosahedral protein capsid. Viral particles are up to 76.5 nm in diameter and are not enveloped.

Proteins
There are six viral proteins (VPs) that form the virus particle (virion). These structural proteins are called VP1, VP2, VP3, VP4, VP6 and VP7. In addition to the VPs, there are six nonstructural proteins (NSPs), that are only produced in cells infected by rotavirus. These are called NSP1, NSP2, NSP3, NSP4, NSP5 and NSP6. At least six of the twelve proteins encoded by the rotavirus genome bind RNA. The role of these proteins play in rotavirus replication is not entirely understood; their functions are thought to be related to RNA synthesis and packaging in the virion, mRNA transport to the site of genome replication, and mRNA translation and regulation of gene expression.

Structural proteins
VP1 is located in the core of the virus particle and is an RNA polymerase enzyme. In an infected cell this enzyme produces mRNA transcripts for the synthesis of viral proteins and produces copies of the rotavirus genome RNA segments for newly produced virus particles.

VP2 forms the core layer of the virion and binds the RNA genome.

VP3 is part of the inner core of the virion and is an enzyme called guanylyl transferase. This is a capping enzyme that catalyses the formation of the 5' cap in the post-transcriptional modification of mRNA. The cap stabilises viral mRNA by protecting it from nucleic acid degrading enzymes called nucleases.

VP4 is on the surface of the virion that protrudes as a spike. It binds to molecules on the surface of cells called receptors and drives the entry of the virus into the cell. VP4 has to be modified by a protease enzyme (found in the gut) into VP5* and VP8* before the virus is infectious. It determines how virulent the virus is and it determines the P-type of the virus.

VP6 forms the bulk of the capsid. It is highly antigenic and can be used to identify rotavirus species. This protein is used in laboratory tests for rotavirus A infections.

VP7 is a glycoprotein that forms the outer surface of the virion. Apart from its structural functions, it determines the G-type of the strain and, along with VP4, is involved in immunity to infection.

Nonstructural viral proteins
NSP1, the product of gene 5, is a nonstructural RNA-binding protein.

NSP2 is an RNA-binding protein that accumulates in cytoplasmic inclusions (viroplasms) and is required for genome replication.

NSP3 is bound to viral mRNAs in infected cells and it is responsible for the shutdown of cellular protein synthesis.

NSP4 is a viral enterotoxin to induce diarrhoea and was the first viral enterotoxin discovered.

NSP5 is encoded by genome segment 11 of rotavirus A and in virus-infected cells NSP5 accumulates in the viroplasm.

NSP6 is a nucleic acid binding protein, and is encoded by gene 11 from an out of phase open reading frame.

This table is based on the simian rotavirus strain SA11. RNA-protein coding assignments differ in some strains.

Replication
Rotaviruses replicate mainly in the gut, and infect enterocytes of the villi of the small intestine, leading to structural and functional changes of the epithelium. The triple protein coats make them resistant to the acidic pH of the stomach and the digestive enzymes in the gut.

The virus enter cells by receptor mediated endocytosis and form a vesicle known as an endosome. Proteins in the third layer (VP7 and the VP4 spike) disrupt the membrane of the endosome, creating a difference in the calcium concentration. This causes the breakdown of VP7 trimers into single protein subunits, leaving the VP2 and VP6 protein coats around the viral dsRNA, forming a double-layered particle (DLP).

The eleven dsRNA strands remain within the protection of the two protein shells and the viral RNA-dependent RNA polymerase creates mRNA transcripts of the double-stranded viral genome. By remaining in the core, the viral RNA evades innate host immune responses called RNA interference that are triggered by the presence of double-stranded RNA.

During the infection, rotavirus produces mRNA for both protein biosynthesis and gene replication. Most of the rotavirus proteins accumulate in viroplasm, where the RNA is replicated and the DLPs are assembled. Viroplasm is formed around the cell nucleus as early as two hours after virus infection, and consists of viral factories thought to be made by two viral nonstructural proteins: NSP5 and NSP2. Inhibition of NSP5 by RNA interference results in a sharp decrease in rotavirus replication. The DLPs migrate to the endoplasmic reticulum where they obtain their third, outer layer (formed by VP7 and VP4). The progeny viruses are released from the cell by lysis.