Rotavirus infection and diagnosis

Rotavirus is a widely recognised cause of acute gastroenteritis in young children all around the world. Almost every child under the age of five is at risk of contracting group A rotavirus (RVA). The World Health Organization (WHO) estimates that Rotavirus causes around 450,000 fatalities, more than 2 million hospitalizations, and 25 million outpatient cases each year, with more than 90 percent of mortality happening in underdeveloped Asian and African nations.

Both people and animals can get diarrhea from rotavirus. Rotavirus was identified as the cause of human sickness in 1973. (Bishop et al., 1973). The virus was detected using EM in intestinal biopsy tissues and feces from children suffering from acute gastroenteritis.

Morphology

Rotaviruses are named after the Latin word rota, which meaning “wheel.” When viewed with negative-stain EM, intact rotavirus particles show a characteristic appearance of a well-defined rim with small spikes emanating from a broad hub. The virus is made up of three capsids: an inner core, an intermediate capsid, and an outside capsid with small radiating spikes. Rotaviruses have a double-stranded RNA (dsRNA) that is included inside the core structure. Under the microscope, three kinds of rotavirus particles may be identified:

  • the complete infectious or triple-layered particles,
  • the double-layered particles, and
  • the core or single-layered particles.

Rotaviruses are dsRNA viruses that non-enveloped with a segmented genome. Its genome is approximately 18.5 Kb in size and is made up of 11 doublestranded RNA (dsRNA) segments that encode six structural (VP1, VP2, VP3, V41, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Except for gene segment 11, which is bicistronic and encodes two proteins in some Rotavirus strains, the gene segments are between 667 and 3302 BP in length and are monocistronic (NSP5 and NSP6).

Fig: A schematic diagram of rotavirus virion—the virion consists of an inner VP2 protein layer surrounding the RNA segments and several molecules of VP1 and VP3 proteins, a middle VP6 protein capsid, and an outer layer containing VP4 protein spikes embedded in a VP7 capsid.
GenomeEncoded ProteinFunction
1VP1RdRp
2VP2Core protein
3VP3Methyltransferase
4VP4Protease sensitive
5VP6Inner capsid
6VP7Glycosylated
7NSP1Interferon antagonist
8NSP2NTPase
9NSP3Translation enhancer
10NSP4Enterotoxin
11NSP5/NSP6Phosphoprotein

Pathogenesis

In several systems, the protein product of RNA segment 4, VP4, has been identified to be a key predictor of pathogenicity, although products of additional structural genes (VP3, VP7) and nonstructural genes (NSP1, NSP2, NSP4) have also been involved. Extensive cellular necrosis of the gut epithelium causes villous atrophy, loss of digestive enzymes, decreased absorption, and elevated osmotic pressure in the gut lumen, leading in diarrhea. This is followed by reactive crypt-cell hyperplasia, which is accompanied by increased fluid output and adds to the severity of the diarrhea.

A rotavirus infection causes symptomatic or asymptomatic illness. Although rotavirus infection is primarily confined to the small intestine, extraintestinal (systemic) dissemination has been recorded in a variety of organs. The pathogenesis of rotavirus diarrhea is influenced by both host and viral variables, and both have an impact on the disease’s prognosis. Age is the most obvious host factor that influences the disease’s clinical outcome. The reduction in maternal antibody titer correlates to the age of greatest rotavirus exposure in children between the ages of 3 and 24 months.

Rotavirus diarrhea-inducing ability has been attributed to several different mechanisms, including malabsorption secondary to enterocyte destruction or disruption of enterocyte absorptive functions, villus ischemia, enterotoxin activity and mobilization of intracellular fluid (calcium and chloride ion) secretion by NSP4, and activation of the enteric nervous and vascular systems that stimulate indirect secretion.

Disease manifestation

Rotavirus infections cause a variety of symptoms in children, ranging from asymptomatic illness to mild, watery diarrhea of limited duration to severe diarrhea with vomiting and fever, which can lead to dehydration, shock, electrolyte imbalance, and death. Symptoms of rotavirus infection are often accompanied with a triad of vomiting, diarrhea, and fever in both developed and developing environments.

Other clinical symptoms related with the diarrhea include irritability, fatigue, pharyngeal erythema, rhinitis, and palpable cervical lymph nodes. Rotavirus infections in young children incubate for 24 to 78 hours following infection, whereas adults incubate for 1 to 4 days. Rotavirus infection lasts 3 to 7 days on average; however, more severe infections might last up to 14 days. Rotavirus infection in newborns and young children can cause severe dehydration (10–20 bowel movements per day), electrolyte imbalance, and metabolic acidosis.

Rotavirus infection may persist longer in immunocompromised children due to congenital immunodeficiency diseases. Although rotavirus proteins and RNA have been found in various tissues, including blood and cerebrospinal fluid, the clinical relevance of these results is uncertain.

Laboratory Diagnosis Rotavirus

Timely detection of viruses linked with diarrhea is critical for proper patient care and the prevention of AGE outbreaks. To identify rotavirus in stool samples, a number of diagnostic procedures have been developed. Electron microscopy (EM), virus isolation in cell culture, polyacrylamide gel electrophoresis (PAGE) of viral RNA segments, enzyme immunoassays (EIAs), passive particle agglutination tests, immunochromatographic tests, coupled RT-PCR, and qRT-PCR are all instances of these procedures.

Electron Microscopy

Due to the unique morphologic appearance of a triple-layered virus with spikes, direct EM analysis of feces following negative staining with phosphotungstic acid is a very precise approach for detecting rotavirus particles. In a nutshell, fecal samples are suspended in phosphate buffered saline and centrifuged. The pellet is then resuspended in Tris buffer and negatively stained with phosphotungstic acid on formvar-coated grids. The grids are then viewed with a high magnification (×40,000) EM. The EM detection threshold for RVA is around 107 virus particles/mL of feces. EM, on the other hand, necessitates the purchase of a pricey instrument, is too labor demanding for regular detection of rotavirus in large numbers of stool samples, and necessitates highly skilled experts.

Cell culture isolation

Rotavirus is cultured from clinical fecal specimens using a variety of primary cell types and continuous cell lines. Rotaviruses may be recovered from African green monkey kidney cells as well as continuous cell lines such as Rhesus monkey kidney (MA104). Using cesium chloride gradients, virions may be isolated from RV-infected cell lysate. To confirm rotavirus recovery, purified virus is subjected to sodium dodecyl sulfate (SDS) –PAGE analysis. Based on the rotavirus’s cytopathic action in cultured cells, a plaque test may be used to estimate the viral titer in plaque-forming units per milliliter of virus. The fluorescence focus test, which is faster than the plaque assay, might also be used to quantify rotavirus.

This technique identifies viral antigen using polyclonal antibodies raised against intact virions, which cross-react with different rotavirus strains and provide a bright fluorescence signal. The detection limit of rotavirus by this approach is 5×102 infectious units per mL of sample. Human rotaviruses are difficult to isolate and cultivate from clinical fecal specimens, and adaptation to develop rotavirus in vitro often necessitates numerous rounds of passage in primary cells. The recovery of viruses from feces samples using cell culture is significantly less efficient. Because technique is time consuming, labor expensive, and prone to contamination, diagnosing rotavirus in stool samples using cell culture is rarely done on a regular basis.

Polyacrylamide gel electrophoresis

Rotavirus nucleic acid segments may be seen instantly following PAGE separation from virus particles and silver treatment. Human rotavirus groups A, B, and C show unique patterns of gene-segment distribution after electrophoresis, known as electropherotypes. The PAGE test may identify rotavirus in feces samples while also providing information on the viral electropherotype. RVAs in humans and animals have two types of electropherotypes: long and short. A brief electrophoretic pattern shows a bigger segment 11 (encoding NSP5) that migrates more slowly and is situated between gene segments 9 and 11. The sensitivity of the PAGE test is equivalent to that of EM and ELISA, but it is labor expensive and time consuming, hence it is not frequently used to identify rotavirus in stool samples nowadays.

Enzyme immunoassays

The most extensively used approach for rotavirus detection is EIA, which employs broadly reactive antibodies against VP6 epitopes shared by Rotavirus ‘A’. For routine laboratory identification of rotavirus in stool specimens, EIA provides a simple, quick, and highly sensitive approach. Rotavirus antigen is identified in EIA employing a colorimetric reaction in a solid-phase sandwich EIA configuration. A plate reader may be used to simply record the optical density. EIAs are tens to hundreds of times more sensitive than EM tests, although their sensitivity and specificity are more variable. For regular laboratory detection of rotavirus antigen in stool specimens, several commercial EIA kits are available.

Passive particle agglutination tests

Latex agglutination is a rapid diagnostic wherein latex particles coated with rotavirus antibodies interact in the presence of rotavirus antigen to form macroscopically apparent aggregates. The latex agglutination test for rotavirus antigen detection is direct, simple, and faster than EM and EIAs, and it may be utilized in diagnostic laboratories, emergency care, and physicians’ offices. Although the latex agglutination test is the least complicated, it has the lowest sensitivity and specificity when compared to other diagnostic tests such as EM, ELISAs, and PAGE.

Immunochromatographic tests

Immunochromatographic assays, also known as lateral flow tests or strip tests, can be used to identify rotavirus antigens in patient feces. Immunochromatographic techniques are based on the sandwich immunochromatography concept, in which antibodies against RVA-specific VP6 protein are utilized to identify rotavirus antigen in stool samples. The rotavirus antigen in the stool sample initially interacts with the antihuman antibody in the membrane strip before being collected by the recombinant antibody in immunochromatography strips; the presence of a colored line in the test window indicates a positive result.

When compared to EIA and latex agglutination tests, the immunochromatographic approach is faster, has higher sensitivity and specificity, and may be conducted routinely in clinical laboratories. The immunochromatographic tests are more sensitive and specific than latex agglutination tests and have equivalent sensitivity and specificity to EIA assays. Rapid screening for Rotavirus A in fecal material can be accomplished using immunochromatographic techniques.

PCR

Rotavirus may be detected in stool samples using a combination of RT and PCR tests at far lower concentrations (×1000 less) than those necessary for detection by EM and EIA. RT-PCR may be used to confirm that RNA extracts include intact rotavirus RNA. Rotavirus RT-PCR tests can be done in a singleplex or multiplex format, in conjunction with gel electrophoresis, probe hybridization, or real-time fluorescence capture. qRT-PCR tests provide various benefits over classic RT-PCR assays, including greater sensitivity, better throughput, shorter turnaround time, and viral load measurement.

Treatment

Antibiotics and other medications are ineffective against rotavirus. The treatment’s primary goal is to replenish fluids and electrolytes lost due to vomiting and diarrhea. Mild rotavirus infections can be efficiently treated in the same way as other types of diarrhea are, by administering fluids and salts until the sickness has run its course. Children with severe rotavirus diarrhea, on the other hand, require IV fluids immediately or risk dying from dehydration. This sort of urgent health treatment is frequently inaccessible or unavailable in low-income nations, making rotavirus vaccine crucial.

References

  • Principles and Practice of Clinical Virology, Sixth Edition
  • Esona MD, Gautam R. Rotavirus. Clin Lab Med. 2015 Jun;35(2):363-91. doi: 10.1016/j.cll.2015.02.012. PMID: 26004648.
  • Sadiq A, Bostan N, Yinda KC, Naseem S, Sattar S. Rotavirus: Genetics, pathogenesis and vaccine advances. Rev Med Virol. 2018 Nov;28(6):e2003. doi: 10.1002/rmv.2003. Epub 2018 Aug 29. PMID: 30156344.
  • Rivero-Calle I, Gómez-Rial J, Martinón-Torres F. Systemic features of rotavirus infection. J Infect. 2016 Jul 5;72 Suppl:S98-S105. doi: 10.1016/j.jinf.2016.04.029. Epub 2016 May 12. PMID: 27181101.

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