# Narin KORKMAZ - ***Rotavirus*** -Virology by P. Saravanan (2017)

**ROTAVIRUSES**
Rotavirus diarrhoea is a major public health problem throughout the world causing more than 125 million cases of infantile diarrhoea and about 1 million deaths per year.

**History**
Rotavirus was first discovered in 1973 by Bishop et al. and the association of the 70 nm rotavirus with severe diarrhoea in infants and young children was also found. The identification relied on direct visualization by thin section electron microscopy (EM) of duodenal biopsies, obtained from children with acute non-bacterial gastroenteritis.
>Source:https://www.historyofvaccines.org/content/articles/rotavirus

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**Structure**
The term rotavirus is derived from the Latin word, ‘Rota’ which means ‘wheel’, and was suggested because, the sharply defined circular outline of the outer capsid gives the appearance of the rim of a wheel placed on short spokes radiating from a wide hub.

Figure 1. Appearance of rotavirus
> Source: https://www.medicana.com.tr/saglik-rehberi-detay/12267/rota-virusu

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**Characteristics of Rotaviruses**

Figure 2. Characteristic of Rotaviruses
> Source: Virology by P. Saravanan (2017)

**Epidemiology**
Epidemiology Numerous studies on the distribution of group A rotavirus ‘G’ serotypes have demonstrated a high prevalence of serotypes G1 to G4 globally. However, unusual ‘G’ serotypes can be common in some parts of the world, especially developing countries e.g. G8 and G9 in India, and G5 and G10 in Brazil. Presently, rotavirus serotype can also be predicted by nucleotide sequence analysis of the VP7 gene and VP4 gene, because there is a high degree of conservation of sequence among rotaviruses belonging to the same serotype. The use of reverse transcriptase-polymerase chain reaction (RT-PCR) technique has enabled the genotyping of rotavirus specimens that could not be typed by ELISA. Since this method is based on genomic RNA, the term ‘genotyping’ is used in place of serotyping. This method is 1000 times more sensitive than ELISA and 10,000 times more sensitive than RNA-PAGE.

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**Clinical Features**
Rotavirus infection produces a spectrum of responses that vary from subclinical infection to mild diarrhoea to a severe and occasionally fatal dehydrating illness. Three major clinical features observed in rotavirus gastroenteritis were vomiting, diarrhoea and dehydration. The incubation period in humans and most animals appeared to be 2–3 days. In a study conducted by Rodriguez et al. (1977), the clinical
 Fıgure 3. Rotavıruses and gastroenteritis
> Source:https://www.historyofvaccines.org/content/articles/rotavirus

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**Assays for Rotavirus Detection**
Many assays have been developed for the detection of rotaviruses in stools. Specimens from the first to fourth day of illness are ideal for rotavirus detection.
Initially, visualization of stool material by direct electron microscopy (DEM) was employed for rotavirus detection which permits detection of rotavirus in 80% to 90% of the virus positive specimens. DEM has the advantage of high specificity because rotaviruses have a distinctive morphologic appearance. DEM continues to be a mainstay in the diagnosis of rotavirus disease, and is frequently used as the final arbiter when discrepancies occur with other techniques.
* **ELISA** is the most widely used method for the diagnosis of HRV infections. The method has the advantage of screening samples on large scale rapidly. Presently, Mab ELISA has been used in the detection of rotavirus from faecal specimens.
Figure 4. Sandwich Elisa
> Source: https://nordicbiosite.com/blog/elisa-principles-101

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* **Polyacrylamide gel electrophoresis (PAGE)** All rotaviruses contain a genome of 11 double-stranded RNA segments contained within the core of the virus. The genomic RNA segments can be resolved by PAGE and visualized by ethidium bromide or silver staining. The resulting RNA profiles are called genome pattern or electropherotypes (E-types). E-types have been widely used to characterize isolates from outbreaks.
Figure 5 : ResearchGate SDS-PAGE analysis of purified VLPs of rotavirus from silkworm.
> Source: https://www.researchgate.net/figure/SDS-PAGE-analysis-of-purified-VLPs-of-rotavirus-from-silkworm-5-mg-VLPs-was-loaded-on_fig5_221689547

* **RT-PCR** PCR amplification of rotavirus nucleic acid from stool specimens has been described and its sensitivity has been reported to be 1000 times higher than ELISA and 10,000 times higher than PAGE. Most PCR studies on group A rotaviruses are limited to characterization into G and P genotypes using gene segment 9 and 4 respectively. The use of RT-PCR as a diagnostic tool for group A rotavirus and its comparison with other routine methods such as ELISA and PAGE has been reported. The investigators reported RT-PCR as the most sensitive and specific assay for detection of group A rotaviruses than PAGE and ELISA.
Figure 6 : PCR typing of four representative bovine rotavirus strains.
> Source: https://www.researchgate.net/figure/SDS-PAGE-analysis-of-purified-VLPs-of-rotavirus-from-silkworm-5-mg-VLPs-was-loaded-on_fig5_221689547
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* **Rapid and sensitive detection of rotavirus by surface-enhanced Raman scattering immunochromatography** A surface-enhanced Raman scattering (SERS) immunochromatographic assay (ICA) has been developed for rapid, ultrasensitive, and quantitative detection of rotavirus in feces using double Raman molecule-labeled Au-core Ag-shell nanoparticles. The Raman signals are generated by 5,5′-dithiobis-(2-nitrobenzoic acid) and the intensity of the characteristic peak at 1334−1 cm was detected as the analytical signal. The Raman signals were enhanced by the SERS-enhanced effect of both Au and Ag, the large amount of Raman molecules, and the hot-spot effect in the narrow gap between the Au core and Ag shell. The SERS ICA can quantitatively detect rotavirus in a concentration range of 8- 40,000 pg/mL, with detection limits of 80 pg/mL and 8 pg/mL based on naked eye observation and SERS signal detection, respectively. No cross-reaction was observed from other common pathogens. The standard deviation of the intra- and inter-batch repetitive tests is less than 10%, and the coincidence between SERS ICA and RT-qPCR as well as commercial colloidal gold ICA is 100%. The results indicated that this SERS ICA is able to quantitatively detect rotavirus in feces in 20 min with high sensitivity, selectivity, reproducibility, and accuracy and might be a promising method for the early detection of rotavirus in clinical analysis.
Figure 7 : surface-enhanced Raman scattering immunochromatography
> Source: https://www.x-mol.com/paper/1345903446199832576

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**Cultivation of Rotavirus in MA 104 Cell Line**
Important progress has been made in the cultivation of the fastidious HRVs directly in tissue culture from clinical specimens by pretreatment of the specimen with trypsin and incorporation of trypsin into the maintenance medium, use of roller tube cultures of MA 104 cells, and incubation at 37 C. The pretreatment of the virus with trypsin greatly augmented the ability of the virus to infect cells. Culture is most frequently performed with the monkey kidney epithelial cell line MA 104, although a variety of epithelial cells are permissive for rotavirus growth.