·Along with the Picornaviridae and the Caliciviridae, the Astroviridae comprise a third family of nonenveloped viruses whose genome is composed of plus-sense, single-stranded RNA.

·Astrovirus has a non-segmented, single stranded, positive sense RNA genome within a non-enveloped icosahedral capsid.

·         Transmission electron microscope (laborious, time consuming, subjective, involve personal experience) 

·         Immune Electron Microscope (IEM)  or solid phase microscope (use antibody to enhance sensitivity of essay and enable confirmation of viral agent. Limitation: limited availability of antibody) – IEM and EIA is less time consuming

·         RT-PCR (offer threshold of detection as low as 10-100 genome copies per gram of stool. Sensitivity: depends on primer/probe, enzyme amplification reaction condition. Differentiation of classic and novel Human Astrovirus (HAstV) 

·         Microarray (distinguish representative of eight serotype of classic HAstVs

·         Acid sequence-based amplification (NASBA) 

·         RT-quantitative PCR (RT-qPCR) – high speed, sensitive, reproducibility, reduction of contamination risk in the HAstV. (quantification technique) 

 

2.    From the above detection techniques, can you classify your virus into respective family?  Explain your answer with a phylogenetic tree.

First, use the above detection techniques to obtain the genome sequence to determine the relatedness between each genotype based on difference in complete capsid sequence which is the capsid protein level. Within genotype, strain can be grouped in serotype based on their antigenicity. Strain can also be classified based on their ORF2 variability. 

(phylogenetic tree picture)

 

3.    Illustrate, in detail, the structure and the life cycle of the virus.

-       Main cellular receptor remains unknown. 

-       Have dimeric surface spike that can recognize a polysaccharide molecule. 

-       However, there are several substance that can prevent the infectivity which is dextran sulphate, heparin sulphate 

-       The mechanism of entry is via clathrin-mediated endocytosis. 

-       Entry depends on acidification and maturation of endosome, 

-       Binding time of the virus to host cell surface is about 10 minutes while the uncoating process is 130 minutes. 

-       Interaction of virus and host cell will activate extracellular signal-regulated kinase (ERK1/2) and phosphoinositide3-kinase (P13K pathway). 

-       VPg protein on viral genome plays a role in infectivity. The cleavage of VPg using proteolytic treatment will inactivate the viral genome infectivity.

-       VPg protein has a role in recruitment cellular translation initiation factors. 

-       When the genome is translated, proteins formed are nsP1a1b and nsP1a polyprotein. The polyprotein will be cleaved into individual nonstructural proteins via serine proteases. 

-       The cleavage process of viral nonstructural polyprotein is unknown. 

-       Proteolytic processing of polyprotein nsP1a1b produces nsP1b protein and nsP1a protein which is cleaved to yield matured products. 

-       Endoplasmic reticulum derived intracellular membrane will be the cellular site for genome replication. 

-       However, the genome replication process remains unknown. 

-       The negative-sense genomic RNA will be synthesized and use as template to produce positive-sense genomic RNA and positive-sense subgenomic RNA. 

-       VP90 structural polyprotein initially assembles in immature virion in association with intracellular membrane. 

-       VP90 polyprotein is further cleaved into VP70 polyprotein. 

-       VP70 containing viral particles would be released from the cell and proteolytically processed by trypsin to enhance infectivity.

-       The process was carry out without cell lysis. 

(Life cycle picture)

 

4.    4. Describe how the virus transmitted from a patient to another. Does it also involve age factor?  

The virus is transmitted via fecal-oral route, contamination of food and contamination of water. The transmission via fecal-oral route often happen at nursery, daycare center, hospitals, schools and military facilities. Transmission of virus via contamination of water occurs from freshwater, drinking water and marine water. The waste water treatment practices could not completely remove the virus. While the transmission of food occurs due to the production of food using contaminated water or animals that have consumed the virus. Also, it can be due to poor hygiene during post-harvest and cooking utensils. 

This virus targets children, elderly and immunocompromised individual. 

5. 

- control of transmission route (include virus detection and virus inactivation in food and  disinfection of contaminated fomites.)

- prevention and control of the disease at host level. 

- anti-viral using synthetic flavonoids. 

- insertion of fluid to prevent dehydration 

- treatment with IgG to treat immunocompromised host.