Index
They are a major cause of viral gastroenteritis, especially in children under 5 years of age.
Astroviruses (AstV) are non-enveloped, positive sense, single-stranded RNA viruses of icosahedral particle symmetry that cause a variety of clinical diseases.
Occasionally, it is the cause of outbreaks in the elderly in hospitals and nursing homes. Adults are likely to be protected from antibodies acquired in childhood, although infection in healthy adults can sometimes occur.
Virology of astroviruses
Astroviruses have been assigned to their own family, the Astroviridae , despite their physical similarity to caliciviruses, due to their unique genomic organization and antigenic structure.
Like Norwalk virus, astroviruses form 28-30 nm particles that contain a single positive-stranded polyadenylated genomic RNA. During cellular infection, a large number of subgenomic RNA copies are made that encode the structural proteins of the virus capsid.
About 10% of the particles in the stool show a characteristic solid five- or six-pointed star by electron microscopy (EM), in contrast to the hole-centered stars often seen in caliciviruses .
Astroviruses contain three major structural proteins and can be grown in tissue culture with added trypsin under conditions similar to those used to propagate rotavirus.
Immunological studies have identified eight different serotypes of human astroviruses, all of which appear to cause diarrheal illness. Two new distinct groups of human astroviruses have recently been described – MLB and -VA / HMO.
Although both groups were originally associated with pediatric gastroenteritis , their full role in human disease requires further study.
epidemiology
Astroviruses have been found to be an important pathogen in young children with diarrhea and predominantly infect children under 2 years of age.
Several studies have suggested that very young babies are particularly susceptible. Astroviruses have also been associated with foodborne gastroenteritis outbreaks in Japanese adults and school-age children.
Astroviruses appear to account for 4% to 8.6% of all young children with diarrhea, depending on geographic location. Infections occur primarily in the winter in temperate climates, as has been reported for rotavirus.
Studies have shown that astroviruses can cause sporadic outbreaks of diarrhea in elderly patients and that it is significantly associated with diarrheal illness in immunocompromised AIDS patients, solid organ transplant recipients, and bone marrow transplant recipients.
Transmission of the AstV
AstV is transmitted essentially via the fecal-oral route, as has been shown in studies with human volunteers. However, in these studies, few adult recipients developed gastroenteric disease with viruses in their stool, probably due to their age.
Food and water can act as vehicles for the transmission of the human enteric virus. Several large outbreaks of AstV have been associated with the consumption of contaminated food.
Additionally, fecally contaminated fomites have been implicated in reported outbreaks in institutions such as day care centers, hospitals, day care centers, schools, and military installations.
Similarly, waterborne transmission of AstV has been suggested as a risk of digestive morbidity for the general population, and AstV was recovered in surface waters of an area in which an outbreak of gastroenteritis occurred.
The environmental transmission of AstV is supported by the stability of the virus in drinking water, fresh water and marine water.
Among the foods susceptible to being contaminated with AstV in the pre-harvest stage are bivalve molluscs that grow in contaminated water and fresh products irrigated with contaminated water, such as lettuce, green onions and other vegetables, as well as soft fruits such as raspberries and strawberries.
These products generally undergo little or no processing prior to consumption and are therefore likely to act as vehicles for the transmission of the human enteric virus.
Surface and groundwater are used as sources of drinking water for public consumption, and poor water quality is recognized as a major threat to health.
WHO estimates that almost 90% of the global burden of gastroenteritis is attributable to unsafe water and poor sanitation and hygiene, and the pediatric population in developing areas is the most vulnerable group.
As an example of this situation, AstV genomes have been detected in tap water in Ghana.
Although the risk from recreational activities in sewage-contaminated water is much lower than the consumption of contaminated water, an outbreak of gastroenteritis occurred in children and adults who bathed in a children’s pool in Helsinki that had norovirus contamination and astrovirus.
Furthermore, a seroprevalence study in the UK showed that 93% of surfers had antibodies against AstV, while only 23% of age-matched controls had these antibodies.
Chlorine and other disinfectants are commonly used for drinking water treatment and are effective in the inactivation of AstV in water.
Poor hygiene practices with improper food handling are often responsible for post-harvest contamination of food products, particularly ready-to-eat foods such as salads, sandwiches and deli meats, which are consumed without cooking or lightly cooked.
AstV causes asymptomatic infections in children and adults, and asymptomatic food workers are more involved in outbreaks of gastroenteritis than are symptomatic food handlers.
It is essential to determine the importance of asymptomatic, asymptomatic and post-symptomatic AstV in the transmission of foodborne infections.
Fomites (fomite is any inanimate object that can become contaminated with infectious agents and serve as a mechanism to transfer infectious agents from there to a new host, a healthy person). These play an important role in the institutional spread of AstV infections.
Fomites include serving and cutting utensils, and the virus’s ability to persist drying on these surfaces long enough and high enough to pose a risk is a major public health concern.
Disinfection practices, as well as improved hygiene measures, particularly in high-risk settings such as hospitals, nursing homes, nurseries, or restaurants, are of great importance in preventing the spread of infection.
Antiseptics and disinfectants should be used for the inactivation of pathogens on the skin (notably the hands) and fomites, respectively.
Although astroviruses were initially considered to be so species specific that it led to their classification by host species, concern has recently been raised about possible zoonotic transmission of astroviruses to humans.
As discussed above, the advent of pyrosequencing allowed the characterization of previously unrecognized AstV, termed non-classical AstV.
Some of these non-classical AstVs have been shown to have common ancestry with the recently described rat AstV, while others are phylogenetically related to mink and sheep AstV.
As mentioned above, there is evidence of recombination events between human and animal AstV. Recombination in the ORF2 variable region has been shown to occur between human and porcine strains of AstV.
These AstV recombinants were described in areas where human and porcine strains were in close contact.
Since the genetic variability of AstV strains appeared to be much greater than that of porcine strains, it was concluded that zoonotic transmission probably took place from humans to pigs rather than vice versa, as previously suggested.
High genetic variability, along with the occurrence of interspecific infections and recombination events, makes AstV a serious candidate for emerging zoonotic infections.
Clinical data manifestations
The clinical illness caused by astroviruses in children is similar to that caused by rotavirus, with a high incidence of fever and diarrhea.
The disease is generally considerably milder, however; and it causes less dehydration than rotavirus infection.
The range of symptoms also seems to vary from place to place; an outbreak in Guatemala was characterized by a lower frequency of vomiting and fever than an outbreak in Thailand.
Astrovirus disease is considerably less pathogenic in young adult volunteers, with only 5% of infected individuals developing clinical symptoms, probably due to pre-existing immunity.
The increased incidence of astrovirus disease in elderly patients suggests that immunity is acquired in childhood and disappears later.
In premature infants, the infection has been associated with necrotizing enterocolitis, but it is still unknown whether this association is causal.
Laboratory diagnosis
Early developments: electron microscopy and immunological tests.
Although cell culture propagation of some classic AstVs is possible with the help of trypsin, virus isolation in cell culture is not applicable for the diagnosis of astroviruses in fecal samples.
In the past, AstVs were routinely detected by direct transmission electron microscopy (EM) in negatively stained stool samples. This procedure is laborious, time-consuming, and subjective and involves highly experienced personnel.
Other limitations were that, as mentioned above, only about 10% of the particles show the characteristic surface structure of a star and that the sensitivity of EM requires concentrations of around 107 particles per gram of fecal matter for visualization. of the virus.
These levels are usually detected only 12 to 48 hours after the onset of symptoms, and the numbers are often too low for the detection of MS.
The use of antibodies in immune electron microscopy (IEM) or solid phase immune electron microscopy (SPIEM) increases the sensitivity of the assay and allows confirmation of the identity of the viral agent.
However, the limited availability of antibodies to the different types of AstV makes it difficult to use these techniques.
Enzyme immunoassays (EIA) were developed for the detection and typing of AstV.
Herrmann et al. Developed in 1990 an EIA based on the use as a capture antibody of a monoclonal antibody (8E7) directed towards the conserved domain of the capsid and a peroxidase-labeled polyclonal antibody as the detection antibody.
Another refinement was labeling the detection antibody with biotin.
At least two commercial EIAs are available for the classical detection of AstV in stool samples. Although the assays do not allow the typing of isolates, they are useful for rapid virus detection when large numbers of samples must be tested.
EIA-based techniques have been reported to have the same sensitivity as direct observation of MS, but are obviously much less time consuming.
In addition, several rapid immunochromatographic tests against astroviruses are commercially available, and some of them have been evaluated.
Molecular assays
The advent of molecular detection techniques, initially based on probe hybridization and later on genome amplification, opened the possibility of developing assays with exquisite sensitivity and specificity for the diagnosis of AstV in feces.
Although the sensitivity of probe hybridization has been reported to be on the same level as EIA, reverse transcriptase PCR (RT-PCR) offers detection thresholds as low as 10 to 100 copies of the genome per gram of feces.
However, the sensitivity of these molecular assays is largely based on several variables, such as primer / probe selection, enzyme amplification reaction conditions, and last but not least, the efficiency of acid extraction. target nucleic.
In any case, studies using EIA and RT-PCR in parallel reported a greater number of positive samples scored by the latter technique, as well as a longer period of viral spread that extended beyond resolution of symptoms.
Conventional RT-PCR methods are still widely used today in many research laboratories, both for the detection and typing of additional isolates.
Some of the most commonly used common-type and common-type primers for the detection of 8 classic AstV serotypes have been previously reviewed.
Also the primers Mon244 / Mon245 (and Mon269 / Mon270) described by Noel et al., Pointing to the conserved 5 ‘end of ORF2 and allowing typing by sequencing, are some of the most used.
Following the discovery of new AstVs, consensus astrovirus RT-PCR primers targeting ORF1b were also published. While these primers should be able to detect all known human and animal AstVs, they have thus far enabled the detection of classic and novel human AstVs.
Primers designed to specifically detect AstV-MLB and AstV-VA / HMO have also been described.
Finally, microarrays have also proven to be an especially useful option for virus detection and typing.
One of the described microarray-based methods is capable of distinguishing representatives of the eight known serotypes of classical AstVs, and another is a combiner for the simultaneous detection of several enteric viral pathogens, including AstVs.
Furthermore, other less conventional molecular techniques, such as nucleic acid sequence based amplification (NASBA), have also been described for AstV.
Another improvement was the development of quantification techniques, such as real-time RT quantitative PCR assays (RT-qPCR), which provide high speed, sensitivity and reproducibility and a reduction in the risk of contamination in the diagnosis of AstVs.
Similarly, a real-time reverse transcription loop-mediated isothermal amplification has been developed for the rapid and quantitative detection of AstV-1.
Interestingly, some of the published RT-qPCR methods already come in a multiplex format that allows the simultaneous detection of classical AstV and other targets such as norovirus, rotavirus, sapovirus, adenovirus, and / or bocavirus.
One of these methods includes up to 19 targets, including bacteria, protozoa, and helminths.
Interestingly, that assay also includes the detection of two extrinsic controls to monitor extraction and amplification efficiencies, which can be highly relevant in surveillance or food virology applications.
Commercial real-time monoplex and multiplex RT-qPCR kits are also available, although most of them are especially relevant to the clinical diagnostic environment.
Notable among these is the FilmArray gastrointestinal panel (BioFire Diagnostics, Inc., USA), which is currently under development and aims to simultaneously detect 23 gastrointestinal pathogens in feces, including AstV and 4 other enteric viruses.
Regarding the new AstVs, although they are not designed for use in surveillance studies, two RT-qPCR assays targeting AstV-MLB have been applied for the quantification of viral loads in feces and serum.
If the importance of the new AstVs in human health is clinically confirmed, molecular methods should be updated soon to incorporate reactive primers / probes in general to universally detect all AstVs.
Finally, although the classic AstVs are somewhat delicate to grow in vitro and the new AstVs have not been propagated in any cell line, the combination of molecular techniques with infectivity assays can help to finally analyze the importance that positive results can have in terms of genomic copies.
The use of CaCo-2 cells combined with pretreatment of virus inocula with trypsin remains the gold standard for classical isolation of AstV from clinical and environmental samples, and various integrated cell culture (ICC) methods combined with detection by RT-PCR has been described.
Whether these methods allow the isolation of new AstVs or whether these viruses show significant differences in cell tropism remains to be clarified.
Next-generation technologies, including whole genome sequencing and viral metagenomics, can rapidly generate sequence-based data that will be instrumental in optimizing currently used primers and will also help identify additional genomic regions for analysis.
Furthermore, whole genome sequencing will provide genetic information that can also be correlated with virulence factors and can help us better understand the basis of viral pathogenesis, and metagenomics will be helpful in identifying new emerging strains.
Astrovirus pathogenesis
The pathogenesis of astroviruses has not been well studied in humans. Viral particles have been visualized by EM in intestinal epithelial cells and in epithelial cells located in the lower part of the villi, suggesting that the intestine is the site of replication.
The pathogenesis of astroviruses has been studied in lambs and calves, using isolates of ovine and bovine astroviruses, respectively. Infection of lambs induces a mild transient diarrhea that occurs 2 days after infection and lasts approximately 2 days.
Infection of calves with at least one strain of bovine astrovirus did not induce diarrheal disease, although the infection was associated with shedding of infected hair cells.
Susceptibility
No model has been established for astrovirus susceptibility testing. Several attempts to infect small laboratory animals with human astrovirus isolates have been unsuccessful.
Murine astroviruses are common in laboratory mice, but have not been associated with diarrheal diseases.
Prevention and treatment
The prevention of AstV infections is essentially based on the control of the transmission routes and on the prevention and control of the disease at the host level.
The control of transmission routes includes the detection and inactivation of viruses in water and food and the disinfection of contaminated fomites.
Methods for the detection and quantification of AstV are available, but the threshold of acceptable genome copy numbers in food and water matrices has not yet been defined not only for AstV but also for other gastroenteritis agents.
Although the survival of classic AstV in drinking water is high, 2-hour disinfection treatments with 1 mg / ml of free chlorine are quite effective.
However, genogroup B is somewhat more resistant to chlorine disinfection than genogroup A, suggesting that there may be differences in environmental persistence between strains.
Survival and inactivation of astroviruses in food matrices have not been extensively studied, probably because only a few foodborne AstV outbreaks have been described and because efforts have been devoted to the application of emerging technologies for the inactivation of other agents. viral.
These viral agents can be the norovirus and the hepatitis A virus.
With regard to disinfecting contaminated fomites, 90% alcohol has proven useful. Unfortunately, inactivation information for non-classical AstVs is not yet available.
The prevention of the development of diseases in the host includes vaccination and the enhancement of natural defenses, such as the intestinal microbiota.
Vaccines for AstV have not been developed despite various descriptions of virus-like particle (VLP) production in different systems.
This lack of commercial interest in the production of vaccines may be due to the low clinical impact of astrovirus infection in healthy patients and the need for a multivalent vaccine to cover all circulating serotypes and strains or at least the most prevalent ones, since apparently no exists.
Some authors have speculated that probiotics, which can interfere with the life cycle of the enteric virus in many different steps, can be used as a measure to prevent and / or treat intestinal viral infections.
Furthermore, the antiviral activity of some synthetic flavonoids in the replication of astroviruses has been described.
The usual therapy applied to patients (young children or adults) suffering from severe gastroenteritis consists of oral or intravenous fluid substitution to avoid dehydration.
Intravenous IgG treatment has also been proposed for immunocompromised hosts with severe or persistent diarrhea, although the efficacy of this treatment has not yet been established in large-scale studies.
Conclusions
In recent years, AstV’s global vision of diversity has changed dramatically. Although traditionally considered as agents of acute gastroenteritis in children, it is now known that viruses can spread systemically, especially in immunocompromised individuals.
Viremia has been documented in some cases in humans, pigs, and turkeys, and AstVs have been isolated from neurological tissue in association with neurological disorders in humans, cattle, and mink.
The appearance of so many different viruses infecting so many different species indicates the possibility of zoonotic transmission of viruses to humans.
In this sense, the diversity of AstV found in species of birds, pigs and bats is remarkable, with these species frequently associated with zoonotic transmission and the appearance of new strains of influenza and coronavirus viruses.
In fact, the new AstV-VA / HMO viruses (MAstV 8 and MAstV 9 genotypes) are phylogenetically closer to bat isolates than to classic AstVs (MAstV 1), and some of these new viruses have been associated with extraintestinal manifestations.
All of these concerns highlight the need to improve broadly reactive diagnostic techniques that allow the detection of all AstVs.
A better understanding of the replicative cycle of AstV, particularly new viruses, and its role in clinical disease and its pathogenesis mechanisms is required to prevent and treat potential new infections.
