The Association of Human Astrovirus with Extracellular Vesicles Facilitates Cell Infection and Protects the Virus from Neutralizing Antibodies.

Viral gastroenteritis has a global distribution and represents a high risk for vulnerable population and children under 5 years due to acute diarrhea, fever and dehydration. Human astroviruses (HAstV) have been identified as the third most important cause of viral gastroenteritis in pediatric and immunocompromised patients. Furthermore, HAstV has been reported in biopsies taken from patients with encephalitis, meningitis and acute respiratory infection, yet it is not clear how the virus reaches these organs. In this work we have tested the possibility that the released astrovirus particles could be associated with extracellular vesicles. Comparison between vesicles purified from HAstV Yuc8 infected and mock-infected cells showed that infection enhances production of vesicles larger than 150 nm. These vesicles contain CD63 and Alix, two markers of vesicular structures. Almost 70% of the extracellular virus present in clarified supernatant at 18 h postinfection was found associated with vesicular membranes, and this association facilitates cell infection in the absence of trypsin activation and protects virions from neutralizing antibodies. Our findings suggest a new pathway for HAstV spread and might represent an explanation for the extra-intestinal presence of some astrovirus strains. IMPORTANCE Astroviruses are an important cause of diarrhea in vulnerable population, particularly children; recently some reports have found these viruses in extra-intestinal organs, including the central nervous system, causing unexpected clinical disease. In this work, we found that human astrovirus strain Yuc8 associates with extracellular vesicles, possibly during or after their cell egress. The association with vesicles doubled astrovirus infectivity in less susceptible cells and rendered virus particles insensitive to neutralization by antibodies. These data suggest that extracellular vesicles could represent a novel pathway for astrovirus to disseminate outside the gastrointestinal tract.

Structures of Two Human Astrovirus Capsid/Neutralizing Antibody Complexes Reveal Distinct Epitopes and Inhibition of Virus Attachment to Cells.

Human astrovirus is an important cause of viral gastroenteritis worldwide. Young children, the elderly, and the immunocompromised are especially at risk for contracting severe disease. However, no vaccines exist to combat human astrovirus infection. Evidence points to the importance of antibodies in protecting healthy adults from reinfection. To develop an effective subunit vaccine that broadly protects against diverse astrovirus serotypes, we must understand how neutralizing antibodies target the capsid surface at the molecular level. Here, we report the structures of the human astrovirus capsid spike domain bound to two neutralizing monoclonal antibodies. These antibodies bind two distinct conformational epitopes on the spike surface. We add to existing evidence that the human astrovirus capsid spike contains a receptor-binding domain and demonstrate that both antibodies neutralize human astrovirus by blocking virus attachment to host cells. We identify patches of conserved amino acids which overlap or border the antibody epitopes and may constitute a receptor-binding site. Our findings provide a basis for developing therapies to prevent and treat human astrovirus gastroenteritis. IMPORTANCE Human astroviruses infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies block astrovirus infection. Here, we determined the crystal structures of the astrovirus capsid protein in complex with two virus-neutralizing antibodies. We show that the antibodies bind to two distinct sites on the capsid spike domain, however, both antibodies block virus attachment to human cells. Importantly, our findings support the use of the human astrovirus capsid spike as an antigen in a subunit-based vaccine to prevent astrovirus disease.

Gut tissue-resident memory T cells in coeliac disease.

This mini-review describes observations of the 1990ies with culturing of gluten-specific and astrovirus-specific CD4+ T cells from duodenal biopsies from subjects who presumably had a long time between the exposure to gluten or astrovirus antigens and the sampling of the biopsy. In these studies, it was also observed that antigen-specific CD4+ T cells migrated out of the gut biopsies during overnight culture. The findings are suggestive of memory T cells in tissue which are resident, but which also can be mobilised on antigen stimulation. Of note, these findings were made years before the term tissue-resident memory T cells was invoked. Since that time, many observations have accumulated on these gut T cells, particularly the gluten-specific T cells, and we have insight into the turnover of CD4+ T cells in the gut lamina propria. These data make it evident that human antigen-specific CD4+ T cells that can be cultured from gut biopsies indeed are bone fide tissue-resident memory T cells.

Transcriptome Sequencing of the Spleen Reveals Antiviral Response Genes in Chickens Infected with CAstV.

Astrovirus infections pose a significant problem in the poultry industry, leading to multiple adverse effects such as a decreased egg production, breeding disorders, poor weight gain, and even increased mortality. The commonly observed chicken astrovirus (CAstV) was recently reported to be responsible for the "white chicks syndrome" associated with an increased embryo/chick mortality. CAstV-mediated pathogenesis in chickens occurs due to complex interactions between the infectious pathogen and the immune system. Many aspects of CAstV-chicken interactions remain unclear, and there is no information available regarding possible changes in gene expression in the chicken spleen in response to CAstV infection. We aim to investigate changes in gene expression triggered by CAstV infection. Ten 21-day-old SPF White Leghorn chickens were divided into two groups of five birds each. One group was inoculated with CAstV, and the other used as the negative control. At 4 days post infection, spleen samples were collected and immediately frozen at -70 °C for RNA isolation. We analyzed the isolated RNA, using RNA-seq to generate transcriptional profiles of the chickens' spleens and identify differentially expressed genes (DEGs). The RNA-seq findings were verified by quantitative reverse-transcription PCR (qRT-PCR). A total of 31,959 genes was identified in response to CAstV infection. Eventually, 45 DEGs (p-value < 0.05; log2 fold change > 1) were recognized in the spleen after CAstV infection (26 upregulated DEGs and 19 downregulated DEGs). qRT-PCR performed on four genes (IFIT5, OASL, RASD1, and DDX60) confirmed the RNA-seq results. The most differentially expressed genes encode putative IFN-induced CAstV restriction factors. Most DEGs were associated with the RIG-I-like signaling pathway or more generally with an innate antiviral response (upregulated: BLEC3, CMPK2, IFIT5, OASL, DDX60, and IFI6; downregulated: SPIK5, SELENOP, HSPA2, TMEM158, RASD1, and YWHAB). The study provides a global analysis of host transcriptional changes that occur during CAstV infection in vivo and proves that, in the spleen, CAstV infection in chickens predominantly affects the cell cycle and immune signaling.

Murine astrovirus tropism for goblet cells and enterocytes facilitates an IFN-λ response in vivo and in enteroid cultures.

Although they globally cause viral gastroenteritis in children, astroviruses are understudied due to the lack of well-defined animal models. While murine astroviruses (muAstVs) chronically infect immunodeficient mice, a culture system and understanding of their pathogenesis is lacking. Here, we describe a platform to cultivate muAstV using air-liquid interface (ALI) cultures derived from mouse enteroids, which support apical infection and release. Chronic muAstV infection occurs predominantly in the small intestine and correlates with higher interferon-lambda (IFN-λ) expression. MuAstV stimulates IFN-λ production in ALI, recapitulating our in vivo findings. We demonstrate that goblet cells and enterocytes are targets for chronic muAstV infection in vivo, and that infection is enhanced by parasite co-infection or type 2 cytokine signaling. Depletion of goblet cells from ALI limits muAstV infection in vitro. During chronic infection, muAstV stimulates IFN-λ production in infected cells and induces ISGs throughout the intestinal epithelium in an IFN-λ-receptor-dependent manner. Collectively, our study provides insights into the cellular tropism and innate immune responses to muAstV and establishes an enteroid-based culture system to propagate muAstV in vitro.

Immune-related gene expression in the kidneys and spleens of goslings infected with goose nephritic astrovirus.

Goose nephritic astrovirus (GNAstV) was first isolated in 2018, causing great economic losses to the goose industry. However, little is known about host immune response to GNAstV infection. In this study, forty 2-day-old goslings were randomly divided into 2 groups: infection and negative control groups. Each gosling in the infection group was challenged with 0.5 mL GNAstV-JSHA intramuscularly, whereas the gosling in the negative control group was inoculated with the same amount of PBS. Histopathological changes and virus location in the spleen and kidney were examined, and the expression of immune-related genes was determined by qPCR at 7 and 14 d after infection. Our results showed that GNAstV infection induced degeneration and necrosis of splenic lymphocytes and renal epithelial cells, and these cells were positive for the virus. In addition, GNAstV infection induced the activation of pattern recognition receptors (RIG-I, MDA-5, and TLR3) and key adaptor molecules (MyD88, MAVS, and IRF7) in the spleen and kidney, and upregulated the gene expression of interferon-α in the spleen and antiviral proteins (MX1, OASL, and IFITM3) in the spleen and kidney. Moreover, high expression levels of interleukin (IL)-1ß and IL-8 in the spleen and iNOS in the spleen and kidney were found. These results indicated that GNAstV infection activated host innate immune response. Furthermore, GNAstV infection increased the expression levels of CD8+, MHCI, and MHCII, indicating that adaptive immune response was activated. Besides, TGF-ß was highly expressed in the spleen and kidney, which may be an immune evasion strategy of GNAstV to cause infection. Interestingly, both IL-1ß and IL-6 mRNA levels were decreased in the kidney, which may help reduce kidney lesions. This is the first study to report changes in immune-related gene expression in response to GNAstV infection, and our results provide insights into viral pathogenesis.

OASL Triggered by Novel Goose Astrovirus via ORF2 Restricts Its Replication.

Although astroviruses causes enteric diseases and encephalitis in humans and nephritis and hepatitis in poultry, astrovirus infection is thought to be self-limiting. However, little is known about its molecular mechanism. In this study, we found that a novel goose astrovirus (GAstV), GAstV-GD, and its open reading frame 2 (ORF2) could efficiently activate the innate immune response and induce a high level of OASL in vitro and in vivo The truncation assay for ORF2 further revealed that the P2 domain of ORF2 contributed to stimulating OASL, whereas the acidic C terminus of ORF2 attenuated such activation. Moreover, the overexpression and knockdown of OASL could efficiently restrict and promote the viral replication of GAstV-GD, respectively. Our data not only give novel insights for elucidating self-limiting infection by astrovirus but also provide virus and host targets for fighting against astroviruses.IMPORTANCE Astroviruses cause gastroenteritis and encephalitis in human, and nephritis, hepatitis, and gout disease in poultry. However, the host immune response activated by astrovirus is mostly unknown. Here, we found that a novel goose astrovirus, GAstV-GD, and its ORF2 protein could efficiently induce a high level of OASL in vitro and in vivo, which could feed back to restrict the replication of GAstV-GD, revealing novel innate molecules triggered by astroviruses and highlighting that the ORF2 of GAstV-GD and OASL can be potential antiviral targets for astroviruses.

Astrovirus reverse genetics systems, a story of success.

Astroviruses are one of the main causes of gastroenteritis of medical and veterinary relevance worldwide. Recently, these viruses were associated with neurological disease in mammals, including humans. Reverse genetics systems are the most powerful tool to improve our understanding of the virus replication, and eventually to develop safe vaccine candidates. In the present review, it is summarized the current knowledge on the different strategies used to develop reverse genetics systems for mamastroviruses and avastroviruses, and some of the biological answers that have provided are discussed.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Astrovirus and the microbiome.

Although astroviruses are most commonly associated with acute gastrointestinal illness in humans, their ability to infect a broad range of hosts and cause a spectrum of disease makes them widespread and complex pathogens. The precise mechanisms that dictate the course of astrovirus disease have not been studied extensively but are likely driven by multifactorial host-microbe interactions. Recent insights from studies of animal astrovirus infections have revealed both beneficial and detrimental effects for the host. However, further in-depth studies are needed to fully explore the consequences of astrovirus-induced changes in the gut microenvironment as well as the role of the microbiota in astrovirus infection.

Characterizing a Murine Model for Astrovirus Using Viral Isolates from Persistently Infected Immunocompromised Mice.

Human astroviruses are single-stranded RNA enteric viruses that cause a spectrum of disease ranging from asymptomatic infection to systemic extragastrointestinal spread; however, they are among the least-characterized enteric viruses, and there is a lack of a well-characterized small animal model. Finding that immunocompromised mice were resistant to human astrovirus infection via multiple routes of inoculation, our studies aimed to determine whether murine astrovirus (MuAstV) could be used to model human astrovirus disease. We experimentally infected wild-type mice with MuAstV isolated from immunocompromised mice and found that the virus was detected throughout the gastrointestinal tract, including the stomach, but was not associated with diarrhea. The virus was also detected in the lung. Although virus levels were higher in recently weaned mice, the levels were similar in male and female adult mice. Using two distinct viruses isolated from different immunocompromised mouse strains, we observed virus strain-specific differences in the duration of infection (3 versus 10 weeks) in wild-type mice, indicating that the within-host immune pressure from donor mice shaped the virus kinetics in immunocompetent recipient hosts. Both virus strains elicited minimal pathology and a lack of sustained immunity. In summary, MuAstV represents a useful model for studying asymptomatic human infection and gaining insight into the astrovirus pathogenesis and immunity.IMPORTANCE Astroviruses are widespread in both birds and mammals; however, little is known about the pathogenesis and the immune response to the virus due to the lack of a well-characterized small-animal model. Here we describe two distinct strains of murine astrovirus that cause infections in immunocompetent mice that mirror aspects of asymptomatic human infections, including minimal pathology and short-lived immunity. However, we noted that the duration of infection differed greatly between the strains, highlighting an important facet of these viruses that was not previously appreciated. The ubiquitous nature and diversity of murine astroviruses coupled with the continuous likelihood of reinfection raise the possibility of viral interference with other mouse models of disease.

Isolation of Neutralizing Monoclonal Antibodies to Human Astrovirus and Characterization of Virus Variants That Escape Neutralization.

Human astroviruses (HAstVs) cause severe diarrhea and represent an important health problem in children under two years of age. Despite their medical importance, the study of these pathogens has been neglected. To better understand the astrovirus antigenic structure and the basis of protective immunity, in this work we produced a panel of neutralizing monoclonal antibodies (Nt-MAbs) to HAstV serotypes 1, 2, and 8 and identified the mutations that allow the viruses to escape neutralization. We first tested the capacity of the recombinant HAstV capsid core and spike domains to elicit Nt-Abs. Hyperimmunization of animals with the two domains showed that although both induced a potent immune response, only the spike was able to elicit antibodies with neutralizing activity. Based on this finding, we used a mixture of the recombinant spike domains belonging to the three HAstV serotypes to immunize mice. Five Nt-MAbs were isolated and characterized; all of them were serotype specific, two were directed to HAstV-1, one was directed to HAstV-2, and two were directed to HAstV-8. These antibodies were used to select single and double neutralization escape variant viruses, and determination of the amino acid changes that allow the viruses to escape neutralization permitted us to define the existence of four potentially independent neutralization epitopes on the HAstV capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies and tools to explore the possibility of developing a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.IMPORTANCE Human astroviruses (HAstVs) are common etiological agents of acute gastroenteritis in children, the elderly, and immunocompromised patients; some virus strains have also been associated with neurological disease. Despite their medical importance, the study of these pathogens has advanced at a slow pace. In this work, we produced neutralizing antibodies to the virus and mapped the epitopes they recognize on the virus capsid. These studies provide the basis for development of subunit vaccines that induce neutralizing antibodies, as well as tools to explore the development of a specific antibody therapy for astrovirus disease. Our results also establish a platform to advance our knowledge on HAstV cell binding and entry.

Astrovirus infections induce age-dependent dysbiosis in gut microbiomes of bats.

Astroviruses (AstV) are a major cause of diarrhoea in children. Interestingly, some wildlife species, including bats, remain phenotypically asymptomatic after infection. Disease symptoms, however, may only be less visible in bats and enteric viruses may indeed perturb their gut microbial communities. Gut microbiomes represent an important driver of immune defence mechanisms but potential effects of enteric virus-host microbiome interactions are largely unexplored. Using bats as a natural model system, we show that AstV-infections affect the gut microbiome, with the strength of the effect depending on host age. The gut microbial α- and ß-diversity and the predicted microbial functional orthologs decreased in young bats but surprisingly increased in adult AstV + bats. The abundance of bacterial taxa characteristic for healthy microbiomes was strongly reduced in young AstV+ bats, possibly attributable to their immature immune system. Regardless of age, pathogen-containing genera exhibited negative interactions with several commensal taxa and increased after AstV-infection, leading to pathobiont-like shifts in the gut microbiome of all infected bats. Thus, in apparently healthy bats, AstV-infections disturb gut bacterial homeostasis, possibly increasing previously suppressed health risks by promoting co-infections. If similar processes are present in humans, the effects of enteric virus infections might have longer-term impacts extending beyond the directly observed symptoms.

Development of a multiplex serological assay reveals a worldwide distribution of murine astrovirus infections in laboratory mice.

Laboratory mice play a tremendous role in biomedical research in studies on immunology, infection, cancer and therapy. In the course of standardization of mice used in animal experiments, health monitoring constitutes an important instrument towards microbiological standardization. Infections with murine astroviruses (MuAstV) were only recently discovered and are, therefore, still relatively unknown in laboratory animal science. In rodent health monitoring viral infections within a population are commonly assessed in terms of specific antibodies by serological testing, as active infection and excretion of virus is often temporary and can easily be missed. So far only ongoing infections with astroviruses can be detected by PCR. The objective of this work was the development of a sensitive and specific MuAstV multiplex serological assay with a high-throughput capability to be used in routine testing of laboratory mice. Four different MuAstV proteins were recombinantly expressed and used as antigens. The best reacting antigen, the capsid spike protein VP27, was selected and tested with a panel of 400 sera of mice from units with a known MuAstV status. Assay sensitivity and specificity resulted in 98.5% and 100%, respectively, compared to RT-PCR results. Eventually this assay was used to test 5529 serum samples in total, during routine diagnostics at the German Cancer Research Center (DKFZ) in Heidelberg between 2015 and 2017. High sero-prevalence rates of up to 98% were detected in units with open cages indicating that the virus is highly infectious and circulates within these populations virtually infecting all animals regardless of the mouse strain. In addition, data collected from 312 mice purchased from commercial breeders and from 661 mice from 58 research institutes in 15 countries worldwide allowed the conclusion that MuAstV is widespread in contemporary laboratory mouse populations.

Enteric viruses in HIV-1 seropositive and HIV-1 seronegative children with diarrheal diseases in Brazil.

Diarrheal diseases (DD) have distinct etiological profiles in immune-deficient and immune-competent patients. This study compares detection rates, genotype distribution and viral loads of different enteric viral agents in HIV-1 seropositive (n = 200) and HIV-1 seronegative (n = 125) children hospitalized with DD in Rio de Janeiro, Brazil. Except for group A rotavirus (RVA), which were detected through enzyme immunoassay, the other enteric viruses (norovirus [NoV], astrovirus [HAstV], adenovirus [HAdV] and bocavirus [HBoV]) were detected through PCR or RT-PCR. A quantitative PCR was performed for RVA, NoV, HAstV, HAdV and HBoV. Infections with NoV (19% vs. 9.6%; p<0.001), HBoV (14% vs. 7.2%; p = 0.042) and HAdV (30.5% vs. 14.4%; p<0.001) were significantly more frequent among HIV-1 seropositive children. RVA was significantly less frequent among HIV-1 seropositive patients (6.5% vs. 20%; p<0.001). Similarly, frequency of infection with HAstV was lower among HIV-1 seropositive children (5.5% vs. 12.8%; p = 0.018). Among HIV-1 seropositive children 33 (16.5%) had co-infections, including three enteric viruses, such as NoV, HBoV and HAdV (n = 2) and NoV, HAstV and HAdV (n = 2). The frequency of infection with more than one virus was 17 (13.6%) in the HIV-1 negative group, triple infection (NoV + HAstV + HBoV) being observed in only one patient. The median viral load of HAstV in feces was significantly higher among HIV-1 positive children compared to HIV-1 negative children. Concerning children infected with RVA, NoV, HBoV and HAdV, no statistically significant differences were observed in the medians of viral loads in feces, comparing HIV-1 seropositive and HIV-1 seronegative children. Similar detection rates were observed for RVA, HAstV and HAdV, whilst NoV and HBoV were significantly more prevalent among children with CD4+ T lymphocyte count below 200 cells/mm3. Enteric viruses should be considered an important cause of DD in HIV-1 seropositive children, along with pathogens more classically associated with intestinal infections in immunocompromised hosts.

The first serological investigation of Chicken astrovirus infection in China.

Chicken astrovirus (CAstV) is associated with 'white chick' syndrome, which increases embryo mortality and reduces hatchability in chickens. In the present study, 1760 sera were collected from 21 provinces in China to detect antibodies directed against CAstV with an ELISA. The sera were from different varieties of chicken in 85 flocks and all the flocks produced positive reactions. The overall seroprevalence in the birds tested was 60.68%. The prevalence increased from 34.17% to 74.44% with the increase of age. The positivity rates in layer flocks, layer parent flocks, broiler flocks, broiler parent flocks, and domestic chicken flocks were 70.17%, 89.00%, 31.67%, 59.05%, and 45.79%, respectively. These data indicate that CAstV infections are very common in China. This is the first report of the seroprevalence of CAstV infections in China.

Development and validation of an immunohistochemistry procedure for the detection of a neurotropic bovine astrovirus.

Members of the Astroviridae family are best known to cause diarrhea in different mammalian species. Lately, some strains have been associated with encephalitis in humans, minks and cattle. In this study, we developed an immunohistochemistry (IHC) procedure for the detection of a neurotropic bovine astrovirus (BoAstV-CH13/NeuroS1), which is associated with non-suppurative encephalitis in cattle. We expressed five recombinant antigens corresponding to different putative viral proteins of BoAstV-CH13/NeuroS1. Antigens were then used for the production of hyperimmune sera in rabbits. Out of the five hyperimmune sera, the one directed against the conserved N-terminus of the viral capsid protein, termed ORF2-con, clearly surpassed the others in the detection of viral antigens in IHC in terms of strong signal intensity and low background staining. The accuracy of the ORF2-con IHC protocol was then evaluated using different sets of brain tissue samples: 30 samples from 9 animals with confirmed BoAstV-CH13/NeuroS1 infection, 30 samples from 8 animals with non-suppurative encephalitis of another etiology and 30 samples from apparently healthy slaughtered animals. The IHC was positive only with tissue samples from animals with a known positive BoAstV-CH13/NeuroS1 status, but not with those from negative ones, indicating a good diagnostic sensitivity and specificity of the assay. The ORF2-con IHC procedure is therefore an adequate tool for the detection of BoAstV-CH13/NeuroS1 infections in cattle.

The Immune Response to Astrovirus Infection.

Astroviruses are one of the leading causes of pediatric gastroenteritis worldwide and are clinically importantly pathogens in the elderly and immunocompromised populations. Although the use of cell culture systems and small animal models have enhanced our understanding of astrovirus infection and pathogenesis, little is known about the immune response to astrovirus infection. Studies from humans and animals suggest that adaptive immunity is important in restricting classic and novel astrovirus infections, while studies from animal models and cell culture systems suggest that an innate immune system plays a role in limiting astrovirus replication. The relative contribution of each arm of the immune system in restricting astrovirus infection remains unknown. This review summarizes our current understanding of the immune response to astrovirus infection and highlights some of the key questions that stem from these studies. A full understanding of the immune response to astrovirus infection is required to be able to treat and control astrovirus-induced gastroenteritis.

Development of an indirect enzyme-linked immunosorbent assay test for detecting antibodies to chicken astrovirus in chicken sera.

The development of an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of Group B chicken astrovirus (CAstV) infections is described. The test was based on the use of an affinity-purified capsid antigen, specific to CAstV isolate 11672, produced as a glutathione-S-transferase N-terminal fusion protein by a recombinant baculovirus. Strongly positive ELISA signals were elicited against experimentally produced antisera raised to CAstVs from Group B (subgroups i and ii) but were negative for antisera raised to a Group A CAstV. Using a panel of 240 selected serum samples, 99% agreement was observed when the results obtained by ELISA were compared to those from an indirect immunofluorescence test for CAstV 11672. The ELISA test was applied to 68 serum sets comprising 1864 samples, which were obtained from parent and grandparent flocks originating mainly in the UK. Of the 52 sets containing ELISA-positive samples, 24 sets had >75% samples positive and nine sets had <25% samples positive and were regarded as having high and low seropositivities, respectively. Of the 1864 serum samples tested 1090 (58.5%) were ELISA positive and of these, 234 sera (21.5%) produced strongly positive signals, whereas moderately positive and weakly positive signals were produced by 562 (51.5%) and 294 (27%) sera. When used for flock screening purposes, this ELISA test can be used to (i) investigate the occurrence of first-time CAstV infections of parent flocks during lay and the possible adverse effects caused by vertically transmitted CAstV infections on broiler hatchability and performance and (ii) diagnose Group B CAstV infections within specific pathogen free flocks.

Type I interferon response is delayed in human astrovirus infections.

Type I interferon (IFN) activation and its subsequent effects are important in the response to viral infections. Here we show that human astroviruses (HAstVs), which are important agents of acute gastroenteritis in children, induce a mild and delayed IFN response upon infecting CaCo-2 cells. Although IFN-ß mRNA is detected within infected cells and supernatant from infected cells show antiviral activity against the replication of other well-known IFN-sensitive viruses, these responses occur at late stages of infection once genome replication has taken place. On the other hand, HAstV replication can be partially reduced by the addition of exogenous IFN, and inhibition of IFN activation by BX795 enhances viral replication, indicating that HAstVs are IFN-sensitive viruses. Finally, different levels of IFN response were observed in cells infected with different HAstV mutants with changes in the hypervariable region of nsP1a/4, suggesting that nsP1a/4 genotype may potentially have clinical implications due to its correlation with the viral replication phenotype and the antiviral responses induced within infected cells.