Development of a label-free photoelectrochemical immunosensor for novel astrovirus detection.

Since 2017, an infectious goose gout disease characterized by urate precipitation in viscera, mainly caused by novel goose astrovirus (GoAstV) infection, has emerged in the main goose-producing region of China. The current challenge in managing goose gout disease is largely due to the absence of a rapid and efficient detection method for the GoAstV pathogen. Notably, the potential application of immunosensors in detecting GoAstV has not yet been explored. Herein, a label-free PEC immunosensor was fabricated by using purchased TiO2 as the photoactive material and antibody against GoAstV P2 proteins as the specific recognition element. First, we successfully expressed the capsid spike domain P2 protein of ORF2 from GoAstV CHSH01 by using the pET prokaryotic expression system. Meanwhile, the polyclonal antibody against GoAstV capsid P2 protein was produced by purified protein. To our knowledge, this is the first establishment and preliminary application of the label-free photoelectrochemical immunosensor method in the detection of AstV. The PEC immunosensor had a linear range of 1.83 fg mL-1 to 3.02 ng mL-1, and the limit of detection (LOD) was as low as 0.61 fg mL-1. This immunosensor exhibited high sensitivity, great specificity, and good stability in detecting GoAstV P2 proteins. To evaluate the practical application of the immunosensor in real-world sample detection, allantoic fluid from goose embryos was collected as test samples. The results indicated that of the eight positive samples, one false negative result was detected, while both negative samples were accurately detected, suggesting that the constructed PEC immunosensor had good applicability and practical application value, providing a platform for the qualitative detection of GoAstV.

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.

Characterization and genomic analysis of emerging astroviruses causing fatal gout in goslings.

Since February 2017, severe outbreaks of fatal gout caused by novel gosling astroviruses (GoAstVs) have occurred in several Chinese provinces, causing a considerable economic impact on the poultry industry. To assess the infection status of GoAstVs causing gout, 165 clinical samples were collected from goslings from seven farms located in different Chinese provinces, and they were screened for viral infection. Seven GoAstV strains were completely sequenced. The positive infection rates of GoAstV, goose parvovirus, reovirus, goose haemorrhagic polyomavirus and Tembusu virus were 100%, 9.69%, 3.64%, 0% and 0%, respectively, indicating the role of GoAstV in gout. The genomes of all seven GoAstV strains were 7170-nt long and encoded three open reading frames (ORFs), namely, ORF1a, ORF1b and ORF2. Sequence and phylogenetic analyses of the seven GoAstV strains showed that these were avastroviruses and were closely related to viruses classified within Avastrovirus 3 and turkey astrovirus 2. Moreover, the mutation rates of ORF1a and ORF2 were high, and ORF1a was highly mutated at amino acid loci 545-580. The tertiary structure of the mutated ORF2 protein was smooth, and its antigenic epitope was highly mutated, which may be related to the pathogenicity of the virus and caused by antibody pressure from the host. These findings enrich our understanding of the evolution of novel GoAstVs causing gout and their circulation as well as lay the foundation for the selection of vaccine strains.

Generation and evaluation of a recombinant goose origin Newcastle disease virus expressing Cap protein of goose origin avastrovirus as a bivalent vaccine in goslings.

Currently, both goose astrovirus (GoAstV) and goose-origin Newcastle disease virus (NDV) are widely infectious agents for goslings. There is no vaccine for GoAstV. Capsid protein can elicit a neutralizing antibody in human astroviruses (HAstV). Molecular analysis of the genomic region encoding the capsid protein(ORF2) of goose astrovirus has revealed that it contains neutralizing epitopes. Goose-origin NDV is also an infectious agent. A wide range of NDV strains exist that can be commonly used as vaccine vectors. In the present study, the fusion protein cleavage site RRQKR↓F in a backbone of the virulent goose-origin NDV SH-12 was changed into an avirulent motif GRQGR↓L. The modified goose-origin NDV recombinant vaccine virus expressing the Capsid protein (Cap) of GoAstV was generated as a bivalent vaccine using a reverse-genetics approach. The recombinant virus, rNDV/GoAstV-Cap, was attenuated and similar growth dynamics, cytopathic effects, and virus titers in vitro were maintained when compared to the LaSota strain. Expression of the GoAstV-Cap protein in rNDV/GoAstV-Cap infected cells was detected by an immunofluorescence assay and Western blotting. Goslings inoculated with rNDV/GoAstV-Cap showed no apparent signs of disease and induced GoAstV-Cap-specific immune responses and NDV-specific serum antibody responses to a LaSota vaccination control. Complete protection against a pathogenic GoAstV challenge and avelogenic NDV challenge was conferred. The results of the study suggested that rNDV/GoAstV-Cap viruses have the potential to be the safe, stable, and effective bivalent vaccines.

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.

Age-Related Susceptibility of Turkeys to Enteric Viruses.

Several different enteric viruses have been identified as the causes of gastrointestinal infections in poultry. Enteric virus infections are well characterized in poults, but limited studies have been conducted in older birds. The susceptibility of 2-, 7-, 12-, 30-, and 52-wk-old turkeys to turkey coronavirus (TCoV) and turkey astrovirus (TAstV) was evaluated, as well as the effect of combined infection of TAstV and TCoV in 2-wk-old poults and turkey hens. From cloacal swabs and intestines, TCoV was consistently detected by reverse transcriptase-PCR throughout the experimental period (1-21 days postinoculation [DPI]) from all age groups. In contrast, the last detection point of TAstV gradually decreased to 21, 16, and 12 DPI in birds inoculated at 2, 7, and 12 wk of age, respectively, and viral RNA was rarely detected from cloacal swabs or intestinal contents in turkey hens within 3 DPI. Infection with TAstV alone did not affect body weight in poults or egg production in hens. The combined infection of TAstV and TCoV did not induce more severe clinical signs and pathology than the TCoV infection alone. However, a severe prolonged decrease in egg production (about 50%) was observed in turkey hens in the combined infection group compared with a transient egg production drop in the TCoV-infected hens alone. The underlying mechanism regarding the age-related TAstV susceptibility and the pathogenesis of the TAstV and TCoV coinfection in layer hens needs to be further elucidated.

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.

The C-terminal end of the capsid protein of Avian Nephritis Virus is antigenic and induces broadly cross-reactive antibodies.

Avian nephritis virus (ANV) has been isolated frequently from commercial broilers in many countries. The prevalence and economic impact of ANV however has been difficult to ascertain due to the lack of convenient serological techniques. In this study the full-length and fragments of the ANV capsid protein were expressed in Baculovirus and affinity purified recombinant proteins used for the detection of ANV antibodies in ELISA. The crystal structure of Human Astrovirus (HAstV) was used as a model to determine potential homologous C-terminal antigenic regions in ANV. The rp37 fragment from three ANV strains NSW_3, ANV-1 and ANV-2, and a shorter NSW_3 fragment (rp33) were compared for their ability to detect ANV antibodies in seven reference chicken sera. The ANV-1 rp37 antigen was the most strain specific whereas the NSW_3 rp37 and rp33 antigens detected antibodies in all heterologous sera, including ANV-1 serum. Irrespective of the strain used, the two NSW_3 protein fragments rp37 and rp33 were found to be superior as antigens for ELISA when compared to the full-length capsid protein rp75. An ELISA designed using the NSW_3 rp33 could reliably differentiate between uninfected and infected commercial broiler flocks, as demonstrated by statistically significant differences between the OD values. This study identified an ANV immunogenic region and successfully used recombinant protein expression of this region to detect cross-reactive ANV antibodies. The results of this study facilitate future studies into the epidemiology and importance of ANV infections in commercial poultry.

Detection of antibodies against Turkey astrovirus in humans.

Astroviruses are a leading cause of gastroenteritis in mammals and birds worldwide. Although historically thought to be species-specific, increasing evidence suggests that astroviruses may cross species barriers. In this report, we used enzyme-linked immunosorbent assays to screen sera from three distinct human cohorts involved in influenza studies in Memphis, TN or Chapel Hill, NC, and Midwestern poultry abattoir workers for antibodies to turkey astrovirus type 2 (TAstV-2). Surprisingly, 26% of one cohort's population was TAstV-2 positive as compared to 0 and 8.9% in the other cohorts. This cohort was composed of people with exposure to turkeys in the Midwestern United States including abattoir workers, turkey growers, and non-occupationally exposed participants. The odds of testing positive for antibodies against turkey astrovirus among abattoir workers were approximately 3 times higher than the other groups. These studies suggest that people with contact to turkeys can develop serological responses to turkey astrovirus. Further work is needed to determine if these exposures result in virus replication and/or clinical disease.

Chicken astrovirus capsid proteins produced by recombinant baculoviruses: potential use for diagnosis and vaccination.

Chicken astroviruses (CAstVs) have been characterized recently. Due to their relatively poor growth in cell culture, virus-specific antigens are not readily available for the development of diagnostic reagents and vaccines. For this purpose two capsid protein antigens, specified by the 11672 isolate of CAstV, were produced in insect cells following infection with recombinant baculoviruses. The GST-11672 capsid protein, a fusion protein comprising the capsid protein and glutathione-S-transferase (GST) as an N-terminal affinity tag, and the 11672 capsid protein alone were detected by western blotting as proteins of ~100 and 70 kDa, respectively. Immunization with the affinity-purified GST-11672 capsid protein produced a polyclonal rabbit antiserum, which reacted by indirect immunofluorescence with Group B CAstVs but which showed no reactivity with the Group A CAstV isolate, 612. When used as part of an immunoperoxidase-based immunohistochemical procedure, this rabbit antiserum facilitated the detection of CAstV antigen in formalin-fixed, paraffin-embedded kidney tissue at the sites of histopathology characteristic of nephritis. Although further evaluation with sera from commercial chickens is required, a prototype indirect antibody-detecting enzyme-linked immunosorbent assay (ELISA) based on affinity-purified GST-11672 capsid protein as coating antigen demonstrated considerable potential with low ELISA absorbance values being generated with sera from specific pathogen free (SPF) chickens, and high absorbance values being generated with serum samples from experimentally infected chickens. Immunization experiments of SPF chickens showed that, when administered as mixtures with oil adjuvant, crude cell lysates containing the GST-11672 capsid protein or the 11672 capsid protein elicited virus-specific antibody responses that were detectable by indirect immunofluorescence and by virus neutralization assays.

Segmentation expression of capsid protein as an antigen for the detection of avian nephritis virus infection in chicken flocks.

Subclinical pathological changes in the kidneys of broiler chickens and suppression of growth caused by the avian nephritis virus (ANV) affect poultry flocks worldwide. A test for detection of virus-specific antibodies in serum would be useful for epidemiological investigations, however the poor propagation in cell cultures has restricted the development of serological tests based on the use of ANV particles as antigens. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of ANV-specific antibodies in chicken serum, using a recombinant protein antigen prepared by segmentation expression of the capsid protein antigen epitope of ANV (HM029238) transfected into Escherichia coli. The expressed fusion protein was detected by Western blotting with ANV-positive serum, and the optimal immunoreactive fusion P1 protein was determined. Using the optimized P1-ELISA, ANV-specific antibodies were detected in commercial chicken flocks aged 10-25 days obtained from the Liaoning Province, China. Out of 960 serum samples, 459 (47.8%) were positive for infection with ANV. These results indicate that the P1-ELISA is helpful for preliminary serological diagnosis of ANV infection, and could be used to for screening in ANV infection and for determining antibodies against ANV.

Molecular characterization of avian astroviruses.

Astroviruses are frequently associated with enteric diseases in poultry, being isolated from cases of runting-stunting syndrome (RSS) of broiler chickens, poult enteritis complex (PEC), and poult enteritis mortality syndrome (PEMS) of turkeys. Currently, five types of avian astrovirus have been identified: turkey astroviruses 1 and 2 (TAstV-1, TAstV-2), avian nephritis virus (ANV), chicken astrovirus (CAstV) and duck astrovirus (DAstV). The objective of this study was to molecularly characterize the different types of avian astroviruses circulating in commercial poultry. Sequence analysis of a region of ORF2, which encodes the capsid precursor protein associated with serotype and viral pathogenesis, revealed extensive variation in amino acid sequence within each subtype: TAstV-2 (81.5%-100%), ANV (69.9%-100%), and CAstV (85.3%-97.9%). However, this region was more conserved in TAstV-1's (96.2%-100%). Furthermore, a novel astrovirus was detected in chicken samples and found to be <64% similar to ANV and <30.6% similar to CAstV. The results of this study underline the great genetic variability of avian astroviruses and indicate that there are most likely multiple serotypes of each avian astrovirus circulating in commercial poultry.

A purified recombinant baculovirus expressed capsid protein of a new astrovirus provides partial protection to runting-stunting syndrome in chickens.

A new viral sequence likely belonging to a virus of the family Astroviridae was determined using the gut content of chickens affected with the runting-stunting syndrome (RSS) in chickens. Since the appropriate virus could not be isolated in cell culture the open reading frame of the viral capsid protein was cloned to generate a recombinant baculovirus. The protein was purified and used as an experimental vaccine in broiler breeders to provide maternal derived antibodies for the protection of the offspring. The presence of specific antibodies was monitored by an ELISA. The offspring of vaccinated breeder hens were partially protected in a RSS challenge model.

A seroprevalence investigation of chicken astrovirus infections.

Two genetically different isolates of chicken astrovirus (CAstV), named CAstV612 and CAstV11672, which share low levels of antigenic relatedness in cross-indirect immunofluorescence (IIF) tests, have been identified recently. In the present study, separate IIF tests for detecting antibodies to the CAstV612 and CAstV11672 isolates have been used to determine the seroprevalences of CAstV infections in four generations of flocks involved in broiler chicken production. CAstV antibodies were detected in 78% (73% CAstV612; 46% CAstV11672) of serum samples from UK broiler flocks and in all 10 flocks tested, indicating that infections were very common. Twenty-three (96%) out of 24 and 26 (93%) out of 28 broiler parent flocks, aged 23 to 26 weeks from three UK organizations, were positive for antibody to CAstV612 and CAstV11672, respectively. Of 718 samples tested from these parent flocks, 415 (53%) were positive for either CAstV612 or CAstV11672 antibody. CAstV infections were also widespread in parent flocks, with screening of pooled serum samples showing that antibodies to both CAstVs were detected in flocks from seven other UK poultry organizations and in flocks from eight other European countries. The seropositivities for CAstVs were substantially less in grandparent (28%) and great grandparent (21%) flocks. Overall, higher seropositivities were observed for CAstV612 than for CAstV11672 in broiler, parent, grandparent and great-grandparent flocks. A limited study of 99 sera from 10 turkey breeder flocks showed low-level seropositivities for CAstV612 (9%) and CAstV11672 (2%), indicating that turkeys were infected with CAstVs or antigenically related viruses.