Detection and genetic characterization of chicken astrovirus and avian nephritis virus from hatchery to farm.

RESEARCH HIGHLIGHTS: Identified the role of the hatchery in astrovirus transmission.Sequenced the avian nephritis virus complete genome.Investigated tissue distribution of astrovirus from egg to chicks.Demonstrated co-infection of ANV/CAstV.

Characterization and pathogenicity of a novel avian nephritis virus isolated in China.

ABSTRACTAvian nephritis virus infections of chicken flocks cause enteric and kidney disease, uneven growth, and runting stunting syndrome, leading to economic losses in the poultry industry. In this study, one ANV strain, designated as AH202017, was isolated from a diseased broiler flock in Anhui province, China, in 2020. Virus production in LMH cell culture was confirmed by real-time RT-PCR and immunofluorescence assay. The complete genome sequencing analysis indicated that AH202017 shares 77.5-85.5% identity with 12 reference strains in GenBank. Phylogenetic analysis of the capsid protein revealed that AH202017 is more closely related to VIC-6a/Australia/2014 belonging to ANV genotype 2. However, the phylogenetic tree, based on the ORF1a protein and ORF1b protein, indicated that AH202017 manifests a close relationship with GXJL815/China/2017 belonging to genotype 8. In infection experiments, four infected chickens showed depression and one chicken died at 6 days post-infection, corresponding to 5% mortality. The virus was shed daily in the faeces of infected chickens, and was found distributed in multiple organs. Macroscopic and microscopic lesions in the kidneys were observed. This is the first paper that describes the genomic characteristics and pathogenicity of a novel ANV strain in China. RESEARCH HIGHLIGHTSA novel ANV strain was isolated for the first time from diseased broilers in China.The ANV strain caused nephritis and 5% mortality rate in 1-day-old SPF chickens.

First description of natural concomitant infection of avian nephritis virus and infectious bronchitis virus reveals exacerbated inflammatory response and renal damage in broiler chicks.

We describe the first report on spontaneous Avian Nephritis Virus (ANV) and Infectious Bronchitis Virus (IBV) concurrent infection in broiler chicks. On necropsy, the kidneys were found swollen with its parenchyma and ureters stuffed with urate flakes. Histopathologically, the renal tubular damage and inflammatory response were severe in concurrently infected birds compared to the cases infected only with ANV, which had direct correlation with significantly (p < 0.001) increased expression of IL-1 ß, IL-4, IL-12, IL-13, iNOS and IFN-γ transcripts in the kidneys of concurrently infected birds. Relative decrease in IFN-ß transcript levels in the concurrently infected birds indicates suppression of antiviral response; the iNOS level was manifold increased which can be attributed to the enhanced macrophage response. Nucleotide sequencing of S1-spike glycoprotein gene of IBV and RNA dependent RNA polymerase gene of ANV confirmed etiologies as Igacovirus of Gammacoronavirus and ANV-2 of Avastrovirus 2, respectively. Both ANV and IBV virus affect kidneys. Our findings suggested that concurrent infections of these two viruses might have enhanced the transcripts of Th1, Th2 and proinflammatory cytokines with reduced IFN-ß transcripts resulting in decreased host innate antiviral mechanisms leading to exacerbated renal lesions. Future experimental co-infection studies could throw more lights on pathology and pathogenesis during concurrent infections of ANV and IBV in poultry.

Prevalence of histopathological intestinal lesions and enteric pathogens in Dutch commercial broilers with time.

Intestinal disease has a major impact on the broiler industry due to economic and welfare reasons. Intestinal disease might occur due to a large number of reasons varying from well-defined pathogens to non-specific enteritis and complex syndromes. However, knowledge about the nature of intestinal disease and presence of enteric viruses in the Dutch broiler industry is largely absent. Therefore, a large-scale field study, in which 98 broiler flocks from 86 farms were sampled weekly, was started to assess the prevalence of histopathological lesions in the jejunum, a number of enterotropic viruses by real-time quantitative reverse transcriptase PCR (RT-qPCR) and coccidia by lesion scoring. Histopathological lesions indicative of intestinal disease were found in all flocks examined. The pathogens investigated were chicken astrovirus (99% of flocks positive), avian nephritis virus 3 (100%), rotavirus A (95%), rotavirus D (52%), reovirus (100%), Eimeria acervulina (94%), E. maxima (49%) and E. tenella (40%). The enteric viruses were more prevalent in the first weeks of the growing period, while coccidiosis was more frequently found at 4 and 5 weeks of age. The abundant presence of the enteric viruses and enteric disorders stresses the need to elucidate the role of these viruses in intestinal disease. Furthermore, the high prevalence of coccidiosis despite the use of anticoccidials shows that the current coccidial management programmes might be insufficient in controlling this disease.

Detection and molecular characterization of enteric viruses in enteritis-affected commercial broiler chickens in India.

A study was conducted to detect and characterize the enteric viruses (chicken astrovirus, avian nephritis virus and avian orthoreovirus) present in flocks of commercial broiler chickens suffering from enteritis in Haryana, India. The intestinal contents were collected from 65 enteritis-affected flocks (cases) and tested by reverse transcription PCR (RT-PCR). Of these 65 cases, 35 (53.80%) were positive for a single virus and 26 (40.00%) for two viruses. The remaining four samples were negative for all three viruses tested. Of the 65 cases, 57 were positive for chicken astrovirus (CAstV) while 30 cases had avian nephritis virus (ANV). None of the cases were positive for orthoreovirus. Comparison of 12 CAstVs of this study with previously published CAstV sequences revealed nucleotide identities ranging from 73.20 to 98.00%. The nucleotide identities ranged between 83.10-95.50% when nine ANVs of this study were compared with previously reported ANV sequences. The amino acid sequences of CAstVs in comparison to previously published sequences revealed certain unique changes. Phylogeny based on polymerase gene revealed that CAstVs and ANVs of this study were under the same monophyletic clade. In conclusion, a large number of broiler chicken flocks experiencing enteritis were positive for CAstV and ANV by RT-PCR. The presence of more than one enteric virus in enteritis-affected flocks and changes at the genetic level in these viruses may affect the severity of disease.

Detection and partial genetic characterisation of a novel variant of Avian nephritis virus in Indian poultry flocks showing diverse clinical signs.

Avian nephritis virus (ANV) infects poultry flocks worldwide, but no confirmed cases have been reported from India so far. In the current study, disease investigation was carried out in 21 broiler flocks at different parts of India with clinical signs of nephritis, uneven and stunted growth, diarrhoea, reduced body weight, and mortality up to 9.72%. Out of the 21 flocks screened, two were found positive for ANV in RT-PCR assay. BLAST analysis revealed that the ANV of Indian origin was closely related to ANV-1 strains reported from Japan, Hungary and China. However, comparison of a small portion (~12% of nucleotides, i.e. ~60 nts, common site for ANV-1 and ANV-3, position 2200-2260 of ORF 1a gene) of the Indian ANV sequence with ANV-3 sequences revealed 89-93% identities with different ANV-3 isolates. Phylogenetically, ANV-1 forms three clades, and the Indian ANV clustered under clade II. This study confirms the existence of ANV in Indian poultry flocks and is the first report on the molecular detection and genetic characterisation of ANV from India.

Spectrophotometric microplate assay for titration and neutralization of avian nephritis virus based on the virus cytopathicity.

INTRODUCTION: Plaque assay (PA) is a gold standard for virus titration and neutralization of various cytopathic viruses, including avian nephritis virus (ANV), the etiological agent associated with kidney disorders in chickens. In this study, as an alternative to the labor-intensive PA, we developed a spectrophotometric microplate assay (MA) for ANV titration and neutralization based on the virus cytopathicity to primary chicken kidney (CK) cells. METHODS: CK cells were infected with ANV in the presence or absence of chicken serum in a 96-well microplate, and the virus-induced cytolysis was quantified by measurement of neutral red uptake using a spectrophotometer. The absorbance values obtained were subjected to a sigmoidal four-parameter logistic regression analysis for the virus titer determination and serum neutralization assessment. Accuracy and reliability of the serum neutralization MA in comparison to the standard PA was statistically evaluated. RESULTS: The ANV-MA was capable of quantifying infectious virus titers based on a virus dose-dependent cytolysis of CK cells, and serum neutralization could be assessed as an inhibition of the virus-induced cytolysis accordingly. Statistical evaluation using a 2 × 2 contingency table and receiver-operating characteristic analyses showed 82 % sensitivity, 99 % specificity and 0.97 area under the curve, supporting an overall diagnostic accuracy of the neutralization MA. CONCLUSION: The newly developed MA using simplified experimental procedures in the microplate format and direct spectophotometric data readout is readily applicable to general laboratories for high-throughput screening of serum neutralization of ANV.

First Isolation and Molecular Characterization of Chicken Astrovirus and Avian Nephritis Virus in Chickens in Bangladesh.

Chicken astrovirus (CAstV) and avian nephritis virus (ANV) are enteric viruses of poultry and have infected a wide range of poultry species worldwide, causing runting-stunting syndrome (RSS), which requires virus screening and results in serious economic damage. No confirmed cases have been reported from Bangladesh. In the present study, CAstV and ANV were monitored in Bangladesh. We monitored samples for CAstV and ANV and compared their genomic sequences to other reference strains. We found 8/31 flocks (25.8%) were positive for CAstV, 6/31 flocks (19.3%) had mixed infection of CAstV and ANV, and 1 flock (3.2%) was positive for ANV. Only ANV and a combination of CAstV and ANV were found in broilers and broiler breeders, but CAstV was found in all types of chickens. We isolated two of each from CAstV and ANV through specific pathogen-free chicken embryonated eggs via the yolk sac route. Phylogenetic analysis based on the ORF1b conserved region of CAstV and ANV suggested that the locally circulating strain was closely related to the strains isolated from India and Brazil. This report is the first molecular characterization of CAstV and ANV in Bangladesh. This study highlights that CAstV and ANV are circulating in Bangladeshi poultry.

The First Whole Genome Sequence and Characterisation of Avian Nephritis Virus Genotype 3.

Avian nephritis virus (ANV) is classified in the Avastroviridae family with disease associations with nephritis, uneven flock growth and runting stunting syndrome (RSS) in chicken and turkey flocks, and other avian species. The whole genome of ANV genotype 3 (ANV-3) of 6959 nucleotides including the untranslated 5' and 3' regions and polyadenylated tail was detected in a metagenomic virome investigation of RSS-affected chicken broiler flocks. This report characterises the ANV-3 genome, identifying partially overlapping open reading frames (ORFs), ORF1a and ORF1b, and an opposing secondary pseudoknot prior to a ribosomal frameshift stemloop structure, with a separate ORF2, whilst observing conserved astrovirus motifs. Phylogenetic analysis of the Avastroviridae whole genome and ORF2 capsid polyprotein classified the first complete whole genome of ANV-3 within Avastroviridae genogroup 2.

Molecular characterization of chicken astroviruses in gout-affected commercial broiler chickens in Haryana, India.

Chicken astroviruses (CAstVs) infect young chicks and are associated with gastroenteritis, stunted growth or visceral gout (gout). True incidence and distribution of CAstVs as well as virus variants circulating in India is not well understood. In this study, 80 gout-affected broiler chicken flocks from Haryana, a north-western state of India, were tested for the presence of astroviruses by targeting the polymerase gene of both CAstV and avian nephritis virus (ANV) and capsid gene of CAstV. Of these, 22 (27.5%) flocks were found positive for CAstV, 7(8.75%) for ANV and 2 (2.5%) for both CAstV and ANV genome by reverse-transcription-polymerase chain reaction. CAstV was isolated by inoculating tissue (kidney) homogenate from gout-affected birds into specific-pathogen free embryonated chicken eggs where the infected embryos showed stunted growth with necrosis of liver and enlarged kidney with urate deposits. Capsid gene-based phylogenetic analysis revealed the clustering of CAstV strains from this study with Indian strains of serogroup Biii suggesting their antigenic relatedness. Thus the present study reveals the presence of chicken astroviruses in broiler chickens affected with gout.

Enteric Virus Diversity Examined by Molecular Methods in Brazilian Poultry Flocks.

Enteric viruses play an important role in the Brazilian poultry industry due to the economic impact of resulting low yields of broilers, layers, and breeders. The most common enteric viruses affecting commercial flocks in Brazil include Fowl Adenovirus of group I (FAdV-I), Chicken Parvovirus (ChPV), Chicken Astrovirus (CAstV), Avian Nephritis Virus (ANV), Infectious Bronchitis Virus (IBV), Avian Reovirus (AReo), and Avian Rotavirus (ARtV). The aim of this study was to identify single and multiple infections using data obtained from 270 samples from eleven Brazilian states, corresponding to the period between 2010 and 2017. This was accompanied by an analysis of the relationship between the age of birds, clinical signs, and geographical distribution, using Polymerase Chain Reaction (PCR) and Reverse Transcription-PCR (RT-PCR) techniques. Twenty-five profiles of virus combinations were detected. Single infections were encountered in 86.3% of samples, and multiple infections were present in the remaining 13.7%. Both single and multiple infections affected all kinds of commercial chickens with digestive problems, stunting syndrome, decreases in egg and meat production, increased mortality, and respiratory signs. FAdV-I, ChPV, CAstV, ANV, and ARtV were mostly detected in young broilers, in contrast with IBV, which was detected in hens from one to greater than 51 weeks of age. These results exhibit the complexity of enteric diseases and the still poorly understood role of each pathogen as a unique etiological agent.

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.