Astroviruses (AstV) and adenoviruses (AdV) are associated with diarrhoea in young animals. However, the epidemiology and genetic diversity of AstVs and AdVs in animals is not well studied. Hence, the present study was conducted to detect and characterize AstVs and AdVs in calves, piglets and puppies from Western Maharashtra, India. Out of the processed porcine (48), canine (80), and bovine (65) faecal samples, the porcine AstV (PAstV), bovine AstV (BAstV), canine AstV (CAstV), and porcine AdV (PAdV) were detected in 12.5%, 7.69%, 3.75% and 4.1% of samples, respectively. In the RNA-dependent RNA polymerase region-based phylogenetic analysis, the detected BAstV strains grouped with MAstV-28, MAstV-33, and MAstV-35, CAstV strains belonged to MAstV-5; PAstV strains belonged to MAstV-24, MAstV-26, and MAstV-31. However, in hexon gene-based phylogeny, both the detected PAdV were of genotype 3, exhibiting 91.9–92.5% nucleotide identity with Ivoirian and Chinese strains. The study reports first-time BAstVs from calves and PAdV-3 from piglets in India. The study revealed diversity in the circulation of AstVs in tested animals and AdVs in pigs, and suggested that they alone might be associated with other diarrhoea or in combination with other enteric pathogens, thus highlighting the necessity of extensive epidemiological investigations to develop diagnostic tools and control measures. Diarrhoea is the leading infectious cause of death among neonates of animals. The farmers and pet owners suffer a huge economic loss from diarrheal disease. Therefore, monitoring of diarrhoeal diseases should be performed on a priority basis. Acute diarrhoea is caused predominantly by rotavirus and norovirus worldwide. Some countries have introduced rotavirus vaccines in animals to provide effective protection. However, studies on other diarrheal viruses such as adenoviruses (AdVs) and astroviruses (AstVs) have been largely neglected ,2,3.Adenoviridae. The AdV genome consists of the origin of replication at both ends , viral packaging sequences at the left-hand side of the genome, five early transcription units (E1-E4), intermediate transcription units  and major late transcripts . All the member genera have similar structures and genus-specific genes; Mastadenoviruses contain , while Atadenovirus contain  but lack gene coding for protein IX and V) [Mastadenovirus and Atadenovirus, with 10 recognized serotypes. The Mastadenovirus genus contains BAdV-1 (BAdV-A), BAdV-2 (Ovine adenovirus-B), BAdV-3 (BAdV-B), BAdV-9 (HAdV-C), BAdV-10 (BAdV-C) serotypes, respectively, whereas the serotypes BAdV-4, -5, -6, -7 and -8 are included in Atadenovirus genus as BAdV-D [Mastadenovirus, divided into two serotypes: CAdV-1 and CAdV-2 [AdVs are double-stranded DNA viruses belonging to the family nits E1-E, intermeX and V) . The bovnits E1-E, intermenits E1-E, intermeAstroviridae are non-enveloped, with single-stranded RNA as a genome arranged in three open reading frames; ORF1a and ORF1b codes for serine protease and RNA-dependent RNA polymerase (RdRp), and ORF2 codes for capsid proteins. The family consist of two genera, Mamastrovirus which includes 19 species, and Avastrovirus, which includes 3 species. The porcine AstVs (PAstVs) group contains five lineages, PAstV1-5, which have been classified into seven genotypes; MAstV-3, MAstV-22, MAstV-24, MAstV-26-27, MAstV-31-32. The bovine AstVs (BAstVs) are divided into seven genotypes MAstV-13, MAstV-28-30, and MAstV-33-35, while CAstVs are classified into MAstV-5 [AstVs belonging to the family The characterization of animal AstV and AdV is of immense importance as India ranks first in cattle and buffalo population; pig farming is emerging as a lucrative business, and close contact with stray and pet dogs . HoweverThe faecal samples were randomly collected from diarrheic and asymptomatic calves, piglets and puppies under six months of age between the 2017 to 2019 period from Western Maharashtra, India. The samples were transported on ice, stored at −80 °C until further processing, and were processed in batches of fifteen at a time. A 30% stool suspension was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected and immediately processed for nucleic acid extraction. The bovine samples were already screened for RVA by RT-PCR [Total RNA and DNA were extracted from 140 µL of stool suspension using an RNA extraction kit , following the manufacturer’s recommendation. The extracted nucleic acids were stored at −80 °C until their use as a template in reverse transcription polymerase reaction (RT-PCR) and PCR. 2, 0.5 µL Taq Polymerase enzyme  and volume made to 25 µL with nuclease-free water (NFW). The nucleic acid from the positive sample confirmed by sequencing was used as positive control and NFW was used as negative control in all the PCR reactions. The thermocycling was carried out with an initial denaturation of 94 °C for 4 min; thereafter, 45 cycles of 94 °C at 1 min, 50 °C at 1 min, 72 °C at 2 min and final extension for 7 min, respectively. The positive samples were visualized onto 1.5% agarose gel and observed by the Gel Documentation system . The conventional PCR targeting hexon gene was used for the detection of MastAdV and AtAdV. The primers were used from the previously published literature . The PCRTaq Polymerase mix (Invitrogen), 1 μL each of forward and reverse primer. The RNA was denatured at 95 °C for 5 min, then immediately followed by a snap chill at 4 °C for 5 min. After that cDNA was synthesized at 42 °C for 45 min, then denaturation at 95 °C for 3 min, followed by 40 cycles of 94 °C for 15 s, 50 °C for 30 s, 68 °C for 2 min, final extension step was performed at 68 °C for 7 min, respectively. The positive samples were visualized as mentioned in The one-step RT-PCR used primers from a previously published panAstV assay, which amplifies a 420 bp fragment of the RdRp region . A 5 μL The purified PCR amplicons were subjected to cycle sequencing using Big Dye Terminator cycle sequencing kit v 3.1 , and the dye terminators from the reaction were cleared with DyeEx 2.0 Spin Kit . After post-purification, the sequencing was performed on an automated Genetic Analyzer ABI-PRISM 3720 . The chromatograms were analysed in Sequencing Analysis 5.2.0 . The BLAST was used for sequence similarity assessment. The study sequences, representative sequences of AstV and AdV genotypes, cognate gene sequences exhibiting maximum identity in BLAST, and Indian sequences were aligned using CLUSTALW with the MEGAv6.06 programme. The Neighbour-joining phylogenetic trees were constructed using the Kimura-2 parameter method, and 1000 bootstraps were used to estimate the confidence of the branch. The nucleotide identity was estimated using the same software by pairwise distance using the p-distance model. The AstV and AdV groups/genotypes were assigned to BAstV , PAstV , CAstV , PAdV 1 sequenceA total of 193 samples were collected from young animals, consisting of 48 samples from piglets , 80 from pups , and 65 from calves . In the rotavirus group A (RVA) screening, bovine (29.23%), and porcine (13.79%) samples were positive while all canine samples were negative .Out of 48 porcine stool samples, two (4.1%) were positive for PAdV, while canine and bovine samples were negative for CAdV and BAdV. The size of hexon gene amplicons was variable in the range of 599–725 bp . The parThe RdRp gene amplicons of AstV were of 422 bp size . The RdRThe detection of AstVs and AdV in the gastrointestinal tract of both asymptomatic and diarrheic animals indicates that they are capable of causing persistent infections, coinfections or part of gut virome. Additionally, they have high genetic diversity, broad tissue tropism and disease spectrum, and form recombinants, making them potential emerging zoonotic viruses ,18. TherThe PAdVs are suspected to be widespread in pigs, and among five PAdVs, PAdV3 is the most prevalent and well-characterized serotype ; howeverFive genotypes of PAstV (PAstV1-5) have been identified with varying prevalences from both healthy and diarrheic pigs, including the Czech Republic, Canada, Columbia, USA, Hungary, China, South Korea and Thailand ,20,21. TCanine AstV has been reported to be associated with acute gastroenteritis or without clinical symptoms across the world. The only recently published study from the Hyderabad region of India reported a 7.2% prevalence of CAstV, and the strain with full genome sequence grouped with Chinese and Brazilian strains . In contMost of the BAstVs detected in faecal samples from North and South America, Asia, Europe & African regions (Egypt) fall into G1, G2, G3, G4 and G5 groups . The seqThe major viruses causing neonatal diarrhoea in bovines are RVA and bovine viral diarrhoea virus; in porcine are RVA, porcine epidemic diarrhoea virus and transmissible gastroenteritis virus; and in canines are parvovirus type 2, coronavirus, and canine distemper virus ,2,26. ThIn conclusion, the study found AstVs circulating in puppies, piglets and calves with significant genetic diversity. However, AdV was detected only in piglets and co-infected with AstV with identity matching with strains from diverse geographical regions. The scarcity of genetic data on AstVs and AdV from the Indian animal population challenge laboratory diagnosis and prophylaxis strategies. In the future, large-scale surveillance targeting astrovirus and adenovirus should be initiated to understand genetic diversity, its contribution to disease, and the eventual exploitation of adenovirus as a xenogeneic vector for gene therapy and vaccine delivery.