Picobirnaviruses (PBVs) was first discovered in 1988 in a study on rotavirus-associated diarrheal disease in Brazil. Human fecal samples, analyzed using polyacrylamide gel electrophoresis (PAGE) and silver staining, showed a typical two-segmented, double-stranded RNA profile that differed from that of rotaviruses (Pereira et al., 1983). Shortly thereafter, PBVs were also found in the feces of wild rodents in Brazil, indicating a potentially wide host range, including animals (Pereira et al., 1983). Since these first reports, PBVs have been found in fecal samples of a broad range of vertebrates, including domesticated animals, wildlife and humans with and without gastrointestinal disease, indicating a broad host range and global distribution (Malik et al., 2014; Malik and Matthijnssens, 2014). PBVs are a genus of viruses within the Picobirnaviridae family (Carstens, 2010; Delmas et al., 2019). The RNA-dependent RNA polymerase (RdRp) gene is encoded in segment 2, while the capsid protein precursor is encoded in segment 1 of the double-stranded RNA viral genome (Boros et al., 2018; Gan and Wang, 2023). The genetic diversity of the PBVs in the RdRp gene has resulted in the assignment of PBVs into genogroups I and II (Li et al., 2015; Rosen et al., 2000; Smits et al., 2014).
       
Despite their common detection, the pathogenic role of PBVs remains unclear. PBVs have been detected in both diarrhoeic and asymptomatic hosts and are commonly detected as coinfecting agents with established enteric viruses such as rotaviruses, astroviruses, caliciviruses and coronaviruses (Bhattacharya et al., 2007; Malik et al., 2014; Malik and Ghosh, 2020; Malik and Matthijnssens, 2014). The absssence of any cell culture system or suitable animal model has hindered experimental investigation and epidemiological studies remain the main source of information regarding their distribution and probable clinical importance (Ganesh et al., 2012; Rosen, 2003). Several studies have reported comparable detection rates of PBVs in diarrhoeic and non-diarrhoeic hosts, while others documented higher prevalence in young animals and during specific seasonal conditions (Berg et al., 2021; Kylla et al., 2017).
       
Diarrhea remains one of the major causes of morbidity and mortality in neonatal pigs and a significant hindrance to swine rearing globally (Kongsted et al., 2018; Morin et al., 1983). Among viral pathogens, rotaviruses and coronaviruses are well-recognized causative agents of diarrhoeal disease in pigs, whereas increasing detections of PBVs have been reported in both diarrhoeic and non-diarrhoeic pigs (Chepngeno et al., 2019; Makimaa et al., 2020; Patel et al., 2022; Puente et al., 2023). Detection of PBVs relies primarily on PAGE and RT-PCR assays targeting the RdRp gene; however, low viral loads and high sequence diversity may lead to underestimation of prevalence (Malik et al., 2013; Nates et al., 2011; Reddy et al., 2026). In India, reports of PBV infection in pigs are limited and geographically restricted. Assam is among the most pig-rich states in the country, yet data on PBV circulation in this region remain scarce. Therefore, the present study aimed to detect and molecularly characterize PBV circulating in pigs from Assam, India.

Faecal samples were collected from diarrhoeic and non-diarrhoeic pigs aged 0-8 months from organized and unorganized farms located in different pig-rearing districts of Assam, including Morigaon, East Karbi Anglong, West Karbi Anglong, North Lakhimpur, Nagaon, Golaghat, Nalbari, Tinsukia, Kokrajhar, Dhemaji and Kamrup Metropolitan District (Fig 1). Animals were categorized into four age groups, namely 0-2, 2-4, 4-6 and 6-8 months. Fresh faecal samples were collected aseptically in sterile screw-cap vials from randomly selected pigs, transported to the laboratory under chilled conditions and stored appropriately until further processing. All laboratory investigations were carried out at the College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati.


       
Approximately 1 g of faeces was suspended in four volumes of 0.06 M phosphate-buffered saline (PBS; pH 7.2) and homogenized using sterile glass beads. The homogenized samples were centrifuged at 12,000 × g for 20 min at 4°C and the clarified supernatant was collected as a 20% faecal suspension and stored at -20°C until further use (Liu and Chassagne, 2023). Detection of picobirnavirus (PBV) nucleic acid was carried out using RNA polyacrylamide gel electrophoresis (RNA-PAGE) and reverse transcriptase polymerase chain reaction (RT-PCR).
       
RNA-PAGE was performed following the method described by (Herring et al., 1982) with slight modifications. Briefly, viral RNA was extracted from clarified faecal supernatants by mixing 100 μL of supernatant with 0.1 M sodium acetate buffer (pH 5.0) containing 1% sodium dodecyl sulphate, followed by phenol chloroform extraction. The mixture was centrifuged at 12,000 × g for 10 min at 4°C and the aqueous phase was collected, mixed with bromophenol blue containing 10% sucrose and stored at -20°C. The extracted RNA was resolved on discontinuous polyacrylamide gels and RNA segments were visualized by silver staining. Samples exhibiting two distinct equimolar RNA segments were considered positive for PBV (Malik et al., 2017; Malik et al., 2014).
       
For RT-PCR analysis, viral RNA was extracted from faecal supernatants using a commercially available RNA extraction kit (RNeasy Plus Universal Mini Kit, Qiagen) according to the manufacturer’s instructions. Complementary DNA synthesis was carried out using random primers, followed by PCR amplification targeting segment 2 of the RNA-dependent RNA polymerase (RdRp) gene, as described by Bhattacharya et al. (2007). Two primer sets specific for PBV genogroup I (201 bp) and genogroup II (369 bp) were used for amplification, as described previously (Rosen, 2003). Primer details are provided in Table 1. PCR reactions were performed in a 25 μL volume and thermal cycling conditions consisted of an initial denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 42°C for 1 min and extension at 72°C for 1 min, with a final extension at 72°C for 7 min. Previously confirmed PBV-positive samples from the repository of the Department of Veterinary Microbiology (CVSc, Khanapara) were used as positive controls, while nuclease-free water was used as the negative control in each PCR run.


       
RT-PCR amplicons were analyzed by electrophoresis on 1.5% agarose gels prepared in Tris–acetate–EDTA buffer containing ethidium bromide, using a 100 bp DNA ladder as a molecular size marker. Amplified products were visualized under ultraviolet illumination using a gel documentation system.
       
RT-PCR-positive amplicons were purified using a commercial PCR purification kit (QIAquick PCR Purification Kit) according to the manufacturer’s protocol. Purified products were verified by agarose gel electrophoresis and subsequently subjected to Sanger sequencing using an Applied Biosystems 3500 Genetic Analyzer. Sequencing was carried out by Eurofins Genomics, Bangalore, India.
       
The obtained nucleotide sequences were submitted to the National Center for Biotechnology Information (NCBI) GenBank database for accession number assignment. Sequence similarity analysis was performed using the Basic Local Alignment Search Tool (BLAST) and phylogenetic analysis was carried out using the neighbor-joining method implemented in MEGA version 6.0.

In the present study, the occurrence of picobirnavirus (PBV) was investigated in faecal samples collected from pigs of different age groups from various districts of Assam. RNA-PAGE analysis detected PBV RNA in 4 out of 148 faecal samples, corresponding to an overall positivity rate of 2.70% (Table 2).


       
All RNA-PAGE-positive samples exhibited two discrete equimolar RNA segments, which is a characteristic electrophoretic pattern of PBV (Fig 2). Age-wise analysis revealed that three of the four positive samples originated from pre-weaned piglets aged 0-4 months, while one positive sample was detected in the weaned age group (4-8 months). Seasonally, all RNA-PAGE-positive samples were detected exclusively during the pre-monsoon period, with no positives observed during the monsoon, post-monsoon, or winter seasons (Table 3). The highest incidence of PBV detection by RNA-PAGE was therefore observed in pre-weaned piglets during the pre-monsoon season.




       
RT-PCR screening of all 148 faecal samples identified six samples positive for PBV, resulting in an overall detection rate of 4.05%. All RT-PCR-positive samples yielded a specific amplification product of 369 bp corresponding to the RNA-dependent RNA polymerase (RdRp) gene, indicating the presence of genogroup II (GGII) PBV (Fig  3). No amplification was observed for genogroup I (GGI) PBV in any of the samples analysed. Among the RT-PCR-positive samples, five were detected in the pre-weaned age group (0-4 months), while one positive sample was identified among weaned pigs (4-8 months). Similar to RNA-PAGE findings, the highest number of RT-PCR-positive samples was recorded during the pre-monsoon season (Table 4). A single positive sample was detected during the monsoon season in the pre-weaned group, while no positives were observed during the post-monsoon or winter seasons. It was evident from the results that the incidence of PBV was highest during the pre-monsoon season, whereas no PBV-positive samples were detected during the other seasons.




       
A combined analysis of RNA-PAGE and RT-PCR results revealed that PBV detection was predominantly associated with younger pigs and specific seasonal conditions. Pre-weaned piglets (0-4 months) exhibited a higher positivity rate than weaned pigs across both detection methods. Seasonally, PBV detection was markedly higher during the pre-monsoon period, irrespective of age group. No PBV-positive samples were detected during the post-monsoon or winter seasons by either method. Graphical representations of age- and season-wise detection patterns are presented in Table 4.
       
Molecular characterisation was performed on two representative RT-PCR-positive PBV samples by sequencing the partial RdRp gene. The obtained nucleotide sequences, designated as PBV1 and PBV2, were aligned and analysed using the CLUSTAL W programme, followed by BLAST analysis against sequences available in the NCBI database. BLAST results demonstrated that PBV sequences from Assam shared nucleotide sequence identity ranging from 82% to 100% with PBV strains reported globally. Interestingly, the highest nucleotide identity was observed with PBV strains reported from Tripura and Mizoram, India (97.62-98.20%). Phylogenetic analysis based on the RdRp gene revealed that both Assam PBV isolates clustered within the genogroup II lineage and grouped closely with PBV strains from neighbouring northeastern states, as well as with other GGII PBVs reported worldwide (Fig 4). These findings confirm the circulation of genogroup II picobirnavirus among pig populations in Assam.


       
Diarrhoea remains one of the major health concerns in swine production worldwide, particularly among piglets, contributing significantly to morbidity and mortality (Canibe et al., 2022). The etiology of pig diarrhoea is complex and multifactorial. Viruses such as rotaviruses and coronaviruses are well-recognized etiological agents of enteritis in pigs; however, emerging evidence suggests that picobirnaviruses may also play a role in enteric infections, either as primary pathogens or as coinfecting agents (Reddy et al., 2023; Wilhelmi et al., 2003).
       
PBVs have a small bi-segmented double-stranded RNA genome and have been found to infect a broad spectrum of vertebrate and invertebrate hosts (Hutton et al., 2025; Karayel-Hacioglu et al., 2022). Although PBVs have been found to infect both symptomatic and asymptomatic hosts, their frequent isolation in cases of gastroenteritis has led to speculations about their possible role in enteric diseases (Mondal and Majee, 2014). In pigs, PBVs have been occasionally reported and their epidemiology, pathogenicity and genetic diversity are not well understood, especially in different geographical areas like northeastern India (Kylla et al., 2017; Malik et al., 2018).
       
In the current study, the detection rate of PBV was low in RNA-PAGE and RT-PCR, which is in agreement with previous studies. The low detection rate in RNA-PAGE can be explained by the low sensitivity of the technique, whereas RT-PCR using the conserved RdRp gene showed higher sensitivity (Ganesh et al., 2012; Malik et al., 2018; Patra et al., 2020). All the RNA-PAGE-positive samples showed the characteristic two-segmented genomic pattern of PBV, thus proving the specificity of the technique despite its low sensitivity. All the RT-PCR-positive samples in this study were found to belong to genogroup II only, without the detection of genogroup I PBV. While this could be due to the circulation patterns of the virus in the region, it is also possible that the small sample size could have affected the distribution of the genogroups. Previous studies conducted in neighbouring northeastern states have found the presence of both genogroups in pigs (Kylla, 2015; Kylla et al., 2017). The absence of genogroup I PBV in this study emphasizes the importance of conducting large-scale surveillance studies to underst and the genetic diversity and distribution of PBVs in Assam.
       
Analysis of the data by age showed that the detection rate of PBV was higher in pre-weaned piglets than in older weaned pigs, which is consistent with previous studies that have shown a higher prevalence of PBV in young animals (Wilburn et al., 2017). This could be due to the fact that young piglets have an immature immune system that makes them more vulnerable to infection or viral shedding. Analysis of the data by season showed that the detection of PBV was limited to the pre-monsoon season, with no positives recorded during the post-monsoon or winter seasons. Although there is limited data on the seasonal pattern of PBV infection, similar trends have been observed in previous studies, which have shown that the detection of PBV is higher during warmer seasons.
       
The molecular study also showed high genetic diversity among the PBV strains, with the Assam isolates having 82-100% nucleotide similarity with the PBVs reported worldwide. The fact that the Assam PBV strains are phylogenetically closely related to those from the adjacent northeastern states of India indicates regional spread of the related viruses. PBVs are emerging viruses with a wide host range and high genetic diversity. The fact that PBVs are frequently isolated from both symptomatic and asymptomatic hosts and that they can co-infect with other enteric viruses, makes it difficult to establish a direct causal link with diarrheal disease. Similar molecular epidemiological investigations of emerging swine viruses in India have highlighted the importance of continuous genomic surveillance for understanding viral evolution and regional circulation patterns (Maan et al., 2023). However, the isolation of PBVs from diarrheic piglets in the current study emphasizes the need to include PBVs in the surveillance programs for enteric viruses, especially in areas where pig rearing is an important livelihood activity.

The present study offers the first molecular evidence of PBV infection in pigs from Assam, India and specifically in genotype group II viruses. The above findings add to the scarce information available so far on the epidemiology of PBVs in pigs from North East India and support the idea that further research with a larger sample size and also screening for co-circulating enteric pathogens could help clarify the role of PBVs in pigs.

I hereby declare on behalt of all the authors, that none of the authors has any competing interests or conflicts of interest related to this manuscript.