Detection and Molecular Analysis of Picobirnavirus and Rotavirus from Animal Faecal Samples

The high rates of morbidity and mortality, the reduction in growth and the treatment of neonatal diarrhoea cause substantial economic losses in both animals and humans. Moreover, diarrhoeic animals may harbour, incubate and act as a source of infection to healthy animals and humans (Holland, 2014). Neonatal diarrhoea is caused by a variety of infectious agents, including viruses, bacteria and protozoa. Acute gastroenteritis is caused by viruses such as enteric Coronaviruses, Bocavirus, Kobuvirus, Rotavirus as well as later recognized viruses such as Picobirnavirus (PBV) (Kattoor et al., 2016). PBVs are genetically distinct, rapidly evolving and spreading worldwide. PBVs may have ambiguous clinical implications, but they may pose a public health risk (Kumar et al., 2020). Genetic relatedness between human and animal PBV warrants a thorough investigation of the potential for zoonotic transmission. As a result, continual surveillance is required to detect the emergence of new PBVs in a different host and to determine their origin, diversity and public health implications (Malik et al., 2018).
       
This study was designed to molecularly characterize Picobirnavirus (PBV) in fecal samples collected from various animal species in and around Jabalpur. RNA-PAGE (Polyacrylamide Gel Electrophoresis) and PCR techniques were employed for the detection of PBV, followed by molecular characterization and phylogenetic analysis on selected samples. Both PBV and rotavirus were simult aneously screened from the clinical samples, with rotavirus detected in some cases.

A total of 150 faecal samples (diarrhoeic and non diarrhoeic) from calves (cattle and buffalo aged between 1-90 days), goat, pig, dog aged between 1-6 month and poultry (broiler) aged between 1-45 days were collected from different farms located in Jabalpur, Madhya Pradesh (subtropical part of India) for virus detection and all the experimental procedures were conducted in the Department of Veterinary Microbiology, Nanaji Deshmukh Veterinary Science University in the year 2021-2023. A 10% faecal suspension was prepared with phosphate buffered saline (PBS, pH 7.2) (Himedia) by dissolving one gram of faeces in 10 ml PBS. Centrifugation was done after thorough vortexing at 7500 rpm for 20 min to remove the coarse debris; the supernatant was used for RNA extraction. The viral RNA was extracted according to procedure described by Boom et al., (1990) with slight modifications. RNA-PAGE was used to detect PBV and Rotavirus by observing 2 and 11 genome segments, respectively and their migration pattern in all faecal samples. For electrophoresis, resolving gel (7.5%) was prepared as per the method of Laemmli (1970). The viral genomic electrophoresis was carried out according to Malik et al., (2011).  Silver staining of the gel was carried out as described by Sevenson et al., (1986) and documented. cDNA synthesis was done using PrimeScriptTM 1st strand cDNA synthesis kit (Takara) as per manufacturer’s instructions. The cDNA was used as a template for PCR to detect both PBV and Rotavirus. Primer details and conditions used for the PCR amplification of viral RNA from the fecal samples are presented in Table 1.


       
PCR amplification was conducted using a GeneAmp PCR System 9700 (Applied Biosystems), followed by gel electrophoresis. Two representative samples (bovine and goat) were sequenced at Eurofins Genomics India Pvt. Ltd., using Sanger sequencing.
 
Sequence and phylogenetic analysis
 
Amplicon size of 201 bp PCR product of RdRp gene (PBV) of one isolate were subjected to sequencing. Amplified RdRp gene sequence was analyzed for evolutionary lineages with various global isolates of PBV obtained from NCBI database. The sequences were analyzed using BLAST  (Basic local Alignment Search Tool) and the Clustal-W (CLUSTAL 2.1 multiple sequence alignment) to generate sequence alignment reports. CLUSTAL O program (version 1.2.4) was used to compare nucleotide sequence with Other cognate PBVs and percent identity matrix was created using CLUSTAL 2.1. A BLASTn search confirmed that the sequence of positive isolate was similar to cognate PBVs from GenBank, so the phylogenetic analysis included these sequences. Representative strains and outgroup sequences used were 1-CHN-97 (AF246939) and 4-GA-91 (AF246940) belonging to GI and GII, respectively. Molecular Evolutionary Genetic Analysis (MEGA 6) version was used for construction of phylogenetic tree. The bootstrapped phylogenetic tree was constructed using Maximum-likelihood method and significant bootstrap values with 1000 replicates.

Nucleotide sequence accession numbers
 
Accession number of the sequence submitted at NCBI for S14 sample (PBV/Goat/India/S14/2021) is OP866965.

In our analysis, we observed species-specific variations in the detection of PBV by RNA-PAGE, as detailed in Table 2. This table outlines the number of samples tested per species, the detection rates of PBV and the presence of VP6 bands in both diarrhoeic and non-diarrhoeic samples.        

                   

Among the 25 samples collected from buffalo, 4 were diarrhoeic, with PBV detected by RNA-PAGE in 3 of these diarrhoeic samples (75%), which notably showed two bands on the gel (Fig 1), resulting in an overall PBV prevalence of 12% in buffalo samples. In dogs, PBV was identified in 3 of the 23 diarrhoeic samples (13.04%) via RNA-PAGE.


       
Samples from cattle, goats, pigs and poultry were negative for PBV by RNA-PAGE. This lack of detection may reflect lower viral loads in these species or the labile nature of PBV, which can reduce detection efficiency, especially in stored samples. Previous studies have shown varying PBV detection rates in bovine samples, such as Takiuchi et al., (2016) with 8.3% in buffalo and Prasad et al., (2018) with 13% in diarrhoeic bovine samples, underscoring the virus’s variability across different hosts and sample conditions.
       
In dog faecal samples, the presence of tripartite bands was detected by RNA-PAGE in 3 out of 23 diarrhoeic samples (13.04%), suggesting the presence of Picotrirnavirus. However, none of these samples were amplified for the RdRp gene by RT-PCR, likely due to the narrow specificity of primers, which may limit the detection of all circulating Picotrirnavirus strains (Malik et al., 2014).
       
Fecal samples from cattle, goats, pigs and poultry were negative for PBV by RNA-PAGE, possibly due to low viral loads or the instability of PBV. During PBV screening, rotavirus was identified in three buffalo and two pig samples, with characteristic 11-segment RNA migration patterns confirmed by RT-PCR targeting the VP6 gene. Similar mammalian-like electropherotypes (4:2:3:2) in avian rotaviruses have been previously documented, suggesting possible interspecies transmission (Balan et al., 2018).
       
We used a different set of primers targeting the RdRp gene for molecular detection and characterization. Further molecular characterization was performed using RT-PCR to detect the partial RdRp gene of PBV and VP6 gene of rotavirus. The results of these tests, including the number of positive detections per species, are summarized in Table 3. This study screened 150 fecal samples (25 each from cattle, buffalo, goat, pig, dog and poultry) for PBV using primers targeting the RdRp gene of segment 2. PBV was detected in 11 bovine and goat samples (7.33%). Diagnostic PCR targeting the VP6 gene confirmed the rotavirus-positive samples identified by RNA-PAGE.


       
To assess the effectiveness of different primer sets in detecting the partial RdRp gene of PBV, RT-PCR was conducted across a range of samples. The specific outcomes, indicating which primer sets were successful in amplifying the RdRp gene in each sample, are compiled in Table 4. This table provides insights into the genetic diversity of PBV and the challenges associated with its detection using a single primer set. In buffalo samples, a 20% PBV prevalence was observed by RT-PCR, consistent with previous findings, such as the 23.4% prevalence in bovine samples reported by Navarro et al., (2018). While RNA-PAGE detection was negative in goat samples, RT-PCR revealed a 24% PBV prevalence, aligning with other studies that noted rates from 20% to 70% in small ruminants. For instance, Malik et al., (2018) reported a 35.75% prevalence in small ruminants, with higher rates in goats than in sheep.


       
In our study, the presence of tripartite RNA segments was detected in diarrhoeic dog samples by RNA-PAGE, though RT-PCR failed to amplify PBV with any primer set, likely due to low viral load or limited primer specificity. Our analysis identified all PBV-positive samples as genogroup I, with no detection of genogroup II, consistent with GenBank data showing genogroup I as predominant (83.11%). Atasoy et al., (2022) similarly found only genogroup I in cattle samples. These findings underscore the importance of using both RNA-PAGE and RT-PCR for PBV detection and suggest that RT-PCR for PBV is limited due to genetic diversity, requiring multiple primers and regular updates to capture emerging strains.
       
For rotavirus detection, the VP6 gene was targeted using GARV-D-VP6-F and GARV-F-VP6-R primers. Diagnostic PCR identified rotavirus in 4 buffalo, 1 goat and 2 pig samples, while RNA-PAGE confirmed rotavirus with an 11-segment pattern in three buffalo and two pig samples, indicating co-infection with PBV in samples B90, B245, B267 and B539. Similar co-infections of PBV and rotavirus have been observed, such as in Duarte Junior et al., (2021) in a toucan, emphasizing the relevance of co-infection screening in various species.
       
The RT-PCR product of an isolate of goat sample was subjected to nucleotide sequencing through outsourcing at Eurofins Genomics India Pvt. Ltd., Bangalore by Sanger sequencing method using forward and reverse primers for partial RdRp gene. The results of the blast search revealed that the isolate S14 (PBV/Goat/India/S14/2021) had similarity with the isolates of PBV sequences in GenBank ranged from 93.40% to 95.73% belonging to different geographical areas.
       
On studying the phylogenetic analysis of a goat PBV sample S14, it was found that it had proximity to human PBV strain (Accession no AB186898) and distantly related to monkey picobirnavirus suggesting interspecies transmission. On comparison with prototype strains, it was found closely related to human genogroup I strain 1-CHN-97 and distantly related to human genogroup II strain 4-GA-91 (Fig 2).


       
PBVs have been detected in both diarrhoeic and clinically healthy animals, as well as in invertebrates and environmental specimens, making their role and transmission mechanisms unclear. This study aimed to investigate PBV infection frequency in diarrhoeic and non-diarrhoeic animals. Most PBV strains identified globally are classified as genogroup I, with reported prevalence rates varying between 0.69% and 23.4%. Our findings align with these trends, showing a 7.33% positivity rate, primarily with genogroup I strains, in agreement with previous studies on PBV distribution.