Detection and Molecular Characterization of Porcine Picobirnavirus in Feces of Domestic Pigs from Kolkata, India

Abstract

Picobirnaviruses (PBVs) are small, non-enveloped, 35–41 nm virion with bisegmented double-stranded RNA genome. PBVs are widespread and were detected in feces of humans and a wide variety of animals. Domestic pig, one of the ubiquitous farm animal reported incessant association with a variety of viral zoonoses. The objective of our study is to find out the incidence of PBV infection in healthy domestic pigs. The study was conducted by collecting feces of healthy/asymptomatic pigs from a piggery located in an urban slum at Kolkata, India to detect PBV infections. All the 11 fecal samples were tested by polyacrylamide gel electrophoresis and reverse transcription–polymerase chain reaction assay. In this study, we report the first incidence of detection and molecular characterization of porcine PBV (BG-Por-2/2010 and BG-Por-7/2010) in feces of domestic pigs from India using the human PBV genogroup I specific primer pair: PicoB25(+) and PicoB43(−). Sequence comparison and phylogenetic analysis of partial RNA-dependent RNA polymerase gene of genome segment 2 revealed genetic relatedness to hitherto reported porcine, murine and human genogroup I PBVs from different geographical regions. This warrants a stringent global surveillance to study the potential zoonotic and emerging PBV infections.

Electronic supplementary material

The online version of this article (doi:10.1007/s13337-012-0106-z) contains supplementary material, which is available to authorized users.

Picobirnavirus (PBV), a non-enveloped, bisegmented double-stranded RNA (dsRNA) virus, with spherical virion of 35–41 nm in diameter. PBV is the only genus in the Picobirnaviridae family [6]. PBV infect vertebrates and is thought to be transmitted via the fecal-oral route. When PBV is shed in large quantity in the feces, its genome segments are visible by polyacrylamide gel electrophoresis (PAGE) and silver staining. The size of the large genome segment ranges from 2.3 to 2.6 kbp, while the small genome segment is 1.5–1.9 kbp [35]. Genome patterns can be classified into large and small profiles [15].

To date, only one complete nucleotide sequence of a PBV strain; the human Hy005102 strain has been published. This strain was detected in a stool specimen of an infant with acute non-bacterial gastroenteritis from Thailand [37]. The genomic segment 1 has two open reading frames (ORF1 and ORF2). ORF1 codes for a hydrophilic 224 amino acids protein of unknown function. ORF2 encodes for the 552 amino acids capsid protein. The ORF1 gene, in the segment 2, encodes for the viral RNA-dependent RNA polymerase (RdRp) (534 amino acids). The virion structure consists of an outer capsid and a simple core inside with distinctive icosahedral arrangement [10].

The serendipitous detection of PBV dates back to late 1980s while rotavirus surveillance during gastroenteritis outbreaks as well as analyzing feces of free-living rats by PAGE assay [32, 33]. Thereafter, PBVs were detected from different countries in feces of humans, a wide range of mammals, birds, reptiles and even from environmental samples [1–5, 7, 8, 12, 14, 16–18, 20–23, 25, 27–30, 34, 36]. PBVs were detected from immunocompromised patients [24] in which implied to be “opportunistic pathogens” and have also been detected from asymptomatic hosts [11]. The question whether PBV is an ‘innocuous agent’ of the intestine remains to be investigated [2, 7, 19, 31, 35].

The detection of bisegmented dsRNA genome of PBV by PAGE and silver staining [26], is one of the standard and reliable laboratory diagnosis. The reverse transcription–polymerase chain reaction (RT–PCR) protocol developed by Rosen et al. [35] enabled the detection and molecular characterization of PBV studies, worldwide. With the two sets of primer pairs that specifically amplify small fragments within the RdRp gene of segment 2, PBV strains have been classified into two genogroups (genogroup I with 201 bp amplicon and genogroup II with 369 bp amplicon) represented by prototype strains 1-CHN-97 and 4-GA-91 isolated in China and USA, respectively.

Pigs are increasingly recognized to harbor a wide range of viruses that apparently establish long-term persistence and of emerging zoonotic potential [9]. In this study, we carried out the detection and molecular characterization assays for porcine PBV in feces of asymptomatic domestic pigs in Kolkata, India. Out of 11 samples, two showed very faint PBV positive (large genome profile) by PAGE and both the samples showed positive for genogroup I PBV by RT–PCR using genogroup specific primer pair developed by Rosen et al. [35]. Further characterization by sequencing and phylogenetic analyses revealed that the genogroup I porcine PBV strain detected during this study clustered with hitherto reported porcine, murine and human PBV strains reported from various countries.

Eleven fecal specimens were collected from domestic pigs (Sus scrofa domesticus) without diarrhea from a piggery located at an urban slum at Kolkata, India during November 2010 as part of an ongoing epidemiological study on PBV infections. The age of hosts ranged from 1 to 6 years. Ten percent clarified fecal suspensions were prepared using 10 mM phosphate buffered saline (PBS) solution (pH 7.4) and stored at 4 °C refrigerator till use as previously described [17]. Briefly, an aliquot of the fecal sample was diluted with 10 mM (1×) PBS solution, vortexed thoroughly and centrifuged at 3,000 rpm (704×g) for 15 min at 4 °C for preliminary clarification. The supernatant was transferred to a fresh microfuge tube and centrifuged again at 7,000 rpm (3,834×g) for 15 min. The supernatant was finally saved in a fresh microfuge tube as clarified fecal suspension and stored at 4 °C.

The dsRNA was extracted from fecal suspension using phenol–chloroform-isoamyl alcohol mixture for PAGE assays as previously described [3] and subsequent visualization of dsRNA migration patterns after PAGE and silver staining was performed according to Herring et al. [26].

Molecular biology grade viral RNA extraction was carried out using the commercially available QIAGEN QIAamp^® Viral RNA mini kit (QIAGEN GmbH, Hilden, Germany) as per manufacturer’s instructions.

The primer pairs described by Rosen et al. [35] were used for molecular characterization experiments. RT–PCR was carried out following the protocol of Bhattacharya et al. [3]. The purification of PCR products, sequencing and sequence analysis were performed as previously described [16].

The sequence data of 201 bp amplicon of one PBV positive sample (nucleotide sequence fragment covering partial RdRp gene of genomic segment 2 of genogroup I porcine PBV strain: genogroup I PBV/Pig/India/BG-Por-2/2010) analyzed during this study was submitted to the DNA Data Bank of Japan (DDBJ; http://www.nig.ac.jp/) DDBJ/EMBL/GenBank nucleotide sequence databases with the following accession number: AB669584.

Picobirnavirus was detected from fecal specimens of asymptomatic pigs using a combination of molecular methods. By PAGE assay, all the 11 porcine fecal samples were negative for rotavirus, except two, which showed very faint PBV positive. Subsequently, all the samples were subjected for RT–PCR targeting RdRp gene with genogroup I specific primers [PicoB25(+) and PicoB43(−)] and genogroup II specific primers [PicoB23(+) and PicoB24(−)]. Genotyping RT–PCR results confirmed the presence of only two genogroup I PBV strains with the amplicon of 201 bp in BG-Por-2 and BG-Por-7. The genogroup nature of strain was further confirmed by direct sequencing in both directions separately. Sequence analysis of one of the PBV strain (BG-Por-2) showed close relatedness to hitherto reported porcine, murine and human PBV strains. Based on the recently proposed nomenclature by the Brazilian research group [13], the genogroup I PBV strain detected and sequenced during this study is designated as: genogroup I PBV/Pig/India/BG-Por-2/2010. The other PBV RT–PCR positive sample (BG-Por-7/2010) did not give a clear sequence even after repetition of assay, so not included for analyses.

The comparison of percentage nucleotide identity (170 bp) and the partial length deduced amino acid identity (given in parentheses) of gene segment 2 (stretch of 56 amino acids) among the porcine genogroup I PBV strain (PBV/Pig/India/BG-Por-2/2011) detected in Kolkata and hitherto reported PBV strains from various hosts and PBV prototype strains were compared (Supplementary Table 1). The comparison of deduced stretch of 56 amino acids of porcine PBV strain BG-Por-2 and hitherto reported PBVs from various hosts showed that 14 amino acids were conserved, whereas distinct amino acid changes were observed in other positions (Supplementary Table 2). The comparison of nucleotide sequence of Indian isolate of Porcine PBV with that of a few randomly selected Hungarian PBV strains from human and porcine hosts was shown in the Supplementary Table 3. The phylogenetic tree (Fig. 1) also indicated that the detected genogroup I porcine PBV strain was distinct as it clustered on separate branches showing close homology to other porcine PBV strains reported from Hungary.

Fig. 1.

Phylogenetic tree showing the porcine PBV strain (genogroup I PBV/Pig/India/BG-Por-2/2010) with cognate stretch of hitherto reported human, porcine, bovine, canine, murine, avian, serpentine and environmental genogroup I PBV strains based on partial amino acid sequence [56 amino acids (aa)] partial RdRp gene of genomic segment 2. The phylogenetic tree was constructed by the neighbor-joining method using the MEGA software (Version 4.1). Phylogenetic distances were measured by the Kimura two-parameter model, and the tree was statistically supported by bootstrapping with 1,000 replicates. The genogroup I porcine PBV strain BG-Por-2 is denoted with a filled circle symbol. The tree was rooted with cognate stretch of gene segment 2 of genogroup II prototype strain Hu/4-GA-91 (USA) defined as the outgroup strain. Bar 0.2 substitutions per nucleotide. Abbreviations: Hu human, Bo bovine, Po porcine, Eq equine, Av avian, Mu murine, Env environmental, ARG Argentina, BRA Brazil, Hun Hungary, Thai Thailand, USA United States of America, Ven Venezuela

Detection of PBV in feces of domestic pigs has been reported for the first time in Kolkata, India. The presence of PBV in asymptomatic swine of 1 and 6 years of age might be consistent with a previous onset of infection and subsequent establishment of viral persistence.

A research group in Argentina published a study on systematic sampling of fecal specimens from 150 various animals and birds in captivity from a zoo [30]. In that study, PBV was detected by PAGE and silver staining in 3.70 % fecal samples (19 out of 513) among mammals and birds. The authors suggested a lack of etiological relation of PBV with disease; since, none of the host species showed any signs of diarrhea. These authors proposed that factors like stress due to captivity and/or isolations might favor the PBV replicative cycle in these hosts.

Likewise, another research group from the same region conducted a study in farm animals in Argentina [29] reported dissimilar excretion of PBV in fecal specimens of pigs at different age groups. Also varied physiological characteristics were observed among pigs during the period of lactation and final stage of pregnancy. The research group reported their observations stating that the conditions for higher PBV excretion was attributed to particular physiological status of the hosts, especially farrowing and lactation. They also concluded that the higher detection rates of PBV in the study population might be due to the stress conditions generated by pig farming practices during these stages [29]. They have also suggested that PBV is acquired early in life and establishes a persistent infection among the hosts which exhibit a unique pattern of virus excretion with periods of high viral activity intermingled with periods of silence. Thus concluding that the host infected with PBV could remain life-long asymptomatic carriers and serve as reservoirs of infection [29].

Picobirnaviruses present wide genetic diversity and are evolving rapidly. However, the PAGE assay detects the PBVs readily as it is not dependent on their genomic sequences. PBVs are known to cause chronic diarrhoea with prolonged shedding of the virus in humans [24] and various animals [30] besides frequent infections among piglets [5, 29]. Moreover, the presence of genogroup I PBVs in humans and different animals, rodents and reptiles, suggests that any specific genogroup is not restricted to specific host [2, 3, 5, 12, 16, 19, 31].

Evidence for genetic relatedness was reported between human and animal PBVs or vice versa [2, 5, 16–18, 22] from around the globe. Genogroup I PBVs detected and sequenced from pigs in Hungary [2] and Venezuela and Argentina [5, 22], were observed to be closely related to human genogroup I PBVs. These results strongly suggest that PBV strains may circulate in the shared environment where humans and pigs live. The question if PBV is transmitted directly through contact between different host species or by using the same sources (e.g. Water) is an objective of future studies [19].

These findings on detection of PBV in Indian (Kolkata) pigs extend our knowledge about their widespread geographical occurrence and reinforce the need to survey parallel PBV infections in humans and animals.