Genogroup I and II Picobirnaviruses in Respiratory Tracts of Pigs

Sequence-independent amplification and specific reverse transcription PCRs identified genogroup I and II picobirnaviruses in respiratory tracts of pigs. These data expand knowledge of picobirnavirus diversity and tropism. Genetic relationships between porcine respiratory and human enteric picobirnaviruses suggest cross-species transmission of picobirnaviruses between pigs and humans.


The Study
We analyzed 197 respiratory tract swab specimens from pigs obtained in slaughterhouses in Hong Kong, China, and Colombo, Sri Lanka. Large-scale molecular RNA virus screening, based on host nucleic acid depletion, sequence-independent amplifi cation, and sequencing of partially purifi ed viral RNA was performed on nucleic acids isolated from 10 respiratory tract swab samples from pigs (4)(5)(6). Most of the 893 analyzed sequences were of unclassifi ed porcine genome origin, unclassifi ed, or bacterial origin. Three clones showed picobirnaviruses, which are double-stranded RNA viruses with a segmented genome belonging to the family Picobirnaviridae (7).
Because picobirnaviruses have been identifi ed only in fecal specimens, diagnostic PCRs for genogroup I and II picobirnaviruses specifi c for the RNA-dependent RNA polymerase (RdRp) gene (6,8,9) were performed on 60 respiratory tract swab specimens to determine whether picobirnaviruses are present in the porcine respiratory tract. Sixteen (26.6%) of 60 samples were confi rmed by sequencing as positive for genogroup I picobirnaviruses, and 4 (6.5%) of 60 were confi rmed by sequencing as positive for genogroup II picobirnaviruses. Three of 60 porcine swab samples that showed evidence of genogroup II picobirnaviruses were also positive for genogroup I picobirnavirus.
To determine genetic relationships between porcine respiratory genogroup I picobirnaviruses with reported genogroup I viruses detected in wastewater and human and porcine feces, a phylogenetic tree was constructed on the basis of a 165-nt fragment of the RdRP gene as described (6,10) (online Appendix Figure, Figure).
To determine genetic relationships between porcine respiratory genogroup II picobirnaviruses with reported genogroup II viruses detected in human feces, a phylogenetic tree was constructed on the basis of a 339-nt fragment of the RdRP gene as described (6,10) (Figure). Porcine genogroup II picobirnavirus partial RdRP nucleotide sequences from samples VS4400017, VS4400028, VS4400041, and VS4400049 showed 69%-96% similarity with each other (Table).  Table). Close genetic relationships could also be observed between genogroup II picobirnaviruses from pigs and humans.

Conclusions
Our results indicated that picobirnaviruses can be commonly found in the respiratory tract of pigs from different locations and identifi ed genogroup II picobirnaviruses in animals. Picobirnaviruses have been regarded as enteric viruses because all described cases were associated with virus shed in feces. Picobirnaviruses have been detected in human patients with and without gastroenteritis and are found in patients co-infected with enteric pathogens such as rotaviruses, caliciviruses, and astroviruses (8,9). Prevalence studies of picobirnaviruses in immunocompromised patients suggest that picobirnaviruses might be opportunistic enteric pathogens (11,12). Picobirnaviruses in the respiratory tracts of pigs suggest that picobirnaviruses might be not only potential enteric pathogens but also respiratory pathogens. The pigs used in our study showed no evidence of overt respiratory or other disease at the time of sampling. Whether these viruses contribute to disease early in life remains unclear.
Genogroup II porcine picobirnaviruses detected in this study were highly diverse. Phylogenetic analysis of porcine and human genogroup II picobirnaviruses indicated that >2 phylogenetic clades of genogroup II picobirnaviruses coexist in these populations. Similar observations were found regarding the high genetic diversity of porcine respiratory genogroup I picobirnaviruses. Genetic relationships between porcine respiratory picobirnaviruses and picobirnaviruses from wastewater in the United States and human feces were observed. These results suggest that there were multiple cross-species transmissions of picobirnavirus strains between swine and humans (8)(9)(10)(13)(14)(15).
Because molecular characterization of picobirnaviruses is limited, mostly to partial RdRP sequences, and only 1 complete genome has been determined, the zoonotic potential of picobirnaviruses awaits further characterization of full-length genomes. Attempts to obtain full-length genomes in this study were undertaken by using nextgeneration sequencing platforms but were unsuccessful. However, the extent of sequence variation along the 165-bp fragments of the RdRP gene of genogroup I picobirnaviruses for which an entire RdRP gene sequence is available (strains 1-CHN-97, HY005102,   GU968924) show good correlation with overall sequence variation observed in the entire RdRP gene (13).
A thorough understanding of the diversity of viruses in animals, virus transmission routes, and virus tropism provides epidemiologic baseline information about potential pathogenic threats from animal reservoirs for human health. Detection of genogroup I and II picobirnaviruses in porcine respiratory tract swab samples is an example of the needed expansion of our knowledge of picobirnavirus diversity and also expands our knowledge of picobirnavirus tropism. To better understand the epidemiology of genogroup II picobirnaviruses in pigs and to defi ne whether zoonotic or reverse-zoonotic transmissions occur, more intensive surveillance on this group of virus in pigs from other regions needs to be conducted. Whether genogroup I and/ or II picobirnaviruses can also be detected in the human respiratory tract and whether they play a causal role in respiratory diseases remain to be determined. This study illustrates how novel molecular techniques can provide new understanding of viral ecology, evolution, and spread.