Novel picornaviruses and tissue tropism in dogs and horses and comparative genome analysis

Abstract

The SARS epidemic has boosted interests in study of zoonotic viruses, which has led to the discovery of several novel viruses including picornaviruses since then. However, little is known about the host diversity and tissue tropism of picornaviruses especially in domestic companion animals such as dogs and horses as the viruses have been confined to a range of animals and sites only. Therefore, extensive surveillance studies were conducted using reverse transcription polymerase chain reaction (RT-PCR) to investigate the molecular epidemiology of picornaviruses in dogs and horses.

In the first and second parts of this study, the molecular epidemiology, characterization and comparative genome analysis of two previously undescribed picornaviruses, canine picodicistrovirus (CPDV) and canine picornaviruses (CanPV), were conducted. In a 22-month prospective study involving 1472 samples of 368 dogs captured at various locations in HKSAR, CPDV was discovered from 47 faecal and urine samples. Complete genome sequencing and phylogenetic analysis of CPDV revealed that three genomes possessed very low amino acid identities (<31%) to those of all other known picornaviruses. Moreover, the genomes of CPDV were distinct from other picornaviruses by containing two putative internal ribosome entry site (IRES) elements and two open reading frames, encoding two polyprotein precursors (844 and 1,406 amino acids respectively), separated by an intergenic region of 588 bases. The functionality of both putative IRES elements was confirmed by a bicistronic reporter assay using DNA and RNA transfection. This is the first report of the natural occurrence of two functional IRES elements in picornaviruses.

Besides CPDV, another novel picornavirus was also identified in dogs from HKSAR, and highlighted previously unrecognized diversity of picornaviruses in dogs. Among 1347 faecal samples of dogs subjected to RT-PCR, CanPV was detected in 15 (1.11%) faecal samples. Upon partial 5 UTR gene analysis, CanPV was found to be distinct from CPDV and formed a separate genetic cluster, representing another previously undetected picornavirus. Apart from Hong Kong, CanPV was further identified in one (0.76%) of the 131 canine faecal samples from Dubai. Complete genome sequencing of two CanPV strains from HKSAR and one strain from Dubai revealed that CanPV represents a novel picornavirus. The high amino acid identities shared among three sequenced strains suggested they are likely from the same lineage despite being found from different geographical areas.

Further genome and phylogenetic analyses indicated that CanPV is closely related to several unclassified picornaviruses including bat picornaviruses 1 to 3, feline picornavirus and Ia io picornavirus 1. CanPV, together with these viruses, may form a new genus in the family Picornaviridae.

For the third part of this study, an attempt to investigate the molecular epidemiology of picornaviruses in faecal samples of horses was made. Of 29 and 47 faecal samples collected from horses in HKSAR and Dubai, four (13.8%) samples from Dubai were found to contain equine rhinitis B virus (ERBV). The complete genome sequences of the four ERBV strains displayed two groups, ERBV2 and ERBV3. This represents the first study to demonstrate previously undescribed tissue tropism of ERBV for the gastrointestinal tract.