Replication of Porcine Astrovirus Type 1-Infected PK-15 Cells In Vitro Affected by RIG-I and MDA5 Signaling Pathways.

The interferon (IFN) system is an extremely powerful antiviral response in animal cells. The subsequent effects caused by porcine astrovirus type 1 (PAstV1) IFN activation are important for the host's response to viral infections. Here, we show that this virus, which causes mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa in piglets, induces an IFN response upon infection of PK-15 cells. Although IFN-ß mRNA was detected within infected cells, this response usually occurs during the middle stages of infection, after genome replication has taken place. Treatment of PAstV1-infected cells with the interferon regulatory factor 3 (IRF3) inhibitor BX795 decreased IFN-ß expression, whereas the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) inhibitor BAY11-7082 did not. These findings indicate that PAstV induced the production of IFN-ß via IRF3-mediated rather than NF-κB-mediated signaling pathways in PK-15 cells. Moreover, PAstV1 increased the protein expression levels of retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in PK-15 cells. The knockdown of RIG-I and MDA5 decreased the expression levels of IFN-ß and the viral loads and increased the infectivity of PAstV1. In conclusion, PAstV1 induced the production of IFN-ß via the RIG-I and MDA5 signaling pathways, and the IFN-ß produced during PAstV1 infection inhibited viral replication. These results will help provide new evidence that PAstV1-induced IFNs may protect against PAstV replication and pathogenesis. IMPORTANCE Astroviruses (AstVs) are widespread and can infect multiple species. Porcine astroviruses produce mainly gastroenteritis and neurological diseases in pigs. However, astrovirus-host interactions are less well studied, particularly with respect to their antagonism of IFN. Here, we report that PAstV1 acts via IRF3 transcription pathway activation of IFN-ß. In addition, the knockdown of RIG-I and MDA5 attenuated the production of IFN-ß induced by PAstV1 in PK-15 cells and increased efficient viral replication in vitro. We believe that these findings will help us to better understand the mechanism of how AstVs affect the host IFN response.

Insertion of exogenous genes within the ORF1b coding region of porcine astrovirus.

Porcine astrovirus (PAstV) is a common cause of diarrhea in swine farms. The current understanding of the molecular virology and pathogenesis of PAstV is incomplete, especially due to the limited functional tools available. Here, ten sites in the open reading frame 1b (ORF1b) of the PAstV genome were determined to tolerate random 15 nt insertions based on the infectious full-length cDNA clones of PAstV using transposon-based insertion-mediated mutagenesis of three selected regions of the PAstV genome. Insertion of the commonly used Flag tag into seven of the ten insertion sites allowed the production of infectious viruses and allowed their recognition by specifically labeled monoclonal antibodies. Indirect immunofluorescence showed that the Flag-tagged ORF1b protein partially overlapped with the coat protein within the cytoplasm. An improved light-oxygen-voltage (iLOV) gene was also introduced into these seven sites, and only one viable recombinant virus that expressed the iLOV reporter gene at the B2 site was recovered. Biological analysis of the reporter viruses showed that these exhibited similar growth characteristics to the parental virus, but they produced fewer infectious virus particles and replicated at a slower rate. The recombinant viruses containing iLOV fused to ORF1b protein, which maintained their stability and displayed green fluorescence for up to three generations after passaging in cell culture. The porcine astroviruses (PAstVs) expressing iLOV were then used to assess the in vitro antiviral activities of mefloquine hydrochloride and ribavirin. Altogether, the recombinant PAstVs expressing iLOV can be used as a reporter virus tool for the screening of anti-PAstV drugs as well as the investigation of PAstV replication and the functional activities of proteins in living cells.

Isolation and Phylogenetic Analysis of a Hunnivirus Strain in Water Buffaloes From China.

In recent years, hunniviruses have been reported in a variety of animal species from many countries. Here, hunnivirus was detected in fecal samples from water buffaloes and named as BufHuV-GX-2106. The samples were inoculated into cultures of MDBK cells supplemented with TPCK trypsin and the BufHuV-GX-2106 strain was stably passaged and replicated. Electron microscopic analysis showed the BufHuV-GX-2106 virus particles were spherical and 20~30 nm in diameter. The complete genome of a plaque purified sample of BufHuV-GX-2106 was determined and analyzed. Genomic analysis revealed that the whole sequence of BufHuV-GX-2106 was ~7,601 nucleotides (nt) in length and consisted of a large open reading frame of 6,759nt, a 5'UTR, a 3'UTR and a poly(A) tail. The complete genome sequence of BufHuV-GX-2106 shares 68-85% nucleotide identities with other known hunnivirus strains, indicating high genetic heterogeneity among these viruses. Phylogenetic analysis showed that BufHuV-GX-2106 belonged to the Hunnivirus A species and was more closely related to ovine hunnivirus than other known viruses of this type. This study describes the first isolation and complete genome sequence of a hunnivirus strain from water buffaloes. In addition, this study will help to understand the mechanisms involved in the pathogenesis of Hunnivirus A among different animal species.

Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus.

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

Identification of a novel protein in porcine astrovirus that is important for virus replication.

Overlapping genes are common in some RNA viruses. It has been proposed that a potential overlapping gene is the ORFX, here termed ORF2b, which overlaps the ORF2 coding sequence in astroviruses. The aim of this study was to determine whether ORF2b is an overlapping gene that encodes a functional protein which is needed for viral replication. Sequence alignment showed that there was an ORF2b in a PAstV type 1 strain of astrovirus, PAstV1-GX1, which was embedded within the larger ORF2. The AUG codon for ORF2b is located 19 nucleotides downstream of the initiation site of ORF2 and contains 369 nucleotides and it codes for a predicted 122-amino-acid protein. A specific polyclonal antibody against the ORF2b protein was raised and used to demonstrate the expression of the new identified gene in virus-infected and pCAGGS-ORF2b-transfected cells. Analysis of purified virions revealed that the ORF2b protein was not incorporated into virus particles. Reverse genetics based on a PAstV type 1 infectious cDNA clone showed that the ORF2b protein was not essential but important for optimal virus infectivity. Knockout of the downstream potential stop codon candidate of ORF2b demonstrated that the C-terminus of the ORF2b protein can be extended by 170 amino acids, suggesting that the C-terminus of the newly identified ORF2b protein may be variable.

Pathogenic Characteristics of a Porcine Astrovirus Strain Isolated in China.

Astroviral infection is considered to be one of the causes of mammalian diarrheal diseases. It has been shown that astrovirus infections cause varying degrees of diarrhea in turkeys and mice. However, the pathogenesis of porcine astrovirus is unknown. In this study, the virulence of a cytopathic porcine astrovirus (PAstV) strain (PAstV1-GX1) isolated from the PK-15 cell line was tested using seven-day-old nursing piglets. The results showed that PAstV1-GX1 infection could cause mild diarrhea, growth retardation, and damage of the villi of the small intestinal mucosa. However, all the above symptoms could be restored within 7 to 10days post inoculation (dpi). To evaluate the innate immunity response of PAstV in vivo, the alteration of inflammatory cytokine expression in piglets infected with PAstV1-GX1 was determined using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The mRNA expression levels of the IFNß and ISG54 were found to be significantly elevated in virus-infected piglets. In contrast, expression of IFNλ was downregulated in piglets infected with PAstV1-GX1. In addition, the mRNA expression of the tight junction protein 1 and 2 and zonula occludin 1, which are associated with the intestinal barrier permeability, were affected after PAstV1 infection. The present study adds to our understanding of the pathogenic mechanism of PAstV and has established an animal model for further study of pig astrovirus infection.