CRISPR-Cas13a-based detection method for avian influenza virus.

Avian influenza virus (AIV) causes huge losses to the global poultry industry and poses a threat to humans and other mammals. Fast, sensitive, and portable diagnostic methods are essential for efficient avian influenza control. Here, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a based platform was developed to detect AIV. This novel method was developed to specifically detect H1-H16 subtypes of AIV with fluorescence and lateral flow-based readouts and exhibited no cross-reactivity with Newcastle disease virus, avian infectious bronchitis virus, or infectious bursal disease virus. The limit of detection was determined to be 69 and 690 copies/µL using fluorescence and lateral flow as readouts, respectively. The developed assay exhibited 100% consistency with quantitative real-time polymerase chain reaction in detecting clinical samples. The heating of unextracted diagnostic samples to obliterate nuclease treatment was introduced to detect viral RNA without nucleic acid extraction. Single-step optimization was used to perform reverse transcription, recombinase polymerase amplification, and CRISPR-Cas13a detection in a tube. These advances resulted in an optimized assay that could specifically detect AIV with simplified procedures and reduced contamination risk, highlighting the potential to be used in point-of-care testing.

PRRSV Promotes MARC-145 Cells Entry Into S Phase of the Cell Cycle to Facilitate Viral Replication via Degradation of p21 by nsp11.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains one of the most economically significant pathogens that seriously affect the global swine industry. Despite sustained efforts, the factors that affect PRRSV replication in host cells are far from being fully elucidated and thus warrants further investigation. In this study, we first demonstrated that PRRSV infection can cause downregulation of endogenous p21 protein in MARC-145 cells in a virus dose-dependent manner. Next, we analyzed the effect of p21 knockdown by RNA interference on cell cycle progression using flow cytometric analysis, and found that knockdown of p21 promotes MARC-145 cells entry into S phase of the cell cycle. Interestingly, we further discovered PRRSV infection is also able to promote MARC-145 cells entry into the S phase. Subsequently, we synchronized MARC-145 cells into G0/G1, S and G2/M phases, respectively, and then determined PRRSV replication in these cells. Results here show that the MARC-145 cells synchronized into the S phase exhibited the highest viral titer among the cells synchronized to different phases. Additionally, to reliably analyze the potential role of endogenous p21 protein in PRRSV replication, we constructed a p21 gene-knockout MARC-145 cell line (p21-/-) using CRISPR/Cas9 technology and evaluated its capability to support PRRSV replication. Our results indicate that knockout of p21 is conducive to PRRSV replication in MARC-145 cells. Furthermore, through construction of a series of eukaryotic plasmids expressing each of individual PRRSV proteins combined with cell transfection, we demonstrated that the nonstructural protein 11 (nsp11) of PRRSV mediates p21 degradation, which was further confirmed by generating a stable MARC-145 cell line constitutively expressing nsp11 using a lentivirus system. Notably, we further demonstrated that the endoribonuclease activity rather than the deubiquitinating activity of nsp11 is essential for p21 degradation via mutagenic analysis. Finally, we demonstrated that nsp11 mediates p21 degradation via a ubiquitin-independent proteasomal degradation manner. Altogether, our study not only uncovers a new pathogenesis of PRRSV, but also provides new insights into development of novel antiviral strategies.

Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells.

The nonstructural protein 10 (nsp10) of porcine reproductive and respiratory syndrome virus (PRRSV) encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10a, was found in PRRSV-infected cells and the production of nsp10a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10a production. Finally, we demonstrated that nsp10a exerted little influence on the growth kinetics of PRRSV in vitro.

Interleukin-2 enhancer binding factor 2 interacts with the nsp9 or nsp2 of porcine reproductive and respiratory syndrome virus and exerts negatively regulatory effect on the viral replication.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failures in sows and respiratory diseases in growing pigs, resulting in huge economic loss for the pig production worldwide. The nonstructural protein 9 (nsp9) and nonstructural protein 2 (nsp2) of PRRSV are known to play important roles in viral replication. Cellular interleukin-2 enhancer binding factor 2 (ILF2) participates in many cellular pathways and involves in life cycle of some viruses. In the present study, we analyzed the interaction of cellular ILF2 with the nsp9 and nsp2 of PRRSV in vitro and explored the effect of ILF2 on viral replication. METHODS: The interaction of ILF2 with the nsp9 or nsp2 of PRRSV was analyzed in 293FT cells and MARC-145 cells by co-immunoprecipitation (Co-IP) and the co-localization of ILF2 with the nsp9 or nsp2 of PRRSV in MARC-145 cell and pulmonary alveolar macrophages (PAMs) was examined by confocal immunofluorescence assay. The effect of ILF2 knockdown and over-expression on PRRSV replication was explored in MARC-145 cells by small interfering RNA (siRNA) and lentivirus transduction, respectively. RESULTS: The interaction of ILF2 with nsp9 or nsp2 was first demonstrated in 293FT cells co-transfected with ILF2-expressing plasmid and nsp9-expressing plasmid or nsp2-expressing plasmid. The interaction of endogenous ILF2 with the nsp9 or nsp2 of PRRSV was further confirmed in MARC-145 cells transduced with GFP-nsp9-expressing lentiviruses or infected with PRRSV JXwn06. The RdRp domain of nsp9 was shown to be responsible for its interaction with ILF2, while three truncated nsp2 were shown to interact with ILF2. Moreover, we observed that ILF2 partly translocated from the nucleus to the cytoplasm and co-localized with nsp9 and nsp2 in PRRSV-infected MARC-145 cells and PAMs. Finally, our analysis indicated that knockdown of ILF2 favored the replication of PRRSV, while over-expression of ILF2 impaired the viral replication in MARC-145 cells. CONCLUSION: Our findings are the first to confirm that the porcine ILF2 interacts with the nsp9 and nsp2 of PRRSV in vitro, and exerts negatively regulatory effect on the replication of PRRSV. Our present study provides more evidence for understanding the roles of the interactions between cellular proteins and viral proteins in the replication of PRRSV.

Epitope mapping and characterization of a novel Nsp10-specific monoclonal antibody that differentiates genotype 2 PRRSV from genotype 1 PRRSV.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of PRRS, has two distinct and highly diverse genotypes (genotype 1 and genotype 2) in the field. Accurate diagnosis and differentiation of the two genotypes of PRRSV are critical to the effective prevention and control of PRRS. The non-structural protein 10 (Nsp10) plays a vital role in viral replication and is one of the most conserved proteins of PRRSV, thus constituting a good candidate for PRRSV diagnosis. RESULTS: In this study, we generated a monoclonal antibody (mAb) 4D9 against Nsp10 by immunizing BALB/c mice with purified recombinant Nsp10 expressed by an Escherichia coli system. Through fine epitope mapping of mAb 4D9 using a panel of eukaryotic expressed polypeptides with GFP-tags, we identified the motif 286AIQPDYRDKL295 as the minimal unit of the linear B-cell epitope recognized by mAb 4D9. Protein sequence alignment indicated that 286AIQPDYRDKL295 was highly conserved in genotype 2 PRRSV strains, whereas genotype 1 PRRSV strains had variable amino acids in this motif. Furthermore, a mutant of the motif carrying two constant amino acids of genotype 1 PRRSV, Cys290 and Glu293, failed to react with mAb 4D9. More importantly, the mAb 4D9 could differentiate genotype 2 PRRSV strains from genotype 1 PRRSV strains using Western blotting and immunofluorescence analysis. CONCLUSION: Our findings suggest that Nsp10-specific mAb generated in this study could be a useful tool for basic research and may facilitate the establishment of diagnostic methods to discriminate between genotype 1 and genotype 2 PRRSV infection.

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray.

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.

Identification of a novel linear epitope on the NS1 protein of avian influenza virus.

BACKGROUND: The NS1 protein of avian influenza virus (AIV) is an important virulent factor of AIV. It has been shown to counteract host type I interferon response, to mediate host cell apoptosis, and to regulate the process of protein synthesis. The identification of AIV epitopes on NS1 protein is important for understanding influenza virus pathogenesis. RESULTS: In this paper, we describe the generation, identification, and epitope mapping of a NS1 protein-specific monoclonal antibody (MAb) D9. First, to induce the production of MAbs, BALB/c mice were immunized with a purified recombinant NS1 expressed in E. coli. The spleen cells from the immunized mice were fused with myeloma cells SP2/0, and through screening via indirect ELISAs, a MAb, named D9, was identified. Western blot assay results showed that MAb D9 reacted strongly with the recombinant NS1. Confocal laser scanning microscopy showed that this MAb also reacts with NS1 expressed in 293T cells that had been transfected with eukaryotic recombinant plasmids. Results from screening a phage display random 7-mer peptide library with MAb D9 demonstrated that it recognizes phages displaying peptides with the consensus peptide WNLNTV--VS, which closely matches the (182)WNDNTVRVS(190) of AIV NS1. Further identification of the displayed epitope was performed with a set of truncated polypeptides expressed as glutathione S-transferase fusion proteins, and the motif (182)WNDNT(186) was defined as the minimal unit of the linear B cell epitope recognized by MAb D9 in western blot assays. Moreover, homology analysis showed that this epitope is a conserved motif among AIV. CONCLUSIONS: We identified a conserved linear epitope, WNDNT, on the AIV NS1 protein that is recognized by MAb D9. This MAb and its epitope may facilitate future studies on NS1 function and aid the development of new diagnostic methods for AIV detection.