Plasmids Expressing shRNAs Specific to the Nucleocapsid Gene Inhibit the Replication of Porcine Deltacoronavirus In Vivo.

Porcine deltacoronavirus (PDCoV) is a novel enteric coronavirus and is becoming one of the major causative agents of diarrhea in pig herds in recent years. To date, there are no commercial vaccines or antiviral pharmaceutical agents available to control PDCoV infection. Therefore, developing a reliable strategy against PDCoV is urgently needed. In this study, to observe the antiviral activity of RNA interference (RNAi), four short hairpin RNAs (shRNAs) specific to the nucleocapsid (N) gene of PDCoV were designed and tested in vitro. Of these, a double-shRNA-expression vector, designated as pSil-double-shRNA-N1, was the most effectively expressed, and the inhibition of PDCoV replication was then further evaluated in neonatal piglets. Our preliminary results reveal that plasmid-based double-shRNA-expression targeting the N gene of PDCoV can significantly protect LLC-PK1 cells and piglets from pathological lesions induced by PDCoV. Our study could benefit the investigation of the specific functions of viral genes related to PDCoV infection and offer a possible methodology of RNAi-based therapeutics for PDCoV infection.

Assembly of three-dimensional ultralight poly(amidoxime)/graphene oxide nanoribbons aerogel for efficient removal of uranium(VI) from water samples.

Assembling graphene oxide nanoribbons (GONRs) into three-dimensional (3D) materials with controllable and desired structure is an effective way to expand their structural features and enable their practical applications. In this work, an ultralight 3D porous amidoxime functionalized graphene oxide nanoribbons aerogel (PAO/GONRs-A) was prepared via solvothermal polymerization method using acrylonitrile as monomer and GONRs as solid matrices for selective separation of uranium(VI) from water samples. The PAO/GONRs-A possessed a high nitrogen content (13.5%), low density (8.5 mg cm-3), and large specific surface area (494.9 m2 g-1), and presented an excellent high adsorption capacity of uranium, with a maximum capacity of 2.475 mmol g-1 at a pH of 4.5, and maximum uranium-selectivity of 65.23% at a pH of 3.0. The results of adsorption experiments showed that U(VI) adsorption on PAO/GONRs-A was a pH-dependent, spontaneous and endothermic process, which was better fitted to the pseudo-second-order kinetic model and Langmuir isotherm model. Both X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) calculations revealed that U(VI) adsorption on PAO/GONRs-A mainly did rely on the amidoxime groups anchored on the aerogel while UO2(PAO)2(H2O)3 was dominant after interaction of uranyl with PAO/GONRs-A. Therefore, as a candidate adsorbent, PAO/GONRs-A has a high potential for the removal of uranium from aqueous solutions.

Porcine Circovirus Type 3 in Pig Farms Experiencing Diarrhea in Jiangxi, China: Prevalence, Genome Sequence and Pathogenicity.

Porcine circovirus 3 (PCV3) infections have been reported in different clinical presentations. However, the prevalence and pathogenicity of PCV3 associated with diarrhea in piglets have been limited. Herein, we present an investigation and genome analyses of PCV3 in piglets experiencing diarrhea, and observed clinical signs, gross lesions, and histological changes in pigs negative for all known pathogens associated with diarrhea but positive for PCV3 alone. Among the feces (n = 141) tested, 16.31% (23/141) were positive for PCV3. Of which, 27.28% (15/55) and 14.29% (5/35) were present in diarrheal samples from suckling and weaned piglets, respectively. Moderate to severe atrophic villi was confined in duodenum, jejunum, and ileum, and significantly decreased average heights of villi, and the depths of crypt were observed in PCV3-infected piglets. The complete genome of a representative strain of PCV3, designated as JX/CH/2018, was determined. Multialignment analysis indicated that JX/CH/2018 had 97.7-99.7% nucleotide identity at the complete genome level, and 97.2-100% at the amino acid level of the capsid protein when compared with reference PCV3 strains. Phylogenetic analysis showed that the PCV3 strain identified in this study belonged to PCV3a lineage. The present study demonstrated that PCV3 is a common virus in diarrheal suckling and weaned piglets.

Evaluation of Cross-Protection between G1a- and G2a-Genotype Porcine Epidemic Diarrhea Viruses in Suckling Piglets.

To date, two genotypes, i.e., genotype 1 (G1) and genotype 2 (G2), of porcine epidemic diarrhea virus (PEDV) have been identified in swine, while the cross protection between the G2a and G1a subgenotypes is undetermined. Hence, in the present study, we attempted to observe a comparative pathogenicity and cross protection of G1a (CV777) and G2a (CH/JX/01) PEDVs. Initially pregnant sows were vaccinated twice with the two kinds of inactivated G1a- and G2a-based PEDV vaccines, respectively and the delivered neonatal piglets were challenged with prototype isolates of G1a and G2a PEDVs, and then the pathogenicity and cross-protection in neonatal piglets were observed. The results showed that CH/JX/01, a highly virulent and dominant G2a PEDV strain currently circulating in China had more severe pathogenicity in vitro and in vivo, and induced more strong immune responses, including higher titers of sIgA in maternal milk than that induced by CV777 PEDV, a prototype of G1a PEDV strain. All piglets from the sows immunized with CH/JX/01 could not only survive when challenged with the homologous PEDV, but also be fully protected when challenged with heterogenous G1a PEDV. In contrast, the piglets from the sows immunized with CV777 could be protected when challenged with homologous PEDV and only partially protected when challenged with heterologous G2a strain of PEDV (CH/JX/01). The findings of this study provide new insights into the pathogenicity, antigenicity, and immunogenicity of currently circulating wild type G2a PEDV, which might be valuable for the development of novel PEDV vaccine candidates with improved efficacy.

Altered Gut Microbiota Profiles in Sows and Neonatal Piglets Associated with Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea virus (PEDV) is a devastating cause of diarrhea in pigs worldwide. Most of studies have focused on molecular and pathogenic characterization of PEDV, whereas there were limited studies in understanding the role of gut microbiota (GM) in viral-associated diarrhea. Here, using the Illumina MiSeq platform, we examined and compared the impact of PEDV infection on the GM of sows and their piglets less than 10 days old. Our results showed that PEDV caused alternations in the structure and abundance of GM from levels of phylum to genus, and even species. For sows, a significant decrease of observed species was found in diarrheal sows than that in healthy sows (p < 0.05). The unweighted and weighted UniFrac distances also revealed considerable segregations of GM structure among healthy, asymptomatic, and diarrheal sows. For piglets, Bacteroidetes, the dominant bacteria in healthy piglets, were replaced by Firmicutes in asymptomatic and diarrheal piglets. The abundances of Fusobacteria and Proteobacteria were also remarkably increased in asymptomatic piglets and diarrheal piglets when compared to those of the healthy piglets. Our findings demonstrated that PEDV infection caused severe perturbations of GM, reduced probiotic bacteria, and enriched pathogenic bacteria.

Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D.

Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.

Complete Genome Sequence of a Porcine Kobuvirus Variant Strain from Jiangxi, China.

The complete genome sequence of a porcine kobuvirus (PKoV) variant strain, CH/KB-1/2014 from Jiangxi, China, with a 90-nucleotide deletion in the 2B gene, was determined and characterized. This study provides a better understanding of the molecular characteristics and evolution of PKoV in Jiangxi, China.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea viruses associated with outbreaks of severe diarrhea in piglets in Jiangxi, China 2013.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is a highly contagious, acute enteric viral disease of swine characterized by vomiting, watery diarrhea, dehydration and death. To identify and characterize the field PEDVs associated with the outbreaks of severe diarrhea in piglets in Jiangxi, 2013, the complete genome sequences of two representative strains of PEDV, designated CH/JX-1/2013 and CH/JX-2/2013, were determined and analyzed. The genome sequences of both emergent Jiangxi PEDV strains, CH/JX-1/2013 and CH/JX-2/2013, were 28,038 nucleotides in length excluding 3' poly (A) tail. Compared to the PEDV CV777 strain, CH/JX-1/2013 and CH/JX-2/2013 had some unique genetic characteristics in the proximal region of the 5´-UTRs. Phylogenetic analysis of the complete genomes and the structural proteins revealed that CH/JX-1/2013 and CH/JX-2/2013 had a close relationship with post-2010 Chinese PEDV strains and US strains identified in 2013. The nucleotide identity between the two Jiangxi strains (CH/JX-1/2013 and CH/JX-2/2013) and 30 strains of PEDV identified ante-2010 and post-2010 ranged from 96.3-97.0% and 97.3-99.7%, respectively. Multiple nucleotide and deduced amino acid mutations were observed in the ORF1a/b, S, ORF3, E, M and N genes among the current field PEDV strains when compared to the CV777 strain. Some of the mutations altered the amino acid charge and hydrophilicity, and notably, there was an amino acid substitution in the middle of one neutralizing epitope (L1371I) of the S gene of both CH/JX-1/2013 and CH/JX-2/2013. Taken together, the accumulated genetic variations of the current field PEDV strains might have led to antigenic changes of the viruses, which might confer the less effectiveness or failure of the CV777-based vaccines currently being widely used in Jiangxi, China.