In 2011, porcine epidemic diarrhea virus (PEDV) infection rates rose substantially in vaccinated swine herds. To determine the distribution profile of PEDV outbreak strains, we sequenced the ...full-length spike gene from samples from 9 farms where animals exhibited severe diarrhea and mortality rates were high. Three new PEDV variants were identified.
Porcine reproductive and respiratory syndrome virus (PRRSV) causes severe economic losses to current swine production worldwide. Highly pathogenic PRRSV (HP-PRRSV), originated from a genotype 2 ...PRRSV, is more virulent than classical PRRSV and further exacerbates the economic impact. HP-PRRSV has become the predominant circulating field strain in China since 2006. CD163 is a cellular receptor for PRRSV. The depletion of CD163 whole protein or SRCR5 region (interaction site for the virus) confers resistance to infection of several PRRSV isolates in pigs or cultured host cells. In this study, we described the generation of a CD163 knockout (KO) pig in which the CD163 protein was ablated by using CRISPR/Cas9 gene targeting and somatic cell nuclear transfer (SCNT) technologies. Challenge with HP-PRRSV TP strain showed that CD163 KO pigs are completely resistant to viral infection manifested by the absence of viremia, antibody response, high fever or any other PRRS-associated clinical signs. By comparison, wild-type (WT) controls displayed typical signs of PRRSV infection and died within 2 weeks after infection. Deletion of CD163 showed no adverse effects to the macrophages on immunophenotyping and biological activity as hemoglobin–haptoglobin scavenger. The results demonstrated that CD163 knockout confers full resistance to HP-PRRSV infection to pigs without impairing the biological function associated with the gene.
•We establish knockout pigs devoid of CD163 expression via CRISPR/Cas9 gene editing combined with SCNT.•The modified pigs are completely protected from challenge with highly pathogenic PRRSV infection.•Deletion of CD163 shows no adverse effects to the other biological functions associated with this gene.
Broccoli (Brassica oleracea var. italica) is an important B. oleracea cultivar, with high economic and agronomic value. However, comparative genome analyses are still needed to clarify variation ...among cultivars and phylogenetic relationships within the family Brassicaceae. Herein, the complete chloroplast (cp) genome of broccoli was generated by Illumina sequencing platform to provide basic information for genetic studies and to establish phylogenetic relationships within Brassicaceae. The whole genome was 153,364 bp, including two inverted repeat (IR) regions of 26,197 bp each, separated by a small single copy (SSC) region of 17,834 bp and a large single copy (LSC) region of 83,136 bp. The total GC content of the entire chloroplast genome accounts for 36%, while the GC content in each region of SSC,LSC, and IR accounts for 29.1%, 34.15% and 42.35%, respectively. The genome harbored 133 genes, including 88 protein-coding genes, 37 tRNAs, and 8 rRNAs, with 17 duplicates in IRs. The most abundant amino acid was leucine and the least abundant was cysteine. Codon usage analyses revealed a bias for A/T-ending codons. A total of 35 repeat sequences and 92 simple sequence repeats were detected, and the SC-IR boundary regions were variable between the seven cp genomes. A phylogenetic analysis suggested that broccoli is closely related to Brassica oleracea var. italica MH388764.1, Brassica oleracea var. italica MH388765.1, and Brassica oleracea NC_0441167.1. Our results are expected to be useful for further species identification, population genetics analyses, and biological research on broccoli.
•A novel reassortant mammalian orthoreovirus 3 (MRV3), designated MRV-ZJ2013, was isolated from diarrheic piglets.•Genomic and phylogenetic analysis shows that MRV-ZJ2013 may have originated from ...reassortments among mink, bat, and pig MRVs.•Experimental infection study showed that MRV-ZJ2013 had low, if any, pathogenesis in newborn piglets.•A seroepidemiological survey of MRV3 revealed a high seroprevalence (77%) from diarrheic pigs of different ages in east China.
Mammalian orthoreoviruses (MRVs), which cause gastrointestinal and respiratory illness, have been isolated from a wide variety of mammalian species including bats, minks, pigs and humans. Here we report the isolation and genetic and pathogenic characterization of a novel MRV type 3 (MRV3), named MRV-ZJ2013, from the diarrheic feces of piglets in Zhejiang province, China. Genomic and phylogenetic analysis shows that MRV-ZJ2013 may have originated from reassortments among mink, bat, and pig MRVs, suggesting the hypothesis that interspecies transmission has occurred in pig herds. Neonatal piglets infected with MRV-ZJ2013 displayed mild clinical signs such as poor appetite and soft feces, but vomiting and diarrhea were not observed. Fecal virus shedding was detected only in three out of six piglets, each for one- or two-day post-infection. In contrast, piglets inoculated with a virulent porcine epidemic diarrhea virus (PEDV) strain as the control group had severe signs characterized by acute vomiting and watery diarrhea. These findings suggest that the virulence of MRV-ZJ2013, if any, was likely not significant compared to that of PEDV. A seroepidemiological survey of MRV by means of an indirect enzyme-linked immune-sorbent assay (ELISA) based on a recombinant MRV3 capsid protein sigma1 as antigen revealed a high seroprevalence (77%) in 1037 samples from diarrheic pigs of different ages from 24 herds in seven provinces of east China between 2015 and 2016, indicating that MRV3 is endemic in pig herds in China, and may contribute collectively to enteric disease along with other porcine pathogens.
An outbreak of diarrhea in pigs started in Guangdong, South China in January 2011. Cases were characterized by watery diarrhea, dehydration and vomiting, with 80-100% morbidity and 50-90% mortality ...in suckling piglets. The causative agent of the diarrhea was ultimately identified as porcine epidemic diarrhea virus (PEDV). In this study, we isolated a PEDV strain designated CHGD-01 from piglet intestines using Vero cell cultures, and its specific cytopathic effects were confirmed in susceptible cells by direct immunofluorescence testing and electron microscopy. The complete genome of CHGD-01 was shown to be 28,035 nucleotides in length, with a similar structure to that of PEDV reference strains. Phylogenetic analyses based on the whole genome revealed that CHGD-01 shared nucleotide sequence identities of 98.2-98.4% with two other Chinese isolates reported in the same year, thus constituting a new cluster. Amino acid sequence analysis based on individual virus genes indicated a close relationship between the spike protein gene of CHGD-01 and the field strain KNU0802 in Korea. Its ORF3 and nucleoprotein genes, however, were divergent from all other sequenced PEDV isolate clusters and therefore formed a new group, suggesting a new variant PEDV isolate in China. Further studies will be required to determine the immunogenicity and pathogenicity of this new variant.
Apoptin, a chicken anemia virus‐derived, p53‐independent, bcl‐2‐insenstive apoptotic protein with the ability to specifically induce apoptosis in tumor or transformed cells, is a promising tool for ...cancer gene therapy. In this study, pseudotype baculovirus, a recently developed alternative gene delivery system, was used as a vector to express Apoptin. The resultant recombinant baculovirus (BV‐Apoptin) efficiently expressed the Apoptin protein and induced apoptosis in HepG2 and H22 cells. Studies in vivo showed that intratumoral injection of BV‐Apoptin into a xenogeneic tumor (derived from H22 murine hepatoma cells in C57BL/6 mice) significantly suppressed tumor growth, and significantly prolonged the survival of tumor‐bearing mice compared to a control pseudotype baculovirus that expressed EGFP. Taken together, these results suggest that Apoptin, expressed from the pseudotype baculovirus vector, has the potential to become a therapeutic agent for the treatment of solid tumors.
Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) infection still remains today as the most significant health threat to swine and poses a challenge to current vaccination ...strategies. To develop a new generation of vaccine against PRRSV, a live attenuated pseudorabies virus (PRV) was used as vaccine vector to express the two major membrane-associated proteins (GP5 or M) of PRRSV in various forms. Four PRV recombinants, rPRV-GP5 (expressing native GP5), rPRV-GP5m (expressing GP5m, a modified GP5), rPRV-GP5-M (co-expressing GP5 and M proteins), rPRV-GP5m-M (co-expressing GP5m and M proteins) were generated. Mouse immunized with all these recombinants developed comparable PRV-specific humoral immune responses and provided complete protection against a lethal PRV challenge. However, the highest level of PRRSV-specific neutralizing antibodies and lymphocyte proliferative responses was observed in mice immunized with rPRV-GP5m-M. The immunogenicity and protective efficiency of rPRV-GP5m-M were further evaluated in the piglets. Compared to commercial PRRSV killed vaccine, detectable PRRSV-specific neutralizing antibody and higher lymphocyte proliferative responses could be developed in piglets immunized with rPRV-GP5m-M before virus challenge. Furthermore, more efficient protection against a PRRSV challenge was obtained in piglets immunized with rPRV-GP5m-M, as showed by the balanced body-temperature fluctuation, shorter-term viremia, lower proportion of virus load in nasal and oropharyngeal scrapings and tissues, and milder lung lesions. These data indicate that the recombinant rPRV-GP5m-M is a promising candidate bivalent vaccine against both PRV and PRRSV infection.
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