Yongchang Cao

The combination of deoxynivalenol and zearalenone at permitted feed concentrations causes serious physiological effects in young pigs

This study was to investigate the effects of the combination of deoxynivalenol (DON) and zearalenone (ZON) on pigs. Twenty-four weaning piglets were divided into a control group fed a diet free of mycotoxins and a toxin group fed a diet containing 1 mg/kg DON and 250 µg/kg ZON. The results showed that supplementation of DON and ZON in diets had extensive effects on pigs. More specifically, DON and ZON caused levels of total protein, albumin, and globulin in sera to decrease (p < 0.05) by 14.5%, 6.5% and 11.3%, respectively, and at the same time increased (p < 0.05) the serum enzyme activities of γ-glutamyltransferase, aspartate aminotransferase and alanine aminotransferase by 72.0%, 32.6% and 36.6%, respectively. In addition, DON and ZON decreased (p < 0.05) the level of anti-classical swine fever antibody titers by 14.8%. Real-time PCR showed that DON and ZON caused the mRNA expression levels of IFN-γ, TNF-α, IL-2, to decrease (p < 0.05) by 36.0%, 29.0% and 35.4%, respectively. Histopathological studies demonstrated that DON and ZON caused abnormalities in the liver, spleen, lymph nodes, uterus, and kidney. The concentrations of DON and ZON used in this study are in line with the published critical values permitted by BML. Our study clearly put the standard and adequacy of safety measures for these toxins into question. The authors suggest that with the increasing availability of cellular and molecular technologies, it is time to revisit the safety standards for toxins in feeds so as to make feeds safer, providing consumers with safer products.

Marek's disease virus type 1 microRNA miR-M3 suppresses cisplatin-induced apoptosis by targeting Smad2 of the transforming growth factor beta signal pathway.

ABSTRACT Viruses cause about 15% of the cancers that are still the leading causes of human mortality. The discovery of viral oncogenes has enhanced our understanding of viral oncogenesis. However, the underlying molecular mechanisms of virus-induced cancers are complex and require further investigation. The present study has attempted to investigate the effects of the microRNAs (miRNAs) encoded by Mareks disease virus 1 (MDV1), a chicken herpesvirus causing acute T-cell lymphomas and solid visceral tumors in chickens, on anti-cancer drug-induced apoptosis and identify the targets of the miRNAs. The results showed that of the total 14 miRNAs encoded by MDV1, MDV1-miR-M3 significantly promoted cell survival under treatment with cisplatin, a widely used chemotherapy drug. MDV1-miR-M3 suppressed cisplatin-induced apoptosis by directly downregulating expression at the protein but not the mRNA level of Smad2, a critical component in the transforming growth factor β signal pathway. Our data suggest that latent/oncogenic viruses may encode miRNAs to directly target cellular factors involved in antiviral processes including apoptosis, thus proactively creating a cellular environment beneficial to viral latency and oncogenesis. Furthermore, the knowledge of the apoptosis resistance conferred by viral miRNAs has great practical implications for improving the efficacy of chemotherapies for treating cancers, especially those induced by oncogenic viruses.

ISOLATION AND GENETIC ANALYSIS REVEALED NO PREDOMINANT NEW STRAINS OF AVIAN INFECTIOUS BRONCHITIS VIRUS CIRCULATING IN SOUTH CHINA DURING 2004-2008

Abstract Twenty-seven strains of avian infectious bronchitis virus (IBV) were isolated from dead or diseased chickens at different chicken farms in South China during 2004–2008, of which the S1 gene was sequenced. Phylogenetic analysis of the S1 gene sequences of the isolated 27 strains together with 29 strains published in Genbank revealed that all IBV strains except for one isolated and one published were clustered into six distinct genotypes I-VI. 26 isolated strains belong to genotypes I, II, and III, forming a big phylogenetic branch without new predominant strains, whereas all five vaccine strains belong to genotype V that is evolutionarily distant from genotypes I, II, and III. The study of the protease cleavage motif within the S1 protein found 12 different cleavage motifs, of which 3 motifs are shared by both isolated and published strains, 2 motifs unique to isolated strains, and 7 motifs unique to published strains, further bolstering the notion of no new predominant strains. Alignment analysis of the S1 amino acid sequences indicated that the amino acid substitutions, insertions, and deletions are polymorphic and diverse, showing no sign of predominant genetic changes among the isolated strains. Taken together, there was no predominant new strain circulating in South China during 2004–2008. Nonetheless, circulating IBV strains have been continuously evolving with genetic compositions distant from vaccine strains; this explains why there have been constant but infrequent outbreaks in commercial flocks in South China during 2004–2008. Furthermore, in order to safe guard against the sudden emergence of new predominant strains, continuing surveillance of IBV strains circulating in the field is of extreme importance.

Genomic analysis of two Chinese strains of porcine reproductive and respiratory syndrome viruses with different virulence

Two strains of porcine reproductive and respiratory syndrome virus (PRRSV) were isolated, designated GDQJ and GDBY1. Experimental inoculation showed that GDBY1, caused 100% morbidity and 67% mortality, while GDQJ, caused 100% morbidity but no death. Full-length genomes were sequenced. Homologic and phylogenetic analyses indicated that these two strains were closely related to Chinese highly pathogenic PRRSV strains. Surprisingly, identical 30 amino acids (aa) deletion in the NSP2-coding region, a presumed high virulence marker, was present in low virulent strain GDQJ. Further comprehensive analysis of GDQJ genome in comparison with Chinese highly pathogenic PRRSV strains revealed multiple genomic variations, distributing in 5′ UTR, NSP1b, NSP2, NSP3, NSP5, NSP7, NSP9, NSP10, GP5, and N regions. Data present in this article confirm that the 30 aa deletion in the NSP2-coding region alone is not a reliable genomic indicator for the high virulence of PRRSV strains emerged in China. The genomic variations of GDQJ strain provided the basis for further studies of virulence determinants for PRRSVs.

Rapid detection of porcine reproductive and respiratory syndrome virus by reverse transcription loop-mediated isothermal amplification assay

A rapid detection assay based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) has been developed for detecting porcine reproductive and respiratory syndrome virus (PRRSV). The RT-LAMP assay utilized a set of six primers to amplify the open reading frame 6 (ORF6) of the PRRSV. The amplified products were analyzed by agarose gel electrophoresis or visualized by colorimetric method. The results demonstrated that the RT-LAMP assay detected all 22 different PRRSV isolates, had no cross-reaction with four other swine viruses (i.e., PCV2, SIV, CSFV, and PEDV), and obtained a 91.3% sensitivity in 23 positive clinical samples in reference to the permissive cells-based virus isolation procedure. Therefore, the RT-LAMP assay provides a specific and sensitive means for detecting PRRSV in a simple, fast, and cost-effective manner. Furthermore, the RT-LAMP assay can be performed in less well-equipped laboratories as well as fields.

Profiling of cellular proteins in porcine reproductive and respiratory syndrome virus virions by proteomics analysis

BackgroundPorcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped virus, bearing severe economic consequences to the swine industry worldwide. Previous studies on enveloped viruses have shown that many incorporated cellular proteins associated with the virions membranes that might play important roles in viral infectivity. In this study, we sought to proteomically profile the cellular proteins incorporated into or associated with the virions of a highly virulent PRRSV strain GDBY1, and to provide foundation for further investigations on the roles of incorporated/associated cellular proteins on PRRSVs infectivity.ResultsIn our experiment, sixty one cellular proteins were identified in highly purified PRRSV virions by two-dimensional gel electrophoresis coupled with mass spectrometric approaches. The identified cellular proteins could be grouped into eight functional categories including cytoskeletal proteins, chaperones, macromolecular biosynthesis proteins, metabolism-associated proteins, calcium-dependent membrane-binding proteins and other functional proteins. Among the identified proteins, four have not yet been reported in other studied envelope viruses, namely, guanine nucleotide-binding proteins, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase, peroxiredoxin 1 and galectin-1 protein. The presence of five selected cellular proteins (i.e., β-actin, Tubulin, Annexin A2, heat shock protein Hsp27, and calcium binding proteins S100) in the highly purified PRRSV virions was validated by Western blot and immunogold labeling assays.ConclusionsTaken together, the present study has demonstrated the incorporation of cellular proteins in PRRSV virions, which provides valuable information for the further investigations for the effects of individual cellular proteins on the viral replication, assembly, and pathogenesis.

Effects of combinations of ochratoxin A and T-2 toxin on immune function of yellow-feathered broiler chickens

The study was to investigate the effects of combinations of ochratoxin A (OTA) and T-2 toxin on immune function of yellow-feathered broiler chickens. Three-hundred sixty 21-d-old broiler chickens were randomly assigned to 3 groups, each group consisting of 4 duplicates each with 30 chickens. The 3 groups were fed the following diets for 3 wk: C, basal diet (control, mycotoxin-free); L, basal diet + 0.25 mg/kg of OTA, 0.5 mg/kg of T-2 toxin; and H, basal diet + 0.5 mg/kg of OTA, 1 mg/kg of T-2 toxin. Body weight and feed consumption of chickens in the H group decreased significantly (P < 0.05) during the study, but their efficiency of feed utilization was not affected. The feeding of OTA-T-2 toxin diets decreased not only the relative weight of spleen, thymus, and bursa of Fabricius, but also serum concentrations of total protein, albumin, and globulin. Meanwhile, the feeding of OTA-T-2 toxin diets elevated the activities of serum gamma-glutamyltransferase, asparate aminotransferase, and alanine aminotransferase. The results of methyl thiazolyl tetrazolium reduction assay indicated that the mitogenic responses of peripheral blood lymphocytes were diminished significantly (P < 0.05 for L group; P < 0.01 for H group). Flow cytometry was employed to determine 3 indexes in peripheral blood lymphocyte of broilers, including CD4(+)/CD3(+), CD8(+)/CD3(+), and CD4(+)/CD8(+). Both toxin treatments significantly decreased (P < 0.01) CD4(+)/CD3(+) and CD4(+)/CD8(+) ratios. In summary, the combination of OTA and T-2 toxin impaired chick immune function even at combined concentrations as low as 0.25 mg/kg of OTA and 0.5 mg/kg of T-2 toxin.

Bioinformatics insight into the spike glycoprotein gene of field porcine epidemic diarrhea strains during 2011-2013 in Guangdong, China

Three strains of porcine epidemic diarrhea virus (PEDV) were isolated from dead or diseased pigs at different swine farms in Guangdong during 2011–2013, and their S genes were sequenced. In the same period, seven PEDV strains were also isolated in Guangdong by other laboratories. The spike sequences of 10 Guangdong isolates were compared with vaccine strains and reference pathogenic isolates using six bioinformatics tools. The results revealed that 10 Guangdong strains, excluding strain GDS03, had distinct characteristics in terms of primary structure, secondary structure, high-specificity N-glycosylation sites, potential phosphorylation sites, and palmitoylation sites. Phylogenetic analysis also confirmed these findings and revealed that all PEDV strains were clustered into three distinct groups. Ten Guangdong strains, not including GDS03, belong to Group 1, whereas four vaccine strains and GDS03 belong to Group 3, which is evolutionarily distant from Group 1. Alignment analysis of the neutralizing region amino acid sequences indicated that the amino acid substitutions of Y/D766S, T549S, and G594S that are present in the Guangdong strains, not including GDS03, were a sign of predominant genetic changes among the isolated strains. GDS03 is closely related to the 83P-5 vaccine strain, which suggests that it might represent re-isolation of the vaccine strain or vaccine variants. Taken together, these results indicate that there have been predominant new strains circulating in Guangdong from 2011 to 2013, and the circulating PEDV strains have a genetic composition that is distant from reference strains, especially the vaccine strains; however, the vaccinations might also provide some level of cross-protection, as there have been no changes in the neutralizing epitopes of SS2 and 2C10. This explains why there have been constant but infrequent outbreaks recently in comparison to late 2010 in which PEDV outbreaks were more frequent and severe. In addition, the USA-Colorado-2013 strain had the same amino acid substitutions in the neutralizing regions as the Guangdong strains except GDS03, which suggests that the information and strategies in this study may play role in PEDV variant research in other countries.

Molecular detection of Muscovy duck parvovirus by loop-mediated isothermal amplification assay

Muscovy duck parvovirus (MDPV) usually causes high morbidity and mortality in 1- to 3-wk-old Muscovy ducklings due to serious infections, which is an imminent threat to the commercial duck industry in China. The objectives of this study were to develop and evaluate a simple, rapid, and inexpensive loop-mediated isothermal amplification (LAMP) method for specific detection of MDPV and to compare it with the PCR method in rapidity, sensitivity, and accuracy. The novel LAMP assay used a set of 4 specific primers to recognize 6 distinct genomic sequences of capsid protein (VP3) from MDPV, which could be completed within 50 min at 63 degrees C in a simple water bath. The diagnostic results demonstrated that the LAMP assay detected all 7 preserved MDPV isolates, had no cross-reactivity with other duck pathogens (i.e., goose parvovirus, duck plague virus, H9N2 avian influenza virus, duck hepatitis type virus I, and Muscovy duck reovirus). The LAMP assay was at least 10-fold more sensitive than the routine PCR assay and obtained more sensitivity in 61 clinical samples. Therefore, the newly developed LAMP assay provides a specific and sensitive means for detecting MDPV and can be simply applied both in field conditions and in laboratory operations in a cost-effective manner with primary care facilities.

Identification and pathogenicity of a variant porcine epidemic diarrhea virus field strain with reduced virulence

BackgroundSince 2010, a variant Porcine epidemic diarrhea virus (PEDV), which causes an acute, highly contagious, and devastating viral enteric disease with a high mortality rate in suckling pigs, broke out in China and spread rapidly to neighboring countries, even to the North America. This virus gradually became the main subtype of PEDV worldwide. However, there were no reports of mild pathogenicity of a variant porcine epidemic diarrhea virus in China.FindingsIn 2013, a PEDV-positive sample from a sow with very mild clinical sign was used to inoculate in Vero cells to isolate the virus. This PEDV field strain, designated FL2013 strain, was successfully propagated and genetically characterized. The phylogenetic trees based upon either the complete genome or S gene showed that the FL2013 strain belongs to the genogroup G2b. The S gene of FL2013 has a 7-aa deletion (FEKVHVQ) in the C-terminus comparison with the other G2 PEDV sequences. Further comparative pathology study indicated that the FL2013 strain had reduced virulence to newborn piglets.ConclusionsA novel variant PEDV strain FL2013 with reduced virulence, as determined by the pathological study, was identified from east China. This strain is closely related to the genogroup- 2 PEDV strains prevalent in the U.S. and China currently, but had a short deletion at the 3′- end of the spike gene.