Avian coronavirus infectious bronchitis virus Beaudette strain NSP9 interacts with STAT1 and inhibits its phosphorylation to facilitate viral replication.

Avian coronavirus, known as infectious bronchitis virus (IBV), is the causative agent of infectious bronchitis (IB). Viral nonstructural proteins play important roles in viral replication and immune modulation. IBV NSP9 is a component of the RNA replication complex for viral replication. In this study, we uncovered a function of NSP9 in immune regulation. First, the host proteins that interacted with NSP9 were screened. The immune-related protein signal transducer and activator of transcription 1 (STAT1) was identified and the interaction between NSP9 and STAT1 was further confirmed. Furthermore, IBV replication was inhibited in STAT1-overexpressing cells but inversely affected in STAT1 knock-down cells. Importantly, NSP9 inhibited STAT1 phosphorylation. Finally, the expression of JAK/STAT pathway downstream genes IRF7 and ISG20 was significantly decreased in NSP9-overexpressing cells. These results showed the important role of IBV NSP9 in immunosuppression.

A new H9 influenza virus mRNA vaccine elicits robust protective immunity against infection.

Avian influenza virus (AIV) poses a great threat to the poultry industry and public health. However commercial vaccines only provide limited immunity due to rapid virus mutation and rearrangement. Here, we developed an mRNA-lipid nanoparticle (mRNA-LNP) vaccine expressing AIV immunogenic protein hemagglutinin (HA) and also assessed its safety and immune-protection efficacy in vivo. Specifically, its safety was tested by inoculation of SPF chicken embryos and chicks, and there showed no clinical manifestations and pathological changes in both. As for the immune efficacy, the antibody titers, IFN-γ production levels, and viral loads in various organs were analyzed. The results showed that chickens in the mRNA-LNP-inoculated groups produced higher specific antibody titers compared with that in the control group by hemagglutination inhibition (HI) test. Meanwhile, the ELISpot assay demonstrated that the expression of IFN-γ was markedly induced in the mRNA-LNP group, and the viral loads in multiple organs were decreased. In addition, HE shows no obvious pathomorphological changes in the lungs of the mRNA-LNP-inoculated group. While, there was severe inflammatory cell infiltration in the DMEM-treated group instead. Taken together, the vaccine prepared in this study was safe and could trigger potent cellular and humoral immune response to defend against virus infection.

Preparation and wide band emission characteristics of Eu2+/Eu3+ co-doped Ba3P4O13 phosphors.

A series of phosphors Ba3P4O13:xEu2+/3+ (x = 0-0.1) were synthesized in a CO reduction atmosphere at a relatively low temperature (1113 K) by the solid-phase method. Crystallization and optical properties were investigated by using powder X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and fluorescence spectrophotometry (PL/PLE), respectively. Under the excitation of 394 nm, the emission spectrum of the phosphor presents a broad spectrum band of Eu2+ and characteristic peaks of Eu3+ in the range of 400-750 nm, indicating the coexistence of Eu2+ and Eu3+ in the system. The occupation of Eu2+ and Eu3+ in the Ba3P4O13 matrix position was discussed by Gaussian fitting and PL spectra. The existence of energy transfer between Eu2+ → Eu3+ in the system can be found through PL spectra under excitation of 251 nm. The emission color of the phosphor can be adjusted by changing the Eu ion doping concentration, resulting in the CIE color coordinates changing from (0.213, 0.215) blue to (0.249, 0.261) light bluish white. The results show that Ba3P4O13:Eu2+/3+ phosphors can be used as a potential single-doped single-host white light-emitting material.

Proteomic analyses identify intracellular targets for Japanese encephalitis virus nonstructural protein 1 (NS1).

Japanese encephalitis is a zoonotic disease caused by Japanese encephalitis virus (JEV). JEV nonstructural protein 1 (NS1) is involved in many crucial biological events during viral infection and immune suppression. To investigate the role of JEV NS1 in virus-infected cells, the molecules with which it interacts intracellularly were screened with a pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The interaction between heterogeneous nuclear ribonucleoprotein K (hnRNP K), vimentin and NS1 were verified with coimmunoprecipitation and confocal assays. Our results show that JEV NS1 interacts with vimentin, hnRNP K and colocalizes with cellular vimentin and hnRNP K. Furthermore, our results demonstrate that the expression of vimentin and hnRNP K were up-regulated in both NS1-transfected and JEV-infected cells. Knocking down vimentin and hnRNP K reduced viral replication while conversely, over-expression of vimentin and hnRNP K improved viral replication, suggesting an important role for this protein in the viral life cycle. Also, We found that vimentin also interacted with hnRNP K after overexpression of NS1 or JEV infection. These findings provide insight into the molecular mechanism of JEV replication and highlight the key role the NS1 in JEV infection.

Infectious bronchitis virus inhibits activation of the TLR7 pathway, but not the TLR3 pathway.

Various strains of infectious bronchitis virus (IBV) cause different forms of infectious bronchitis with different clinical signs. Here, primary chicken embryo kidney (CEK) cells and specific-pathogen-free (SPF) chickens were infected with three pathogenic IBV strains, and it was observed that the TLR7-MYD88 pathway was inhibited but the TLR3-TIRF pathway was activated. After treatment with poly(I:C)-LMW, poly (I:C)-LMW/LyoVec, and Imiquimod, the replication of IBV was significantly suppressed after 24 h. However, treatment with TLR3 pathway inhibitors such as Pepinh-TRIF, celastrol, chloroquine, and BX795 resulted in increased replication of IBV after 36 h. These results also showed that chloroquine and celastrol were most effective inhibitors of the antiviral response at 48 hpi.

Isolation and recombinant analysis of variants of porcine epidemic diarrhea virus strains from Beijing, China.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious virus infecting pigs with high morbidity, especially for newborn piglets. Several PEDV strains were isolated from the intestinal tracts of diarrheic piglets from the Beijing area, China. Sequencing of the whole-genome of the PEDV isolates (GenBank numbers MG546687-MG546690) yielded sequences of 28033-28038 nt. The phylogenetic tree revealed that these strains from the Beijing area belonged to group II, while the vaccine strain, CV777, belonged to group I. We also determined the genetic correlation between these strains and CV777 strain. However, it showed that these strains in the Beijing area had unique mutations. The sequence identity of PEDV strains showed that these strains are most similar to these strains LZW, CH/JX-1/2013, USAIllinois972013, USAKansas1252014, CH/GDZQ/2014, SHQPYM2013, AJ1102, CHZMDZY11, KoreaK14JB01, and CHYJ130330, respectively. The possible recombination events indicate that PEDV in this studies were possibly recombinant strain formed by parent strains USAIllinois972013, KoreaK14JB01, CHYJ130330, and CHZMDZY11. These PEDV strains has been genetic recombination and mutations. The variant strains characterized in this study help to the evolutionary analysis of PEDV.

Polymorphisms of chicken TLR3 and 7 in different breeds.

Toll-like receptors (TLRs) mediate immune responses via the recognition of pathogen-associated molecular patterns (PAMPs), thus playing important roles in host defense. Among the chicken (Ch) TLR family, ChTLR3 and 7 have been shown to recognize viral RNA. In our earlier studies, we have reported polymorphisms of TLR1, 2, 4, 5, 15 and 21. In the present study, we amplified TLR3 and 7 genes from different chicken breeds and analyzed their sequences. We identified 7 amino acid polymorphism sites in ChTLR3 with 6 outer part sites and 1 inner part site, and 4 amino acid polymorphism sites in ChTLR7 with 3 outer part sites and 1 inner part site. These results demonstrate that ChTLR genes are polymorphic among different chicken breeds, suggesting a varied resistance across numerous chicken breeds. This information might help improve chicken health by breeding and vaccination.

Immunization with recombinant 3-1E protein in AbISCO®-300 adjuvant induced protective immunity against Eimeria acervulina infection in chickens.

Immunostimulating complexes (ISCOMs), a kind of novel antigen presenting system, could enhance immune protection by antigen presentation. AbISCO®-300 comprising purified saponin, cholesterol and phosphatidyl choline is an effective ISCOM adjuvant. To evaluate the immune protection of recombinant 3-1E protein against Eimeria acervulina infection, chickens were immunized with recombinant 3-1E protein in combination with AbISCO®-300 or recombinant 3-1E protein alone in this study. The protective immunity was assessed with body weight gain, fecal oocyst output, detection of intestinal IgA positive cells and percentages of CD3(+), CD4(+) or CD8(+) intestinal intraepithelial lymphocytes (IELs). Chickens vaccinated with different doses of recombinant 3-1E protein plus AbISCO®-300 showed higher percentages of CD3(+), CD4(+), and CD8(+) intestinal IELs, increased positive expression rate of intestinal IgA, increased body weight gains and decreased oocyst shedding compared with recombinant 3-1E protein-only vaccinated groups. The results showed that immunization with various doses of the recombinant 3-1E protein in AbISCO®-300 adjuvant enhanced immune protection against avian coccidiosis.

Differential expression of the Toll-like receptor pathway and related genes of chicken bursa after experimental infection with infectious bursa disease virus.

Infectious bursa disease virus causes an acute infection in bursal B cells. The Toll-like receptor (TLR) signaling pathway plays a key role in innate immunity during virus infection. In this study, an Agilent microarray was used to investigate different transcriptional profiles of the TLR pathway and related genes of chicken bursa at 48 h after infection with IBDV, compared with simulated infection. Expression of >58 genes changed significantly. Forty-six genes associated with chicken bursa proinflammatory effects, chemotactic effects, and T-cell stimulation were upregulated, which meant enhancement of these features. Twelve genes that are related to proliferation and differentiation of bursal cells were downregulated, implying suppression of these features. These results revealed that genes of the TLR pathway play an important role in the pathogenicity of IBDV infection. The findings are helpful for understanding the molecular basis of viral pathogenesis and the underlying mechanism of the host antiviral response.

Identification of a Toll-like receptor 1 in guinea fowl (Agelastes niger).

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, thus playing important roles in host defense. This study determined the first sequence of a TLR1 type 1 in the guinea fowl (GFTLR1). The open reading frame of GFTLR1 type 1 contains 2,115 nucleotides and encodes 705 amino acids. Amino acid analysis indicated that GFTLR1 type 1 shares 92.3 % homology with the green jungle fowl, 92.1 % with the chicken, 90.4 % with the turkey, and 84.4 % with Cooper's hawk. Genetic patterns were identified within the TLR1 type 1 of the chicken and the guinea fowl. GFTLR1 type 1 was found to have 92 polymorphic amino acid sites, of which 16 were in the leucine-rich repeat (LRR) domain, 3 in a C-terminal LRR domain, and 6 in a Toll/interleukin-1 receptor domain. The data showed that avian TLR1 type 1 genes are under purifying selection and highly conserved, because dN/dS was less than 1.

Different genetic patterns in avian Toll-like receptor (TLR)5 genes.

Toll-like receptors (TLRs) mediate immune response via recognition of pathogen-associated molecular patterns (PAMPs), thus play important roles in host defense. Polymorphisms of TLR5 may affect their recognition of bacterial flagellin, leading to varied host resistance to pathogenic infections. Here, we cloned TLR5 genes from Common Pheasant, Guinea fowl and 9 Chicken breeds and analyzed their sequences. The open reading frames of TLR5 were sequenced. Amino acid analysis indicated that TLR5 from Chicken breeds shared 99.4-99.9% homology. The amino acid homology of TLR5 ranged from 92.1 to 92.5% between Chickens and Guinea fowl, 95.7-96.1% between Chickens and Turkey, 94.3-94.7% between Chickens and Common Pheasant, and 79.9-80.1% between Chickens and Zebra-finch. Different genetic patterns were determined among Chickens, Common Pheasant, Guinea fowl, Turkey and Zebra-finch. It was found that there were 92 amino acid polymorphic sites, among which 5 sites in chicken TLR5, 63 sites in Guinea fowl TLR5 and 44 sites in Common Pheasant TLR5. Our data indicate that the positive Darwinian selection occurred in avian TLR5 genes since frequency of non-synonymous (d ( N )) > frequency of synonymous (d ( S )). These results also demonstrate that avian TLR5 genes are polymorphic among avian breeds, suggesting a varied resistance among breeds of avian. This information might be of help to improve the health of avian by breeding and vaccination.