The Inactivated gE/TK Gene-Deleted Vaccine Against Pseudorabies Virus Type II Confers Effective Protection in Mice and Pigs.

The highly virulent and antigenic variant of Pseudorabies virus (PRV) that emerged from classical Bartha-K61-vaccinated pig herds has caused substantial economic losses to the swine industry in China since 2011. A safe and more effective vaccine is most desirable. In this study, a gE/TK gene-deficient PRV, namely, HD/c, was constructed based on a PRV type II DX strain isolated from a commercial vaccine-immunized farm and the HD/c-based inactivated vaccine was formulated and evaluated for its safety, immunogenicity, and protective efficacy in mice and piglets. The resulting PRV HD/c strain has a similar growth curve to the parental DX strain. After vaccination, the inactivated HD/c vaccine did not cause any visible gross pathological or histopathological changes in the tissues of mice and piglets and provided rapid and potent protection against the challenge of the classical and variant PRVs at day 21 post-vaccination in mice. A single immunization of 108.5TCID50 inactivated PRV HD/c strain-elicited robust immunity with high titer of neutralizing antibody and provided complete protection from the lethal challenge of PRV DX strain in piglets. These results indicated that the inactivated PRV HD/c vaccine with the deletion of gE/TK genes was a safe and effective PRV vaccine candidate for the control of PRV.

Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4, Hunan province, China.

In pigs, three circovirus species within the genus Circovirus have been identified so far, including the non-pathogenic Porcine circovirus 1 (PCV1), the pathogenic Porcine circovirus 2 (PCV2) and the recently identified Porcine circovirus 3 (PCV3). In April 2019, a new circovirus with a distinct relationship to other circoviruses was identified in several pigs with severe clinical disease in Hunan province, China. The size of the viral genome, tentatively designated as porcine circovirus type 4 (PCV4), is 1,770 nucleotides (nt). PCV4 shows the highest genomic identity to mink circovirus (66.9%) and has identities of 43.2%-51.5% to the other PCV genomes. Two major genes, a replicase (Rep) gene spanning 891 nt and a capsid (Cap) gene spanning 687 nt, were predicted. Furthermore, a TaqMan® real-time polymerase chain reaction (PCR) targeting the replicase gene was developed to investigate the prevalence of PCV4 in 187 clinical samples from Hunan province, China. The results revealed an overall PCV4 prevalence of 12.8%, with the highest positive rates in nasal swabs (28.5%, 6/21) followed by serum samples (13.4%, 11/82). The clinical significance and pathogenesis of this virus needs further investigation.

Pathogenicity of an FAdV-4 isolate to chickens and its genomic analysis.

Fowl adenovirus serotype 4 (FAdV-4) strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province, China. The isolate was cultured in primary chicken embryo kidney cells. A study of pathogenicity indicated that SD1511 readily infected 7-35-d-old chickens by intramuscular injection and intranasal and oral routes, causing 50%-100% mortality. The 35-d-old chickens suffered more severe infection than 7- and 21-d-old chickens with mortality highest in the intramuscular injection group. The serum from surviving chickens showed potent viral neutralizing capability. The complete genome of SD1511 was sequenced and analyzed. The strain was found to belong to the FAdV-4 cluster with more than 99% identity with the virulent FAdV-4 strains isolated in China in recent years except for some distinct variations, including deletions of open reading frame 27 (ORF27), ORF48, and part of ORF19. Our findings suggest that SD1511 might be used as a prototype strain for the study of pathogenesis and vaccine development.

A New Segmented Virus Associated with Human Febrile Illness in China.

BACKGROUND: In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected. METHODS: We obtained a blood specimen from the index patient and attempted to isolate and identify a causative pathogen, using genome sequence analysis and electron microscopy. We also initiated a heightened surveillance program in the same hospital to screen for other patients who presented with fever, headache, and a history of tick bites. We used reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cell-culture assays to detect the pathogen and immunofluorescence and neutralization assays to determine the levels of virus-specific antibodies in serum specimens from the patients. RESULTS: We found that the index patient was infected with a previously unknown segmented RNA virus, which we designated Alongshan virus (ALSV) and which belongs to the jingmenvirus group of the family Flaviviridae. ALSV infection was confirmed by RT-PCR assay in 86 patients from Inner Mongolia and Heilongjiang who presented with fever, headache, and a history of tick bites. Serologic assays showed that seroconversion had occurred in all 19 patients for whom specimens were available from the acute phase and the convalescent phase of the illness. CONCLUSIONS: A newly discovered segmented virus was found to be associated with a febrile illness in northeastern China. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China.).

Mitochondrial dynamics controls anti-tumour innate immunity by regulating CHIP-IRF1 axis stability.

Macrophages, dendritic cells and other innate immune cells are involved in inflammation and host defense against infection. Metabolic shifts in mitochondrial dynamics may be involved in Toll-like receptor agonist-mediated inflammatory responses and immune cell polarization. However, whether the mitochondrial morphology in myeloid immune cells affects anti-tumor immunity is unclear. Here we show that FAM73b, a mitochondrial outer membrane protein, has a pivotal function in Toll-like receptor-regulated mitochondrial morphology switching from fusion to fission. Switching to mitochondrial fission via ablation of Fam73b (also known as Miga2) promotes IL-12 production. In tumor-associated macrophages, this switch results in T-cell activation and enhances anti-tumor immunity. We also show that the mitochondrial morphology affects Parkin expression and its recruitment to mitochondria. Parkin controls the stability of the downstream CHIP-IRF1 axis through proteolysis. Our findings identify mechanisms associated with mitochondrial dynamics that control anti-tumor immune responses and that are potential targets for cancer immunotherapy.

Rabies Virus Infection Induces Microtubule Depolymerization to Facilitate Viral RNA Synthesis by Upregulating HDAC6.

Rabies virus (RABV) is the cause of rabies, and is associated with severe neurological symptoms, high mortality rate, and a serious threat to human health. Although cellular tubulin has recently been identified to be incorporated into RABV particles, the effects of RABV infection on the microtubule cytoskeleton remain poorly understood. In this study, we show that RABV infection induces microtubule depolymerization as observed by confocal microscopy, which is closely associated with the formation of the filamentous network of the RABV M protein. Depolymerization of microtubules significantly increases viral RNA synthesis, while the polymerization of microtubules notably inhibits viral RNA synthesis and prevents the viral M protein from inducing the formation of the filamentous network. Furthermore, the histone deacetylase 6 (HDAC6) expression level progressively increases during RABV infection, and the inhibition of HDAC6 deacetylase activity significantly decreases viral RNA synthesis. In addition, the expression of viral M protein alone was found to significantly upregulate HDAC6 expression, leading to a substantial reduction in its substrate, acetylated α-tubulin, eventually resulting in microtubule depolymerization. These results demonstrate that HDAC6 plays a positive role in viral transcription and replication by inducing microtubule depolymerization during RABV infection.

Genetic characterization of H9N2 avian influenza virus in plateau pikas in the Qinghai Lake region of China.

Qinghai Lake is a major migratory-bird breeding site that has experienced several highly pathogenic avian influenza virus (AIV) epizootics. Plateau pikas (Ochotona curzoniae) have previously been implicated in the ecology of avian influenza virus in this region. We first isolated an H9N2 AIV (A/Pika/Menyuan/01/2008) from plateau pikas between November 2008 and October 2009. Sequence analysis showed that the A/Pika/Menyuan/01/2008 AIV was closely related to the H9N2 AIV strain (A/Turkey/Wisconsin/ 1/1966). Our findings suggested that plateau pikas may contribute to AIV epidemiology in the Qinghai Lake region.

Rabies virus matrix protein induces apoptosis by targeting mitochondria.

Apoptosis, as an innate antiviral defense, not only functions to limit viral replication by eliminating infected cells, but also contribute to viral dissemination, particularly at the late stages of infection. A highly neurotropic CVS strain of rabies virus induces apoptosis both in vitro and in vivo. However, the detailed mechanism of CVS-mediated neuronal apoptosis is not entirely clear. Here, we show that CVS induces apoptosis through mitochondrial pathway by dissipating mitochondrial membrane potential, release of cytochrome c and AIF. CVS blocks Bax activation at the early stages of infection; while M protein partially targets mitochondria and induces mitochondrial apoptosis at the late stages of infection. The α-helix structure spanning 67-79 amino acids of M protein is essential for mitochondrial targeting and induction of apoptosis. These results suggest that CVS functions on mitochondria to regulate apoptosis at different stages of infection, so as to for viral replication and dissemination.

Rabies virus inactivates cofilin to facilitate viral budding and release.

Cytoplasmic actin and actin-associated proteins have been identified in RABV particles. Although actin is involved in RABV entry into cells, the specific role of actin in RABV budding and release remains unknown. Our study found that RABV M protein-mediated virion budding depends on intact actin filaments. Confocal microscopy demonstrated a block to virions budding, with a number of M protein-mediated budding vesicles detained in the cell cytoplasm. Furthermore, RABV infection resulted in inactivation of cofilin and upregulation of phosphorylated cofilin. Knockdown of cofilin reduced RABV release. These results for the first time indicate that RABV infection resulted in upregulation of phosphorylated cofilin to facililtate actin polymerization for virus budding.

Evidence of recombinant strains of porcine epidemic diarrhea virus, United States, 2013.

To investigate the evolutionary process by which porcine epidemic diarrhea virus (PEDV) in the United States hypothetically descended from strains in China, we analyzed PEDV-positive samples collected in China during January 2012-July 2013. Recombination in 2 strain sublineages was likely associated with identification of PEDV in the United States in 2013.

Down-regulation of NOD1 in neutrophils of periparturient dairy cows.

Increased susceptibility of periparturient dairy cows to infectious diseases has been linked to neutrophil dysfunction. Cytoplasmic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) mediate numerous aspects of innate immunity in humans and experimental animals. The authors hypothesized that neutrophils in periparturient cows have reduced NLR expression, resulting in declined immune responses to NLR activation. In this study, blood neutrophils from periparturient and mid-lactating cows were isolated and assessed for mRNA and protein expression of NOD1 and NOD2. Neutrophil phagocytosis and oxidative burst activity following NLR agonist-induced activation were also investigated. Periparturient cows demonstrated weak-to-absent NOD1 mRNA expression along with a significant reduction in NOD1 protein in their neutrophils. However, no difference in NOD2 protein expression was observed between groups although NOD2 mRNA abundance in periparturient cows was significantly reduced. Phagocytosis and oxidative burst activity measured by production of reactive oxygen species (ROS) were diminished in periparturient cows. NOD1 activation enhanced phagocytosis and ROS production in both isolated cell populations, regardless of the lactation stage. Nevertheless, NOD1-activated neutrophils showed reduced phagocytosis and ROS generation in periparturient cows with respect to mid-lactating subjects. Collectively, our findings suggest that NOD1 down-regulation in neutrophils may be a mechanism involved in periparturient neutrophil dysregulation.

Development of an immunosensor assay for detection of haptoglobin in mastitic milk.

BACKGROUND: Detection of subclinical mastitis in dairy cows is important, as it represents a major economic challenge for the dairy industry worldwide owing to propagation of mastitis-causing pathogens and to long-term reduction in milk yield and quality. Haptoglobin (Hp) is one of the most sensitive acute phase proteins in milk during udder inflammation and as an indicator of mastitis. OBJECTIVES: The aim of this study was to develop a rapid and sensitive immunosensor assay for measuring Hp concentration in mastitic milk. METHODS: The immunosensor was constructed by immobilizing anti-bovine Hp antibody on a gold electrode through gold nanoparticles fabricated on self-assembled L-cysteine layers. The immunosensor assay was used to measure Hp concentration in 20 milk samples positive for bacteria with a somatic cell count > 5 × 10(5) cells/mL from cows without clinical signs of mastitis. Results were compared with those obtained using a commercial ELISA kit. RESULTS: Reproducibility of Hp measurement and stability after storage for 20 days were good for the immunosensor assay. Measurement of Hp was linear over a range of 15-100 mg/L, with a limit of detection of 0.63 mg/L. Agreement between results obtained with the immunosensor and ELISA methods was satisfactory as analyzed using the Wilcoxon signed-rank test (Z = -1.739, P = .073). CONCLUSION: An immunosensor assay for measuring Hp in milk provided rapid results and was easy to perform, facilitating its potential use in the field for the diagnosis of subclinical mastitis once a cutoff value for Hp concentration is established.

Analysis of putative ORF3 gene within porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic syndrome in pigs, whereas the ubiquitous related porcine circovirus type 1 (PCV1) is nonpathogenic. Corroborating an earlier observation in PCV2, Rep and Rep' proteins encoded by ORF1 are essential for the initiation of PCV2 replication. Cap protein encoded by ORF2 has a potential causative role in the initiation of PCV2 replication and contains a type-specific epitope. The putative ORF3 of PCV2 oriented in the opposite direction within ORF1 is unknown. In this study, ORF3-encoding protein of PCV2 was expressed in vitro as a fusion protein (GST-ORF3 protein), and monoclonal antibodies (MAbs) to the PCV2-ORF3-encoding protein were generated and biologically characterized. The mRNA transcript of ORF3 was characterized during a productive infection in PK-15 cells, and the PCV2 infectious DNA clone lacking ORF3 was constructed. GST-ORF3 protein, with an approximate molecular weight of 37.7 kDa, was obtained from the Escherichia coli transformed with the recombinant vector pGEX-4T-1-F3 after codon optimization of ORF3 DNA sequence. Four MAbs reacted strongly to the ORF3-encoding protein expressed in PK-15 cells in immunohistochemical staining. The mRNA transcript of ORF3 was confirmed in RT-PCR, Northern blot, and sequencing analyses. The progeny PCV2 virions were not revealed in the PK-15 cells transfected by the PCV2 infectious DNA clone without ORF3. These results demonstrate that the ORF3 of PCV2 can be transcribed and expressed and that ORF3-encoding protein plays a pivotal role in viral replication.

Involvement of matrix metalloproteinase-2 in medial hypertrophy of pulmonary arterioles in broiler chickens with pulmonary arterial hypertension.

Medial hypertrophy of pulmonary arterioles during pulmonary arterial hypertension (PAH) in humans is associated with enhanced proliferation of smooth muscle cells (SMCs). Elevated matrix metalloproteinase (MMP)-2 has been found in pulmonary artery SMCs (PA-SMCs) in humans with idiopathic PAH, leading to the hypothesis that MMP-2 contributes to the proliferation and migration of vascular SMCs in the pathogenesis of PAH. Rapidly growing meat-type (broiler) chickens provide a model of spontaneous PAH. The present study was conducted to determine whether MMP-2 is involved in the medial hypertrophy of pulmonary arterioles in this model. Cultured PA-SMCs from normal birds were used to evaluate the effect of MMPs on cell proliferation. Gelatin zymography showed that endothelin (ET)-1-induced proliferation of PA-SMCs was concomitant with increased pro- and active MMP-2 production. Reverse transcription PCR demonstrated upregulation of MMP-2 mRNA. However, PA-SMC proliferation was inhibited by the MMP inhibitors doxycycline and cis-9-octadecenoyl-N-hydroxylamide. In vivo experiments revealed a significant increase of MMP-2 expression in hypertrophied pulmonary arterioles of PAH broiler chickens, which was positively correlated with wall thickness and medial hypertrophy. MMP-2 may contribute to medial hypertrophy in pulmonary arterioles during PAH in broiler chickens by enhancing the proliferation of vascular SMCs.

Genomic characterization of a proventriculitis-associated infectious bronchitis coronavirus.

Transmissible proventriculitis associated with infectious bronchitis virus (IBV) was at first seen in eastern China in mid-1995, and is now endemic in China. Herein, the complete genome sequence of a proventiculitis-associated infectious bronchitis coronavirus (ZJ971) was sequenced and analyzed. Compared with the genome of the vaccine strain H120, ZJ971 had 54 nucleotide substitutions and a deletion in the 3'-UTR. The substitutions were in the regions of nsp2-nsp5, nsp7, nsp12, nsp13, nsp15, S and N genes, and the untranslating region. The results indicated that ZJ971 could be a variant of IBV strain H120.

Interference of porcine circovirus type 2 ORF2 immunogenicity by ORF1 and ORF3 mixed DNA immunizations in mice.

Little is known about the influences of other porcine circovirus type 2 (PCV2) proteins on the immunogenicity of Cap protein. Here we constructed plasmids expressing the ORF1 (pORF1) and ORF3 (pORF3) of PCV2, and mixed either of them with the plasmid expressing ORF2 (pORF2) as combined DNA vaccines, to compare their immunogenicity and protective efficacy. Our data revealed that pORF1 reduced the Cap-specific CD8(+)cell frequency, and both pORF1 and pORF3 attenuated the Cap-specific Th1 and post-challenge-recall VN antibody responses induced by the pORF2 plasmid, despite successful induction of Rep and ORF3 antibodies by pORF1 and pORF3, respectively. Subsequently, protocols with pORF1 or pORF3 showed significantly decreased protective efficacy compared to pORF2 alone. Overall, our data suggested that the ORF1- and ORF3-encoded Rep and ORF3 proteins may interfere with the cellular, humoral and protective immunity of the ORF2-encoded Cap protein in vivo.

[Expressions of CXCL13, CD10 and bcl-6 in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified].

OBJECTIVE: To study the value of immunomarkers CXCL13, CD10, bcl-6 in pathologic diagnosis of angioimmunoblastic T-cell lymphoma (AITL). METHODS: One hundred and fifteen cases of AITL, 30 cases of peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) and 30 cases of reactive lymph nodes with paracortical hyperplasia (RH) encountered during the period from January, 1990 to January, 2008 were retrieved from the archival files of the Department of Pathology, West China Hospital of Sichuan University, China. The morphologic features were reviewed and compared. Immunohistochemical study was performed by SP method for CXCL13, CD10, bcl-6, CD21, CD3epsilon, CD3, CD45RO, CD20 and Ki-67. TCR-gamma gene rearrangement study was also carried out. RESULTS: Regressed follicles were evident in 7.8% (9/115) of AITL cases, 6.7% (2/30) of PTCL, NOS cases and 83.3% (25/30) of RH cases, respectively. A marked increase of number of arborizing venules was shown in 98.3% (113/115) of AITL cases, 63.3% (19/30) of PTCL, NOS cases and 76.7% (23/30) of RH cases, respectively. In lymph nodes with paracortical hyperplasia, the expression of CXCL13, CD10 and bcl-6 were restricted to the germinal centers. In AITL, 96.5% (111/115) of cases showed CXCL13 expression, in contrast to 26.7% (8/30) of PTCL, NOS. Expression of CD10 and bcl-6 were found in the neoplastic cells in 50.4% (58/115) and 78.3% (90/115) of AITL, and 3.3% (1/30) and 3.3% (1/30) of PTCL, NOS, respectively. Irregular meshworks of CD21-positive follicular dendritic cells were found in all the AITL cases. Clonal TCR-gamma rearrangement was detected in 83% (83/100) of the AITL cases. CONCLUSIONS: AITL is a type of lymphoma originated from the follicular helper T cells. Detailed morphologic assessment and use of immunohistochemical markers are essential for accurate diagnosis.

Manganese superoxide dismutase expressed in silkworm larvae, Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo.

Natural killer cell (NK) is known as a major immune system in body through mediating cell death via several possible pathways, and one of three subpopulations of lymphocytes functioning as scavenger of tumor, virus infected cells etc. Our present results found that the SOD-contained silkworm larvae powder caused an enhancement of the effect on NK cell cytotoxicity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of S180 tumor modeled mice treated with silkworm powder including SOD were enhanced significantly ranging from 30% to 48%, respectively, compare to a distilled water feeding control and silkworm powder without SOD. Meanwhile, the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control both in T cells or B cells. The average tumor weight of S180 modeled mice treated with doses of SOD-contained silkworm powder was lighter than that of water control showing the tumor inhibition rates (IR) reached to 22.51% to 37%, respectively. In conclusion, these findings demonstrate that administration of silkworm larvae powder containing SOD results in activation of NK cells and immune T-cell and B-cell, suggesting the silkworm larvae powder containing SOD play a positive role in tumor inhibition.

Fine mapping of antigenic epitopes on capsid proteins of porcine circovirus, and antigenic phenotype of porcine circovirus type 2.

Type 2 porcine circovirus (PCV2) is associated with post-weaning multisystemic wasting syndrome in pigs. In this study, three monoclonal antibodies (mAbs) against the capsid protein (Cap) of PCV2, eight mAbs to Cap of type 1 porcine circovirus (PCV1) and five mAbs specific for Cap of both PCV1 and PCV2, were generated and used to finely map the antigenic sites of PCV1 and PCV2, and to identify the antigenic phenotype of PCV2 with different length of genome. Five linear B-cell epitopes, including the residues 231-233 and 195-202 specific for PCV2, residues 92-103 specific for PCV1, and residues 156-162 and 175-192 shared between PCV1 and PCV2, were finely defined with synthetic peptides, and the critical residue in epitope 231-233 and 156-162 was located at residues 233 ((233)Proline) and 156 ((156)Tyrosine), respectively. The conformational epitopes recognized by mAbs with neutralizing activity against both PCV1 and PCV2 were detected in transfected PK-15 and the residues 231-233 also participated in the formation of conformational epitopes. Analysis of antigenic diversity on these epitopes exhibited three antigenic phenotypes of PCV2, (1766)PCV2, (1767)PCV2 and (1768)PCV2 using mAbs. The results from this study first demonstrated the different antigenic phenotype between PCV2 isolates.

Protective immunity against porcine circovirus 2 by vaccination with ORF2-based DNA and subunit vaccines in mice.

The protective immune response against porcine circovirus 2 (PCV2) infection in mice was characterized using flow cytometric analysis (FCM), assays of antibody (of different IgG isotypes) and viraemia, and histopathological examination. An open reading frame 2 plasmid (pORF2) and the capsid protein (Cap) of PCV2 were used as DNA and subunit vaccines, respectively. In FCM analysis, although pORF2 and Cap alone showed comparable efficacy in eliciting lymphoproliferative responses and Cap-specific CD4(+) T cells, pORF2 was superior to the Cap protein in triggering CD8(+) T cells. A virus neutralization assay showed that pORF2 evoked stronger recall virus-neutralizing (VN) antibody responses than the Cap protein on PCV2 challenge. Correspondingly, VN antibody kinetics coincided with those of Cap-specific IgG2a, but not with the kinetics of IgG and IgG1. Following virus challenge, real-time PCR and histopathological analysis confirmed that only low viral DNA loads and mild microscopic lesions appeared in pORF2-immunized mice. These findings indicate that CD8(+) T cells and VN antibody responses correlating mainly with Cap-specific IgG2a play crucial roles in protecting against PCV2 infection, and that the protective immunity induced by the pORF2 plasmid is superior to that induced by the PCV2 Cap protein.