African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate.

Identification of monoclonal antibody targeting epitope on p72 trimeric spike of African swine fever virus.

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. ASFV p72 protein is a major capsid protein that presents as trimer in the virion. Epitopes on the surface of p72 trimer are considered as protective antigens. In this study, recombinant p72 protein and p72-baculovirus were constructed and obtained. Three monoclonal antibodies (mAbs) specific to ASFV p72 protein, designated as 1A3, 2B5 and 4A5, were generated. Among them, 4A5 showed strong reactivity with ASFV infected cells. Subsequently, the epitope recognized by 4A5 was mapped and identified using a series of overlapping peptides generated from p72 protein. IFA and western blot analyses showed that 4A5 recognized the linear epitope of p72 monomer located between amino acids 245-285 and recognized the conformational epitope located at the surface and top of the p72 trimer. These findings will enrich our knowledge regarding the epitope on p72 protein and provide valuable information for further characterization of the antigenicity and molecular functions of p72 protein.

Immunogenicity of an inactivated novel goose parvovirus vaccine for short beak and dwarfism syndrome in Cherry Valley ducks.

Duck short beak and dwarfism syndrome (SBDS) is a viral infectious disease caused by novel goose parvovirus (NGPV), which has been responsible for serious economic losses to the Chinese duck industry in recent years. Currently, there is no effective vaccine against this disease. In this study, we developed an inactivated virus vaccine candidate for SBDS based on NGPV strain DS15 isolated from a duck in China. Immune efficacy was evaluated in 112 ducks, which were randomly divided into vaccination, challenge-control, vaccination-challenge, and blank control groups (28 per group). Clinical characteristics, antibodies, virus excretion, viremia, and pathological changes were monitored. No morbidity or death was observed in the immunized ducks, which showed normal weight and a good mental state. High levels of serum antibodies (optical density at 450 nm of ~ 0.63) were detected in ducks immunized with the inactivated vaccine at 7 days post-vaccination (dpv), and the titer of virus-neutralizing antibodies increased from 1:23 to 1:28.5 from 7 to 42 dpv. Measurement of the viral load in anal swab, serum, and tissue samples showed that vaccination significantly inhibited the replication of NGPV in immunized ducks. Moreover, NGPV could not be isolated from the spleens of immunized or vaccinated and challenged ducks. Collectively, these results demonstrate that the newly developed inactivated NGPV vaccine, administered in an oil emulsion adjuvant, possesses good immunogenicity and represents a potentially powerful tool for SBDS prevention and control.

Interaction between Translocation-associated membrane protein 1 and σC protein of novel duck reovirus controls virus infectivity.

Novel duck reovirus (NDRV), the prototype strain of the species Avian orthoreovirus (ARV), is associated with high mortality in Pekin ducklings. σC is an outer capsid protein encoded by the S1 genome segment of NDRV which mediates attachment to host cells. Our previous studies using immunoprecipitation and mass spectrometry found that σC coprecipitated with some host proteins including Translocation-associated membrane protein 1 (TRAM1). However, the interaction between σC and TRAM1 has not been further confirmed experimentally. In this study, we utilized coimmunoprecipitation assays, glutathione S-transferase pull-down, and confocal microscopy to confirm the interaction between σC and TRAM1. In addition, knockdown of TRAM1 using siRNA and overexpression of TRAM1 gene were conducted to explore its effect on virus replication. The result showed that TRAM1 silencing benefits while overexpression inhibits viral replication. This study confirms the important role TRAM1 during NDRV infection which can help develop new approaches for NDRV disease prevention and control.

Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virus in vitro.

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.

Construction and characterization of an infectious molecular clone of novel duck reovirus.

Novel duck reovirus (NDRV), the prototype strain of the species Avian orthoreovirus (ARV), is currently an infectious agent for ducks. Studies on NDRV replication and pathogenesis have been hampered by the lack of an available reverse-genetics system. In this study, a plasmid-based reverse-genetics system that is free of helper viruses has been developed. In this system, 10 full-length gene segments of wild-type NDRV TH11 strain are transfected into BSR-T7/5 cells that express bacteriophage T7 RNA polymerase. Production of infectious virus was shown by the inoculation of cell lysate derived from transfected cells into 10-day-old duck embryos. The in vivo growth kinetics and infectivity of the recombinant strains were identical to those of the wild-type strain. These viruses grew well and were genetically stable both in vitro and in vivo. Altogether, these results show the successful production of an infectious clone for NDRV. The infectious clone reported will be further used to elucidate the mechanisms of host tropism, viral replication and pathogenesis, as well as immunological changes induced by NDRV.

Genetic characterization of the complete genome of a mutant canine parvovirus isolated in China.

A field canine parvovirus (CPV) strain, CPV-SH14, was previously isolated from an outbreak of severe gastroenteritis in Shanghai in 2014. The complete genome of CPV-SH14 was determined by using PCR with modified primers. When compared to other CPV-2 strains, several insertions, deletions, and point mutations were identified in the 5' and 3' UTR, with key amino acid (aa) mutations (K19R, E572K in NS1 and F267Y, Y324I and T440A in VP2) also being observed in the coding regions of CPV-SH14. These results indicated that significant and unique genetic variations have occurred at key sites or residues in the genome of CPV-SH14, suggesting the presence of a novel genetic variant of new CPV-2a. Phylogenetic analysis of the VP2 gene revealed that CPV-SH14 may have the potential to spread worldwide. In conclusion, CPV-SH14 may be a novel genetic variant of new CPV-2a, potentially with a selective advantage over other strains.

Evolution of Newcastle Disease Virus Quasispecies Diversity and Enhanced Virulence after Passage through Chicken Air Sacs.

UNLABELLED: It has been reported that lentogenic Newcastle disease virus (NDV) isolates have the potential to become velogenic after their transmission and circulation in chickens, but the underlying mechanism is unclear. In this study, a highly velogenic NDV variant, JS10-A10, was generated from the duck-origin lentogenic isolate JS10 through 10 consecutive passages in chicken air sacs. The velogenic properties of this selected variant were determined using mean death time (MDT) assays, intracerebral pathogenicity index (ICPI), the intravenous pathogenicity index (IVPI), histopathology, and the analysis of host tissue tropism. In contrast, JS10 remained lentogenic after 20 serial passages in chicken eggs (JS10-E20). The JS10, JS10-A10, and JS10-E20 genomes were sequenced and found to be nearly identical, suggesting that both JS10-A10 and JS10-E20 were directly generated from JS10. To investigate the mechanism for virulence enhancement, the partial genome covering the F0 cleavage site of JS10 and its variants were analyzed using ultradeep pyrosequencing (UDPS) and the proportions of virulence-related genomes in the quasispecies were calculated. Velogenic NDV genomes accumulated as a function of JS10 passaging through chicken air sacs. Our data suggest that lentogenic NDV strains circulating among poultry might be a risk factor to future potential velogenic NDV outbreaks in chickens. IMPORTANCE: An avirulent isolate, JS10, was passaged through chicken air sacs and embryos, and the pathogenicity of the variants was assessed. A virulent variant, JS10-A10, was generated from consecutive passage in air sacs. We developed a deep-sequencing approach to detect low-frequency viral variants across the NDV genome. We observed that virulence enhancement of JS10 was due to the selective accumulation of velogenic quasispecies and the concomitant disappearance of lentogenic quasispecies. Our results suggest that because it is difficult to avoid contact between natural waterfowl reservoirs and sensitive poultry operations, circulating lentogenic NDV strains may represent a potential reservoir for emergent velogenic NDV strains that could cause outbreaks in chickens.

The roles of mitochondria in radiation-induced autophagic cell death in cervical cancer cells.

Mitochondria as the critical powerhouse of eukaryotic cells play important roles in regulating cell survival or cell death. Under numerous stimuli, impaired mitochondria will generate massive reactive oxygen species (ROS) which participate in the regulation of vital signals and could even determine the fate of cancer cells. While the roles of mitochondria in radiation-induced autophagic cell death still need to be elucidated. Human cervical cancer cell line, Hela, was used, and the SOD2 silencing model (SOD2-Ri) was established by gene engineering. Cell viability was detected by methyl thiazolyl tetrazolium (MTT) assays, MitoTracker Green staining was used to detect mitochondrial mass, Western blot was used to detect protein expression, and the level of ROS, autophagy, and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. Ionizing radiation (IR) could induce the increase of MAPLC3-II/MAPLC3-I ratio, Beclin1 expression, and ROS generation but decrease the MMP in a time-dependent manner. After SOD2 silencing, the IR-induced changes of ROS and the MMP were significantly enhanced. Moreover, both the radio sensitivity and autophagy increased in SOD2-Ri cells. Whereas, compared with SOD2-Ri, the opposite results were obtained by NAC, an antioxidant. After the treatment with the inhibitor of mitochondrial electron-transport chain complex II, thenoyltrifluoroacetone (TTFA), the rate of autophagy, ROS, and the total cell death induced by IR increased. In addition, the decrease of MMP was more obvious. However, these results were reversed by cyclosporine A (CsA). IR could induce ROS generation and mitochondrial damage which lead to autophagic cell death in Hela cells.

The role of lysosome in cell death regulation.

Lysosome is a highly membrane-bound organelle which possesses a sequence of biological functions including protein degradation, cell signal transduction, plasma membrane repairment, homoeostasis, and autophagy. The lysosome contains more than 50 soluble acid hydrolases, and the acidification of lysosome is the most important biological characteristic. The integrity of lysosome is of vital importance. During the past few years, it was reported that the destabilization of lysosomal membrane can result in the release of lysosomal contents into cytosol and trigger cell death in a caspase-dependent or caspase-independent pathway. Lysosome functions at the late stage of autophagy and degrades cellular components delivered by autophagosome, which is a complicated process. The present article will summarize the current knowledge on the role of lysosome in cell death regulation and the underlying mechanisms.

Hsp90 regulates autophagy and plays a role in cancer therapy.

Nowadays, heat shock protein 90 (Hsp90), a highly conserved molecular chaperone, has become the target of antitumor drugs as a result of its close relationship with the occurrence and development, biological behavior, and prognosis of a tumor. Autophagy has attracted big attention recently for its paradoxical roles in cell survival and cell death, especially in the pathogenesis and treatment of cancer. Moreover, it has been verified that Hsp90 plays a role in autophagy via regulating the stability and activity of signaling proteins, and some Hsp90 inhibitors can induce autophagy. However, the underlying mechanisms for these important processes have not been clarified so far. In this study, we focus on the roles of Hsp90 in the regulation of autophagy, such as toll-like receptor (TLR)-mediated autophagy, Ulk1-mediated mitophagy, and chaperone-mediated autophagy (CMA). The roles of Hsp90 inhibitors in cancer therapy will also be elucidated.

Mutations in the C-terminal region affect subcellular localization of crucian carp herpesvirus (CaHV) GPCR.

G protein-coupled receptors (GPCRs) are known as seven transmembrane domain receptors and consequently can mediate diverse biological functions via regulation of their subcellular localization. Crucian carp herpesvirus (CaHV) was recently isolated from infected fish with acute gill hemorrhage. CaHV GPCR of 349 amino acids (aa) was identified based on amino acid identity. A series of variants with truncation/deletion/substitution mutation in the C-terminal (aa 315-349) were constructed and expressed in fathead minnow (FHM) cells. The roles of three key C-terminal regions in subcellular localization of CaHV GPCR were determined. Lysine-315 (K-315) directed the aggregation of the protein preferentially at the nuclear side. Predicted N-myristoylation site (GGGWTR, aa 335-340) was responsible for punctate distribution in periplasm or throughout the cytoplasm. Predicted phosphorylation site (SSR, aa 327-329) and GGGWTR together determined the punctate distribution in cytoplasm. Detection of organelles localization by specific markers showed that the protein retaining K-315 colocalized with the Golgi apparatus. These experiments provided first evidence that different mutations of CaHV GPCR C-terminals have different affects on the subcellular localization of fish herpesvirus-encoded GPCRs. The study provided valuable information and new insights into the precise interactions between herpesvirus and fish cells, and could also provide useful targets for antiviral agents in aquaculture.

The Role of Deoxycytidine Kinase (dCK) in Radiation-Induced Cell Death.

Deoxycytidine kinase (dCK) is a key enzyme in deoxyribonucleoside salvage and the anti-tumor activity for many nucleoside analogs. dCK is activated in response to ionizing radiation (IR)-induced DNA damage and it is phosphorylated on Serine 74 by the Ataxia-Telangiectasia Mutated (ATM) kinase in order to activate the cell cycle G2/M checkpoint. However, whether dCK plays a role in radiation-induced cell death is less clear. In this study, we genetically modified dCK expression by knocking down or expressing a WT (wild-type), S74A (abrogates phosphorylation) and S74E (mimics phosphorylation) of dCK. We found that dCK could decrease IR-induced total cell death and apoptosis. Moreover, dCK increased IR-induced autophagy and dCK-S74 is required for it. Western blotting showed that the ratio of phospho-Akt/Akt, phospho-mTOR/mTOR, phospho-P70S6K/P70S6K significantly decreased in dCK-WT and dCK-S74E cells than that in dCK-S74A cells following IR treatment. Reciprocal experiment by co-immunoprecipitation showed that mTOR can interact with wild-type dCK. IR increased polyploidy and decreased G2/M arrest in dCK knock-down cells as compared with control cells. Taken together, phosphorylated and activated dCK can inhibit IR-induced cell death including apoptosis and mitotic catastrophe, and promote IR-induced autophagy through PI3K/Akt/mTOR pathway.

Molecular cloning of Peking duck Toll-like receptor 3 (duTLR3) gene and its responses to reovirus infection.

BACKGROUND: Toll-like receptors (TLRs) play an important role in detecting pathogen-associated molecular patterns (PAMPs). Among the TLRs, TLR3 is involved in the recognition of double-stranded RNA. This study was designed to explore the relationship between duTLR3 and duck reovirus (DRV) infection. METHODS: In this study, we cloned and performed a molecular characterization of the complete sequence of Peking duck TLR3 (duTLR3). The expression level of duTLR3 was also determined, along with the relative levels of Mx and IFN-α mRNA after DRV infection. RESULTS: The duTLR3 gene is 2776-bp long and encodes an 895-amino-acid-long protein. Sequence analysis of the product revealed the complete transcript of Peking duck TLR3, including the 88-bp 5'UTR, the 2688-bp coding sequence (ORF), and the 76-bp 3'UTR and poly(A) tail. DuTLR3 was found to share a high amino acid sequence similarity with TLR3 from Jing ding duck (99.6 %), Muscovy duck (97.1 %) and chicken (86.3 %). Additionally, the tissue distribution of duTLR3 suggested that it was abundantly expressed in various tissues, especially in the trachea, esophagus and pancreatic gland. Duck reovirus (DRV) infection resulted in high mRNA expression levels of duTLR3 in the spleen, liver, lung and brain. CONCLUSION: These results suggest that duTLR3 may play an important role in anti-viral defense mechanisms.

Molecular characterization of a novel reovirus isolated from Pekin ducklings in China.

The complete genome sequence of a novel duck orthoreovirus, designated DRV strain TH11(DRV-TH11), was determined and characterized. The DRV-TH11 genome is comprised of 23,417 bp and its genome organization is more similar to that of avian orthoreoviruses (ARVs) of chicken origin than other reoviruses. The results of comparative sequence analysis and dendrograms based on the µB- and σC-encoding genes indicated that TH11 may be derived from the reassortment of ARVs and classic Muscovy duck reovirus (MDRV). A possible recombinant event was identified using the SimPlot program, and it occurred in the M2 segment. The results indicated that reassortment and mutation play a role in the evolution of duck reovirus.

Duck hepatitis A virus serotype 1 minigenome: a model for studying the viral 3'UTR effect on viral translation.

To date, the genetic replication and translation mechanisms as well as the pathogenesis of duck hepatitis A virus type 1 (DHAV-1) have not been adequately characterized due to the lack of a reliable and efficient cell culture system. Although the full-length infections clone system is the best platform to manipulate the virus, it is relatively difficult to assemble this system due to the lack of a suitable cell line. It has been proven that the minigenome system an efficient reverse genetics system for the study of RNA viruses. In some cases, it can be used to displace the infectious clone of RNA viruses. Here, we generated a minigenome for DHAV-1 with two luciferase reporter genes, firefly luciferase (Fluc) and Renilla luciferase (Rluc). The Rluc gene was used as a reference gene for the normalization of the Fluc gene expression in transfected cells, which provided a platform for studying the regulatory mechanisms of DHAV-1. Furthermore, to investigate the role of DHAV-3'UTR in the regulation of viral protein translation, deletions in the 3'UTR were introduced into the DHAV-1 minigenome. Luciferase activity, an indicator of virus translation, was then determined. These results showed that a minigenome system for DHAV-1 was successfully constructed for the first time and that the complete or partial deletion of the DHAV-3'UTR did not affect the expression level of the reporter gene, indicating that DHAV-1 translation may not be modulated by the viral genomic 3'UTR sequence.

Ultrasmall superparamagnetic iron oxide nanoparticles for magnetic resonance imaging contrast agent.

Nanotechnology has given scientists new tools for the development of advanced materials for the detection and diagnosis of various types of diseases. In particular, ultrasmall superparamagnetic iron oxides (USPIOs) have been investigated in many biological applications, both in vitro and in vivo. Due to their small size (diameter < 20 nm), these particles are not immediately removed from the circulation by the reticuloendothelial system (RES), have a longer blood half-life, a wider biodistribution and allow potential targeting with appropriate bioconjugates to specific tissues both normal and tumorous. This review will mainly discuss the synthesis of USPIOs and their applications as MRI contrast agent for disease detection.

Isolation and characterization of a duck reovirus strain from mature ducks in China.

In 2018, a disease characterized by splenic hemorrhage and necrosis killed ducks in a duck farm in Guangxi province, China. A duck reovirus strain was isolated from the tissues of the dead ducks by inoculating duck embryos and BHK-21 cells. Electron microscopy of the cultured the isolate showed that the viral particles were nearly round in shape and approximately 70 nm in diameter, and they were designated DRV-GL18. Sequence analysis showed that the GL18 strain viral genome was 23,419 nt in length and had 10 dsRNA segments. Phylogenetic analysis of cDNA amplicons of segments encoding the protein σC which are outer capsid proteins showed that the isolate belongs to the branch of the epidemic strains of duck reovirus. The Recombination Detection Program (RDP) and SimPlot program analyses suggested potential genetic recombination events in the M2 segments. Pathogenicity experiments revealed that GL18 produced severe hemorrhaging in livers and necrosis in the spleen of infected SPF ducklings. A death rate of 50% in the experimental ducklings was calculated during the first 7 d, and the rest of the ducklings were observed to undergo spleen necrosis. These data suggested that GL18 is a duck reovirus isolate with severer pathogenicity, and it could be a candidate for development of vaccine. This is the first reported isolation of duck reovirus from mature ducks.

Evaluation of an I177L gene-based five-gene-deleted African swine fever virus as a live attenuated vaccine in pigs.

African swine fever (ASF) is a highly contagious disease of domestic and wild pigs caused by the African swine fever virus (ASFV). The current research on ASF vaccines focuses on the development of naturally attenuated, isolated, or genetically engineered live viruses that have been demonstrated to produce reliable immunity. As a result, a genetically engineered virus containing five genes deletion was synthesized based on ASFV Chinese strain GZ201801, named ASFV-GZΔI177LΔCD2vΔMGF. The five-gene-deleted ASFV was safe and fully attenuated in pigs and provides reliable protection against the parental ASFV strain challenge. This indicates that the five-gene-deleted ASFV is a potential candidate for a live attenuated vaccine that could control the spread of ASFV.

Complete genome comparison of duck hepatitis virus type 1 parental and attenuated strains.

Two complete duck hepatitis virus type 1 (DHV-1) genomes, strain SY5 and its chicken embryos passage descendent vaccine strain ZJ-A, were compared and analyzed in order to identify possible sites of attenuation. Of the 205 nucleotide changes, 22 resulted in sense mutations, 174 produced nonsense mutations. Besides, there are 7 consistent nucleotides substitutions in 5'UTR and 2 in 3'UTR. Three of these 22 sense mutations resided in VP0, 6 exists in VP1, one exists in VP3, 3 exists in 2A2, 3 exists in 2C, one was detected in 3B and 5 was in 3D. These results suggested that VP0, VP1, 3D, and 5'/3'UTR may contribute to the attenuation of DHV-1 in chicken/duck/embryos. The results provide a genetic basis for future manipulation of a DHV-1 infectious clone.