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.

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.

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.

Protection Evaluation of a Five-Gene-Deleted African Swine Fever Virus Vaccine Candidate Against Homologous Challenge.

African swine fever virus (ASFV) represents a serious threat to the global swine industry, and there are no safe or commercially available vaccines. Previous studies have demonstrated that inactivated vaccines do not provide sufficient protection against ASFV and that attenuated vaccines are effective, but raise safety concerns. Here, we first constructed a deletion mutant in which EP153R and EP402R gene clusters were knocked out. Based on the deletion mutant, a further deletion from the MGF_360-12L, MGF_360-13L to MGF_360-14L genes was obtained. The five-genes knockout virus was designated as ASFV-ΔECM3. To investigate the efficacy and safety of the ASFV-ΔECM3 virus as a vaccine candidate, the evaluation of the virus was subsequently carried out in pigs. The results showed that the ASFV-ΔECM3 virus could induce homologous protection against the parental isolate, and no significant clinical signs or viremia were observed. These results show that the contiguous deletion mutant, ASFV-ΔECM3 encompassing the EP153R/EP402R and MGF_360-12L/13L/14L genes, could be a potential live-attenuated vaccine candidate for the prevention of ASFV infection.

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.

Isolation and pathogenic characterization of duck adenovirus 3 mutant circulating in China.

Duck adenoviruses (DAdVs) include serotype 1 (DAdV-1) in the genus Atadenovirus and serotypes 2-4 (DAdV-2, 3, and 4) in the genus Aviadenovirus. DAdV-3 was initially isolated from Chinese Muscovy ducks in 2014, whereby the infected ducks exhibited yellowing and hemorrhaging in the liver, along with slight pericardial effusion, swelling, and hemorrhaging in the kidneys. In recent years, duck adenovirus infections have appeared in Muscovy duck farms in Fujian, Zhejiang, Anhui, Guangdong, and other places in China. They have an incidence rate of 40 to 55% and a mortality rate of 35 to 43%, resulting in great losses to the duck breeding industry. In this study, 7 DAdV-3 strains, designated as TZ193, FJPT20161124, GX20170519, FJZZ, GDMM, AHAQ, and GDHS were isolated from Muscovy ducks in different provinces of China during 2016-2019, and their complete genomics were sequenced. Their genomes all exhibited significant deletions in ORF67, which also had G to A transitions at the 41st and 977th nt positions, resulting in a stop codon. The pathogenicity of TZ193, a novel isolate of DAdV-3, was investigated in Muscovy ducks. TZ193 caused characteristic lesions of swelling as well as hemorrhagic liver and kidney in the infected ducklings. Moreover, the mortality rate of TZ193 in 5-day-old domestic ducks was 100%. Our data provide concrete evidence for the identification of the DAdV-3 novel variant mutant in China, which effects increased mortality in ducks. This highlights the necessity for monitoring the specific molecular epidemiology of novel DAdV-3 mutants and the development of new vaccines in the future.

Characterization and pathogenicity of a novel variant infectious bursal disease virus in China.

Infectious bursal disease (IBD) is a highly epidemic and immunosuppressive disease of 3- to 6-week-old chicks caused by infectious bursal disease virus (IBDV). Since 2017, there has been a notable increase in the isolation rates of novel variant IBDV strains in China, of which characteristic amino acid residues were different from those of early antigen variants. In this study, one IBDV strain was isolated from a farm with suspected IBD outbreak in Shandong Province, China, which was designated LY21/2. The strain LY21/2 could replicate in MC38 cells with previous culture adaption in SPF chick embryos. Phylogenetic analysis revealed that LY21/2 formed one branch with novel variant IBDVs and shared 96.8-98.6% nucleotide sequence identity with them. Moreover, LY21/2 serving as the major parent underwent the recombination event of a variant strain (19D69), while the minor parent was a very virulent strain (Harbin-1). SPF chicks inoculated with LY21/2 showed no gross clinic symptom, whereas bursal atrophy was exhibited and apoptosis was occurred in 55.21% of bursal cells. The results of histopathology and immunohistochemical staining showed that lymphocyte depletion and connective tissue hyperplasia and IBDV antigen-positive cells were observed in the bursa of LY21/2-infected chicks. Besides, DNA fragmentation was detected in the LY21/2-infected bursal tissue section by TUNEL assay. Collectivtely, these data presented analysis and evaluation of the genetic characteristics and pathogenicity of a novel variant IBDV strain. This study may help in the development of biosafety strategies for the prevention and control of IBDV in poultry.

Goose nephritic astrovirus infection increases autophagy, destroys intercellular junctions in renal tubular epithelial cells, and damages podocytes in the kidneys of infected goslings.

Goose nephritic astrovirus (GNAstV) has recently been identified, which causes kidney swelling and visceral gout in goslings. However, the pathological changes in kidney tissue due to GNAstV infection have not yet been described. In the study, fifty goslings were orally infected with GNAstV, and fifty goslings received PBS as a control. Kidney tissue was collected at different days following infection (dpi) to assess the injury. GNAstV infection reduced body weight, increased the relative weight of the kidney, and increased serum uric acid and creatinine levels. GNAstV was found within renal epithelial cells, and the viral load in the kidney peaked at 7 dpi. Pale and swollen kidney tissue was observed in infected goslings, especially at 5 and 7 dpi. GNAstV infection caused degeneration and necrosis of renal epithelial cells, structural destruction of the brush border, glycogen deposition in the glomerular mesangium, increased fibrosis, and infiltration of inflammatory cells into the renal interstitium. Moreover, swollen mitochondria, broken mitochondrial ridges, autophagosomes, and autophagolysosomes were observed under ultrahistopathological examination. GNAstV infection increased levels of LC3B, ATG5, and Beclin 1, and decreased p62, and downregulated WT1 mRNA and upregulated desmin mRNA. At early stages, GNAstV infection decreased expression of intercellular junction-related genes, including ZO-1, occludin, claudin-10, and catenin-α2. In conclusion, GNAstV infection causes renal epithelial cell autophagy, destruction of brush border and intercellular junctions, podocyte damage, and increased fibrosis, ultimately resulting in damage to the kidney.

A multiplex nanoparticle-assisted polymerase chain reaction assay for detecting three canine epidemic viruses using a dual priming oligonucleotide system.

A rapid and accurate diagnosis of mixed viral infections is important for providing timely therapeutic interventions. The aim of this study was to develop a highly sensitive and specific method for the simultaneous detection of canine distemper virus (CDV), canine parvovirus (CPV) and canine coronavirus (CCV) in mixed infections by combining the high specificity of a dual priming oligonucleotide (DPO) primer system with the high sensitivity of a nanoparticle-assisted PCR (nanoPCR) assay. Under the optimised assay conditions, the multiplex DPO-nanoPCR assay developed using DPO primers was 100-fold more sensitive than the multiplex PCR assay using conventional primers. The detection limits of the multiplex DPO-nanoPCR assay for the recombinant plasmids containing the cloned CDV, CPV and CCV target sequences were 5.4 × 102, 6.5 × 102 and 1.6 × 102 copies in a 25 µL assay, respectively. No cross-reaction with other canine viruses was observed. This is the first reported use of a multiplex nanoPCR assay with the DPO primer system for the simultaneous detection of CDV, CPV and CCV in mixed infections. The high sensitivity and specificity of the assay indicated its potential for use in clinical diagnosis and field surveillance of animal epidemics.

Zinc finger antiviral protein (ZAP) inhibits small ruminant morbillivirus replication in vitro.

Small ruminant morbillivirus (SRMV) is a highly contagious and economically important viral disease of small domestic and wild ruminants. Difficulty with its stable proliferation in ovis aries-derived cells has led to a relative lag in the study of its natural immunity and pathogenesis. Here we report the antiviral properties of ZAP against SRMV, a single-stranded negative-stranded RNA virus of the genus Morbillivirus. ZAP expression was significantly induced in sheep endometrial epithelial cells following SRMV infection. ZAP inhibited SRMV replication in cells after infection, while its overexpression in Vero-SLAM cells significantly increased their resistance to SRMV replication. The ZAP protein co-localized with SRMV RNA in the cytoplasm and ZAP-responsive elements were mapped to the 5' untranslated region of SRMV nucleocapsid, phosphoprotein, matrix, and fusion. In summary, ZAP confers resistance to SRMV infection by directly targeting viral RNA and inhibiting viral replication. Our findings further extend the ranges of viral targets of ZAP and help elucidate the mechanism of SRMV replication.

Genomic characterization and phylogenetic analysis of a new canine picornavirus variant in the mainland of China.

A new canine picornavirus (CanPV) variant, designated as dog/SH1901/CHN/2019, was detected in a pool of various canine fecal samples in the mainland of China using a viral metagenomic analysis, and its nearly complete genome sequence was determined and analyzed. Sequence analyses revealed that it had a standard picornavirus genome organization, a type I internal ribosome entry site (IRES) in the 5'UTR. However, dog/SH1901/CHN/2019 has generated a serial of unique aa mutations and 7aa insertion when compared with the closely related CanPVs. Phylogenetic analysis and pairwise sequence comparisons based on the P1, 2C, 3C, and 3D protein sequences showed that dog/SH1901/ CHN/2019 was closely related to CanPV strains 244 F, 325 F and 6D, which clustered into an independent evolutionary clade and distantly related to CanPV strain A128thr of the genus Mischivirus, which indicated the unclassified CanPV strains may belong to a novel species or genus in the family Picornaviridae. This study extends our knowledge on the evolution and genetic diversity of CanPVs.

Isolation and complete genome analysis of a novel duck picornavirus in China.

A novel duck picornavirus, designated as duck/AH15/CHN/2015, was isolated and identified from Cherry Valley ducks with short beak and dwarfism syndrome in 2015 in Anhui province of China. Duck/AH15/CHN/2015 has the highest degree of amino acid sequence identity (approximately 43 %) with duck hepatitis A viruses (DHAV) Complete genome analysis revealed that duck/AH15/CHN/2015 possesses a typical picornavirus-like genomic organization, 5' UTR-L-P1 (VP0-VP3-VP1)-P2 (2A1-2A2- 2B-2C)-P3 (3A-3B-3C-3D)-3'UTR-poly (A). The 5'UTR contains a potential type IV internal ribosome entry site, while a conserved "barbell"-like structure is found at the 3'UTR, which is similar to DHAV. Compared to the closest related DHAVs, two unrelated 2A proteins were predicted in duck/AH15/CHN/2015, while three unrelated 2A proteins were presented in DHAVs. Based on the amino acid identity comparison and phylogenetic analysis of P1, 2C, and 3CD (3C and 3D), duck/AH15/CHN/2015 was closely related to but distinct from DHAVs, and it was proposed to be a member of a novel species in the genus Avihepatovirus of the family Picornaviridae.

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.

Hypericin-loaded graphene oxide protects ducks against a novel duck reovirus.

Novel duck reovirus (NDRV) disease is a serious infectious disease for poultry, for which no effective therapy has been established. Therefore, development of novel antivirals against NDRV is urgently needed. In present study, we developed a complex wherein hypericin (HY), which shows broad-spectrum antiviral activity, was loaded onto graphene oxide (GO), which has a high drug-loading capacity and low cytotoxicity. The antiviral activity of the complex (GO/HY) was studied in DF-1 cells and in ducklings infected with the NDRV TH11 strain. GO/HY showed a dose-dependent inhibition of NDRV replication, which may be attributed to direct virus inactivation or inhibition of virus attachment. Western blotting and indirect immunofluorescence assay (IFA) showed markedly suppressed protein expression in GO/HY-treated NDRV-infected DF-1 cells. Moreover, GO/HY prolonged the survival time of the ducklings by reducing pathological lesions caused by the infection and inhibiting viral replication in the liver and lungs. These results suggest that GO/HY has antiviral activity against NDRV both in vitro and in vivo.

A divergent canine parvovirus type 2c (CPV-2c) isolate circulating in China.

The variability and the intrinsically high mutation rate of canine parvovirus type 2(CPV-2) increased the diversity of CPV-2 in canine populations. Since the first occurrence of CPV-2, three antigenic variants (2a, 2b and 2c) were detected and distributed worldwide. CPV-2c infection has been detected and increasingly reported in China. Here, a CPV-2c strain CPV-SH1516 was isolated and its complete genome sequence was first characterized. Compared with other CPV-2c isolates, CPV-2c isolates from China continued to evolve into divergent CPV-2c variants with specific unique amino acid substitutions under purifying selection. Emergence of CPV-2c isolates from China was driven by the unique gradual point mutations in key sites of VP2 rather than introduction from outside China. Combining sequence comparison with phylogenetic analysis based on the amino acid sequences of VP2, the vast majority of CPV-2c isolates from China formed a monophyletic cluster and CPV-SH1516 was a representative isolate of CPV-2c circulating in China. Overall, our study provides valuable insight into the evolutionary mechanism of CPV-2c.

Generation and evaluation of a recombinant goose origin Newcastle disease virus expressing Cap protein of goose origin avastrovirus as a bivalent vaccine in goslings.

Currently, both goose astrovirus (GoAstV) and goose-origin Newcastle disease virus (NDV) are widely infectious agents for goslings. There is no vaccine for GoAstV. Capsid protein can elicit a neutralizing antibody in human astroviruses (HAstV). Molecular analysis of the genomic region encoding the capsid protein(ORF2) of goose astrovirus has revealed that it contains neutralizing epitopes. Goose-origin NDV is also an infectious agent. A wide range of NDV strains exist that can be commonly used as vaccine vectors. In the present study, the fusion protein cleavage site RRQKR↓F in a backbone of the virulent goose-origin NDV SH-12 was changed into an avirulent motif GRQGR↓L. The modified goose-origin NDV recombinant vaccine virus expressing the Capsid protein (Cap) of GoAstV was generated as a bivalent vaccine using a reverse-genetics approach. The recombinant virus, rNDV/GoAstV-Cap, was attenuated and similar growth dynamics, cytopathic effects, and virus titers in vitro were maintained when compared to the LaSota strain. Expression of the GoAstV-Cap protein in rNDV/GoAstV-Cap infected cells was detected by an immunofluorescence assay and Western blotting. Goslings inoculated with rNDV/GoAstV-Cap showed no apparent signs of disease and induced GoAstV-Cap-specific immune responses and NDV-specific serum antibody responses to a LaSota vaccination control. Complete protection against a pathogenic GoAstV challenge and avelogenic NDV challenge was conferred. The results of the study suggested that rNDV/GoAstV-Cap viruses have the potential to be the safe, stable, and effective bivalent vaccines.