Epizootiological surveillance of porcine circoviruses in free-ranging wild boars in China.

Four species of porcine circoviruses (PCV1-4) have been reported to circulate in Chinese domestic pigs, while the epizootiology of these viruses in free-ranging wild boars in China remains unknown. In this study, tissue and serum samples collected from diseased or apparently healthy wild boars between 2018 and 2020 in 19 regions of China were tested for the prevalence of PCV1-4 infections. Positive rates of PCV1, PCV2, and PCV3 DNA in the tissue samples of Chinese wild boars were 1.6% (4/247), 58.3% (144/247), and 10.9% (27/247) respectively, with none positive for PCV4. Sequence analysis of viral genome showed that the four PCV1 strains distributed in Hunan and Inner Mongolia shared 97.5%-99.6% sequence identity with global distributed reference strains. Comparison of the ORF2 gene sequences showed that 80 PCV2 strains widely distributed in 18 regions shared 79.5%-100% sequence identity with reference strains from domestic pigs and wild boars, and were grouped into PCV2a (7), PCV2b (31) and PCV2d (42). For PCV3, 17 sequenced strains shared 97.2%-100% nucleotide identity at the genomic level and could be divided into PCV3a (3), PCV3b (2) and PCV3c (12) based on the phylogeny of ORF2 gene sequences. Serological data revealed antibody positive rates against PCV1 and PCV2 of 11.4% (19/167) and 53.9% (90/167) respectively. The data obtained in this study improved our understanding about the epidemiological situations of PCVs infection in free-ranging wild boars in China and will be valuable for the prevention and control of diseases caused by PCVs infection.

Herpes simplex virus type I glycoprotein L evades host antiviral innate immunity by abrogating the nuclear translocation of phosphorylated NF-κB sub-unit p65.

Nuclear factor (NF)-κB plays an important role in the innate immune response by inducing antiviral genes' expression. However, the herpes simplex virus 1 (HSV-1) virus has developed multiple ways to interfere with NF-κB activity to escape the host antiviral response. Here, we found that HSV-1 envelope glycoprotein L(gL) markedly inhibits interferon (IFN) production and its downstream antiviral genes. Our results showed that ectopic expression of gL inhibited IFN-ß promoter activation, and decreased IFN-ß production, the expression of IFN-stimulated genes (ISGs), and inhibited immunologic stimulant (poly I:C) induced activation of IFN signaling pathway. Depletion of gL by short interfering RNA (siRNA) significantly upregulated IFN-ß and ISG production. Further study showed that the N-terminus of the gL bound to the Rel homology domain (RHD) of the p65 and concealed the nuclear localization signal of p65, thereby impeding the translocation of phosphorylated p65 to the nucleus. In summary, our findings indicated that the N-terminal of HSV-1 gL contributes to immune invasion by inhibiting the nuclear translocation of p65.

Genetic characterization of hepatitis E virus from wild boar in China.

Hepatitis E virus (HEV), the causative agent of hepatitis E (HE), is classified into four major genotypes (1-4), with wild boar being the main natural reservoir for genotypes 3 and 4. However, little is known about the prevalence of HEV infection in wild boars in China. In this study, RT-nested PCR and RT-quantitative PCR were used to detect the HEV RNA in tissue samples taken from 331 free-ranging wild boars collected between 2018 and 2020 from 24 regions across China, and the partial ORF2 genes or complete genomes of the positive samples were sequenced. Furthermore, antibodies against HEV in 216 serum samples from wild boars were tested by ELISA. As a result, HEV RNA was detected in nine out of 331 liver samples of wild boars (2.72%), which were distributed in eight regions. Genetic and evolutionary analysis of partial ORF2 sequences indicated that the HEV strains identified in this study share 83.9%-100% nucleotide sequence identity and belong to subtypes 4d (n = 6), 4g (n = 2), and 4h (n = 1), and similar phylogeny was obtained using the complete genome sequences of seven wild boar HEV strains. Additionally, the HEV viral loads were higher in the liver than in other tissues and blood. Moreover, 61 out of 216 sera (28.2%) from wild boars tested positive for anti-HEV antibodies. To our knowledge, this is the first study to report the epidemiological situations of HEV infections in free-ranging wild boars in China, and the obtained data are valuable for prevention and control of HE.

Dual-peak long period fiber grating coated with graphene oxide for label-free and specific assays of H5N1 virus.

Avian influenza is an acute infectious disease caused by the avian influenza virus (AIV), which has caused enormous economic losses and posed considerable threats to public health. This study aimed to demonstrate an immunosensor based on dispersion turning point long-period fiber grating (DTP-LPFG) integrated with graphene oxide (GO) for the specific detection of a type of AIV H5N1 virus. LPFG was designed to work at DTP, whose dual-peak spacing was very high sensitive to a refractive index. Anti-H5N1 monoclonal antibodies were covalently bonded with the GO film on the fiber surface, thus constructing an immunosensor for the label-free and specific detection of the H5N1 virus. The proposed method was capable of the reliable detection of H5N1 virus with the limit of detection as low as ~1.05 ng/ml within the large range of 1 ng/mL to 25 µg/mL. More importantly, immunoassays of the whole H5N1 virus in clinical samples further confirmed that the GO-integrated DTP-LPFG immunosensor showed very high specificity to the H5N1 virus and demonstrated great potential for clinical use.

CRISPR/Cas12a Based Rapid Molecular Detection of Acute Hepatopancreatic Necrosis Disease in Shrimp.

Acute hepatopancreatic necrosis disease (AHPND), formerly called early mortality syndrome (EMS), causes high mortality in cultured penaeid shrimp, particularly Penaeus vannamei and Penaeus monodon. AHPND is mainly caused by Vibrio species carrying the pVA1 plasmid encoding the virulence genes Photorhabdus insect-related (pir) pir VP A and pir VP B. We developed a new molecular assay that combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology (RPA-CRISPR/Cas12a) to detect pir VP A and pir VP B, with a fluorescent signal result. The fluorescence RPA-CRISPR/Cas12a assay had a detection limit of 20 copies/µL for pir VP A and pir VP B. To improve usability and visualize RPA-CRISPR/Cas12a assay results, a lateral flow strip readout was added. With the lateral flow strip, the RPA-CRISPR/Cas12a assay had a lower limit of detection of 200 copies/µL (0.3 fmol/L). The lateral flow assay can be completed in 2 h and showed no cross-reactivity with pathogens causing other shrimp diseases. In a field test of 60 shrimp samples, the RPA-CRISPR/Cas12a lateral flow assay showed 92.5% positive predictive agreement and 100% negative predictive agreement. As the new RPA-CRISPR/Cas12a assay is rapid, specific, and does not require complicated experimental equipment, it may have important field applications for detecting AHPND in farmed shrimp.

The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice.

BACKGROUND: African swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). Currently, no vaccine is commercially available for prevention, and the epidemic is still spreading. Here, we constructed a recombinant pseudorabies virus (PRV) (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) that expresses the CD2v protein of ASFV and evaluated its effectiveness and safety as a vaccine candidate in mice. METHODS: A homologous recombination fragment containing ASFV CD2v was synthesized and co-transfected into HEK 293 T cells, a knockout vector targeting the PRV TK gene. The transfected cells were infected with PRV-ΔgE/ΔgI, and the recombinant strain (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) was obtained by plaque purification in Vero cells. The expression of ASFV CD2v in the recombinant virus was confirmed by sequencing, Western blotting, and immunofluorescence analysis, and the genetic stability was tested in Vero cells over 20 passages. The virulence, immunogenicity and protective ability of the recombinant virus were further tested in a mouse model. RESULTS: The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain is stable in Vero cells, and the processing of CD2v does not depend on ASFV infection. The vaccination of PRV-ΔgE/ΔgI/ΔTK-(CD2v) causes neither pruritus, not a systemic infection and inflammation (with the high expression of interleukin-6 (IL6)). Besides, the virus vaccination can produce anti-CD2v specific antibody and activate a specific cellular immune response, and 100% protect mice from the challenge of the virulent strain (PRV-Fa). The detoxification occurs much earlier upon the recombinant virus vaccination and the amount of detoxification is much lower as well. CONCLUSIONS: The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain has strong immunogenicity, is safe and effective, and maybe a potential vaccine candidate for the prevention of ASF and Pseudorabies.

Pseudorabies virus (PRV) strain with defects in gE, gC, and TK genes protects piglets against an emerging PRV variant.

The prevalence of an emerging variant of the pseudorabies virus (PRV) has been causing serious losses to farmers in China. Moreover, the commercially available PRV vaccine often fails to provide thorough protection. Therefore, in this study, we generated a PRV-∆gC\gE∆TK strain with defects in gC, gE, and TK of PRV. Compared to the parental PRV strain and the single gene deletion strains (PRV-∆gC, PRV-∆gE, and PRV-∆TK), PRV-∆gC\gE∆TK grew slowly, and exhibited fewer and smaller plaques on swine testis (ST) cells. Furthermore, animal experiment results showed that mice that were immunized intramuscularly with PRV-∆gC\gE∆TK, survived throughout the experiment with no observed clinical symptoms, and were completely protected against PRV challenge. Additionally, deletion of the gC, gE, and TK genes significantly alleviated viral damage in the brain. Furthermore, one-day-old weaned piglets immunized intramuscularly with PRV-∆gC\gE∆TK elicited higher levels of gB antibodies against both the emerging PRV variant and the parental PRV, exhibited full protection against challenge with both variants, and showed neutralization capacity against PRV. These data suggest that PRV-∆gC\gE∆TK is a promising vaccine candidate for the control of pseudorabies.

An Optimized Multilocus Variable-Number Tandem Repeat Analysis Typing Scheme for Listeria monocytogenes from Three Western Provinces in China.

Listeria monocytogenes is a foodborne pathogen worldwide. Multilocus variable-number tandem repeat analysis (MLVA) has been used for listeriosis surveillance and outbreak investigations. MLVA typing schemes have been proposed, but their usefulness for typing isolates from the People's Republic of China has not been assessed. To this aim, all L. monocytogenes strains (79) isolated from 1,445 raw meat and abattoir environmental samples of three western provinces in China were characterized with PCR serogrouping, multilocus sequence typing, and MLVA. The isolates were typed into the four PCR serogroups IIb (38.0%), IIc (26.6%), IIa (24.0%), and IVb (11.4%), with a Simpson's index (SI) of 0.7235. With multilocus sequence typing, they were typed into 18 sequence types (STs), including two new STs, ST1029 and ST1011, with an SI of 0.8880. With the 14 MLVA loci from the previous five schemes, the isolates were typed into 39 MLVA genotypes, with an SI of 0.9656. The typing data indicated that MLVA had the highest typing capability among the three methods. A subsequent optimization analysis identified an optimal combination of eight loci (LMV2, LMV9, LMV1, Lm10, Lm11, Lm15, Lm23, and LMTR6) producing the same SI as that of the 14 loci. The present optimized combination shared only six loci with the optimal nine-loci combination proposed in Australia, verifying for the first time that the optimal combinations varied with the isolates' sets. The current optimal typing scheme was ideal for L. monocytogenes isolates from western China.

Label-free immunoassay for porcine circovirus type 2 based on excessively tilted fiber grating modified with staphylococcal protein A.

Using excessively tilted fiber grating (Ex-TFG) inscribed in standard single mode fiber, we developed a novel label-free immunoassay for specific detection of porcine circovirus type 2 (PCV2), which is a minim animal virus. Staphylococcal protein A (SPA) was used to modify the silanized fiber surface thus forming a SPA layer, which would greatly enhance the proportion of anti-PCV2 monoclonal antibody (MAb) bioactivity, thus improving the effectiveness of specific adsorption and binding events between anti-PCV2 MAbs and PCV2 antigens. Immunoassay experiments were carried out by monitoring the resonance wavelength shift of the proposed sensor under different PCV2 titer levels. Anti-PCV2 MAbs were thoroughly dissociated from the SPA layer by treatment with urea, and recombined to the SPA layer on the sensor surface for repeated immunoassay of PCV2. The specificity of the immunosensor was inspected by detecting porcine reproductive and respiratory syndrome virus (PRRSV) first, and PCV2 subsequently. The results showed a limit of detection (LOD) for the PCV2 immunosensor of ~9.371TCID50/mL, for a saturation value of ~4.801×10(3)TCID50/mL, with good repeatability and excellent specificity.

Multiple amino acid variations in the nonstructural proteins of swine Japanese encephalitis virus alter its virulence in mice.

In a previous study, we performed serial brain-to-brain passages of swine Japanese encephalitis virus in mice and sequenced the complete genomes of the F5 and F20 passaged mouse-adapted variants. In the current study, we analyzed the differences between their genome sequences and found 12 amino acid substitutions in the nonstructural proteins. We also assessed the growth characteristics of these two variants in mammalian cells in vitro and in vivo. Our investigations revealed that the F20 variant had enhanced growth characteristics and modified virulence compared with the F5 variant. We therefore conclude that multiple amino acid substitutions in the nonstructural proteins of swine Japanese encephalitis virus alter its virulence in mice.

Transmission of avian H9N2 influenza viruses in a murine model.

Avian H9N2 influenza viruses have circulated widely in domestic poultry around the world, resulting in occasional transmission of virus from infected poultry to humans. However, it is unknown whether H9N2 influenza virus has acquired the ability to be transmitted from human to human. Here, we report that mouse-adapted H9N2 influenza viruses can replicate efficiently and are lethal for several strains of mice. To evaluate the transmissibility of mouse-adapted H9N2 influenza viruses, we carried out transmission studies in mice using both contact and respiratory droplet routes. Our results indicate that mouse-adapted H9N2 influenza viruses can replicate efficiently and be transmitted between mice. This suggests that once H9N2 influenza viruses adapt to new host, they should present potential public health risks, therefore, urgent attention should be paid to H9N2 influenza viruses.

Multiple amino acid substitutions are involved in the adaptation of H9N2 avian influenza virus to mice.

To explore adaptation of avian influenza virus to mice we previously performed serial lung-to-lung passages of the influenza A/Chicken/Jiangsu/7/2002 (H9N2) strain, resulting in the isolation of a variant influenza strain lethal for mice. We now report that virulence correlates with improved growth characteristics on mammalian cells and extended tissue tropism in vivo. Sequencing of the complete genomes of the wild-type and mouse-adapted viruses revealed 25 amino acid substitutions. Some were found to reiterate known substitutions in human and swine H9N2 influenza isolates. Functions affected include nuclear localization signals and sites of protein and RNA interaction, while others are known determinants of pathogenicity and host specificity such as the viral polymerase PB2 E627K substitution. These observations suggest that enhanced growth characteristics and modified cell tropism may contribute to increased virulence in mice. We conclude that multiple amino acid substitutions are likely to be involved in the adaptation of H9N2 avian influenza virus to mice.