Virologica Sinica

Correction to: Oxymatrine Inhibits Bocavirus MVC Replication, Reduces Viral Gene Expression and Decreases Apoptosis Induced by Viral Infection In Fig. 2B, labels were misnamed in the original article. MVC label and MVC + OMT label were opposite. Now the correct Fig. 2 has been provided below.

Cross-sectional Seroprevalence and Genotype of Hepatitis E Virus in Humans and Swine in a High-density Pig-farming Area in Central China Abstract Hepatitis E virus (HEV) infection is a common public health problem in developing countries. However, the current prevalence of HEV and the relationship of HEV genotype between swine and human within high-density pig-farming areas in central China are still inadequately understood. Here, cross-sectional serological and genotypic surveys of HEV among the 1232 general population, 273 workers occupationally exposed to swine, and 276 pigs in a high-density pig-breeding area, were undertaken by ELISA and nested RT-PCR methods. Anti-HEV IgG was detected in 26.22% of general population and 48.35% of occupational workers. The prevalence of swine serum HEV-Ag was 6.52%. The prevalence of anti-HEV IgG was significantly higher among the workers occupationally exposed to swine than among the general population. An increased HEV seropositivity risk among the general population was associated with either being a peasant or male and was very strongly associated with the increase of age. Among the occupationally exposed group, the prevalence of anti-HEV IgG antibodies increased with age and working years. Among the 30 HEV-IgM-positive people, the infection rates of clerks in the public, peasants, pork retailers, and pig farmers were higher than those of others. A phylogenetic analysis revealed that all the isolates belonged to subgenotype 4d, and four people and four pigs shared 97.04%–100% sequence homology. This study revealed a high HEV seroprevalence among the general population and workers occupationally exposed to swine in the Anlu City, and supports the notion that swine are a source of human HEV infection.

A Recombinant Rabies Virus Expressing Fms-like Tyrosine Kinase 3 Ligand (Flt3L) Induces Enhanced Immunogenicity in Mice Abstract Rabies is a zoonotic disease that still causes 59,000 human deaths each year, and rabies vaccine is the most effective way to control the disease. Our previous studies suggested that the maturation of DC plays an important role in enhancing the immunogenicity of rabies vaccine. Flt3L has been reported to own the ability to accelerate the DC maturation, therefore, in this study, a recombinant rabies virus expressing mouse Flt3L, designated as LBNSE-Flt3L, was constructed, and its immunogenicity was characterized. It was found that LBNSE-Flt3L could enhance the maturation of DC both in vitro and in vivo, and significantly more TFH cells and Germinal Center B (GC B) cells were generated in mice immunized with LBNSE-Flt3L than those immunized with the parent virus LBNSE. Consequently, expressing of Flt3L could elevate the level of virus-neutralizing antibodies (VNA) in immunized mice which provides a better protection from a lethal rabies virus challenge. Taken together, our study extends the potential of Flt3L as a good adjuvant to develop novel rabies vaccine by enhancing the VNA production through activating the DC–TFH–GC B axis in immunized mice.

Novel Recombinant Seneca Valley Virus Isolated from Slaughtered Pigs in Guangdong Province

Seroprevalence of Severe Fever with Thrombocytopenia Syndrome Phlebovirus in Domesticated Deer in South Korea Abstract Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV) has a wide host range. Not only has it been found in humans, but also in many wild and domesticated animals. The infection of breeding deer on farms is a particularly worrisome public health concern due to the large amount of human contact and the diverse use of deer products, including raw blood. To investigate the prevalence of breeding domesticated deer, we examined the SFTSV infection rate on deer farms in South Korea from 2015 to 2017. Of the 215 collected blood samples, 0.9% (2/215) were found to be positive for viral RNA by PCR, and sequence analysis showed the highest homology with the KADGH human isolate. Both SFTSV-specific recombinant N and Gn protein-based ELISAs revealed that 14.0% (30/215) and 7.9% (17/215) of collected blood specimens were positive for SFTSV antibody. These results demonstrate that the breeding farm deer are exposed to SFTSV and could be a potential infection source for humans through direct contact or consumption of byproducts.

A Novel DT40 Antibody Library for the Generation of Monoclonal Antibodies Abstract Early etiological diagnosis is very important for the control of sudden viral infections, and requires antibodies with both high sensitivity and high specificity. Traditional antibody preparation methods have limitations, such as a long and arduous cycle, complicated operation, and high expenses. A chicken lymphoma cell line, DT40, is known to produce IgM-type antibodies and undergo gene conversion and somatic mutation in the variable region of the immunoglobulin gene during culture. Here, the DT40 cell line was developed to produce antibody libraries and prepare antibody rapidly in vitro. Since hypermutation in DT40 cells was regulated by the activation-induced cytidine deaminase (AID) gene, AID expression needs to be controlled to either fix the Ig sequence by stopping mutation or improve affinity by resuming mutation after the antibodies have been selected. In this study, we generated a novel AID-inducible DT40 cell line (DT40-H7), in which the endogenous AID gene was knocked out using the CRISPR/Cas9 genome editing system, and an inducible AID gene, based on the Tet-Off expression system, was stably transfected. AID expression was controlled in DT40-H7 cells in a simple and efficient manner; gene conversion and point mutations were observed only when AID was expressed. Using the antibody library generated from this cell line, we successfully obtained monoclonal antibodies against the NS1 protein of Zika virus. The DT40-H7 cell line represents a useful tool for the selection and evolution of antibodies and may also be a powerful tool for the rapid selection and generation of diagnostic antibodies for emerging infectious diseases.

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats Abstract Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein (NP) and extracellular domain of matrix protein 2 (M2e) genes from the influenza A virus A/Beijing/30/95 (H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34 (PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP- and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD50 of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.

Complementation of Wild-Type and Drug-Resistant Hepatitis B Virus Genomes to Maintain Viral Replication and Rescue Virion Production under Nucleos(t)ide Analogs Abstract As the open reading frames of hepatitis B virus (HBV) genomes are overlapping, resistance mutations (MTs) in HBV polymerase may result in stop codon MTs in hepatitis B surface proteins, which are usually detected as a mixed population with wild-type (WT) HBV. The question was raised how the coexistence of nucleos(t)ide analogs (NAs) resistance MTs and WT sequences affects HBV replication. In the present study, HBV genomes with frequently detected reverse transcriptase (RT)/surface truncation MTs, rtA181T/sW172*, rtV191I/sW182* and rtM204I/sW196*, were phenotypically characterized alone or together with their WT counterparts in different ratios by transient transfection in the absence or presence of NAs. In the absence of NAs, RT/surface truncation MTs impaired the expression and secretion of HBV surface proteins, and had a dose-dependent negative effect on WT HBV virion secretion. However, in the presence of NAs, coexistence of MTs with WT maintained viral replication, and the presence of WT was able to rescue the production of MT HBV virions. Our findings reveal that complementation of WT and MT HBV genomes is highly effective under drug treatment.

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms Abstract Recently the developed single guide (sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector (pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus (BmNPV) in insect cells. We screened the immediate-early-1 gene (ie-1), the major envelope glycoprotein gene (gp64), and the late expression factor gene (lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector (PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening Abstract Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome (SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11 (SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum (ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1 (LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall, these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com