Coronavirus Porcine Epidemic Diarrhea Virus Utilizes Chemokine Interleukin-8 to Facilitate Viral Replication by Regulating Ca2+ Flux.

Chemokine production by epithelial cells is crucial for neutrophil recruitment to sites of inflammation during viral infection. However, the effect of chemokine on epithelia and how chemokine is involved in coronavirus infection remains to be fully understood. Here, we identified an inducible chemokine interleukin-8 (CXCL8/IL-8), which could promote coronavirus porcine epidemic diarrhea virus (PEDV) infection in African green monkey kidney epithelial cells (Vero) and Lilly Laboratories cell-porcine kidney 1 epithelial cells (LLC-PK1). IL-8 deletion restrained cytosolic calcium (Ca2+), whereas IL-8 stimulation improved cytosolic Ca2+. The consumption of Ca2+ restricted PEDV infection. PEDV internalization and budding were obvious reductions when cytosolic Ca2+ was abolished in the presence of Ca2+ chelators. Further study revealed that the upregulated cytosolic Ca2+ redistributes intracellular Ca2+. Finally, we identified that G protein-coupled receptor (GPCR)-phospholipase C (PLC)-inositol trisphosphate receptor (IP3R)-store-operated Ca2+ (SOC) signaling was crucial for enhancive cytosolic Ca2+ and PEDV infection. To our knowledge, this study is the first to uncover the function of chemokine IL-8 during coronavirus PEDV infection in epithelia. PEDV induces IL-8 expression to elevate cytosolic Ca2+, promoting its infection. Our findings reveal a novel role of IL-8 in PEDV infection and suggest that targeting IL-8 could be a new approach to controlling PEDV infection. IMPORTANCE Coronavirus porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric coronavirus that caused severe economic losses worldwide, and more effort is needed to develop economical and efficient vaccines to control or eliminate this disease. The chemokine interleukin-8 (CXCL8/IL-8) is indispensable for the activation and trafficking of inflammatory mediators and tumor progression and metastasis. This study evaluated the effect of IL-8 on PEDV infection in epithelia. We found that IL-8 expression improved cytosolic Ca2+ in epithelia, facilitating PEDV rapid internalization and egress. G protein-coupled receptor (GPCR)-phospholipase C (PLC)-inositol trisphosphate receptor (IP3R)-SOC signaling was activated by IL-8, releasing the intracellular Ca2+ stores from endoplasmic reticulum (ER). These findings provide a better understanding of the role of IL-8 in PEDV-induced immune responses, which will help develop small-molecule drugs for coronavirus cure.

Deletion of a 7-amino-acid region in the porcine epidemic diarrhea virus envelope protein induces higher type I and III interferon responses and results in attenuation in vivo.

Porcine epidemic diarrhea virus (PEDV) leads to enormous economic losses for the pork industry. However, the commercial vaccines failed to fully protect against the epidemic strains. Previously, the rCH/SX/2016-SHNXP strain with the entire E protein and the rCH/SX/2015 strain with the deletion of 7-amino-acid (7-aa) at positions 23-29 in E protein were constructed and rescued. The pathogenicity assay indicated that rCH/SX/2015 is an attenuated strain, but rCH/SX/2016-SHNXP belongs to the virulent strains. Then, the recombination PEDV (rPEDV-EΔaa23-aa29)strain with a 7-aa deletion in the E protein was generated, using the highly virulent rCH/SX/2016-SHNXP strain (rPEDV-Ewt) as the backbone. Compared with the rPEDV-Ewt strain, the release and infectivity of the rPEDV-EΔaa23-aa29 strain were significantly reduced in vitro, but stronger interferon (IFN) responses were triggered both in vitro and in vivo. The pathogenicity assay showed that the parental strain resulted in severe diarrhea (100%) and death (100%) in all piglets. Compared with the parental strain group, rPEDV-EΔaa23-aa29 caused lower mortality (33%) and diminished fecal PEDV RNA shedding. At 21 days, all surviving pigs were challenged orally with rPEDV-Ewt. No pigs died in the two groups. Compared with the mock group, significantly delayed and milder diarrhea and reduced fecal PEDV RNA shedding were detected in the rPEDV-EΔaa23-aa29 group. In conclusion, the deletion of a 7-aa fragment in the E protein (EΔaa23-aa29) attenuated PEDV but retained its immunogenicity, which can offer new ideas for the design of live attenuated vaccines and provide new insights into the attenuated mechanism of PEDV. IMPORTANCE Porcine epidemic diarrhea virus (PEDV) causes high mortality in neonatal piglets and remains a large challenge to the pork industry. Unfortunately, no safe and effective vaccines are available yet. The pathogenesis and molecular basis of the attenuation of PEDV remain unclear, which seriously hinders the development of PEDV vaccines. This study found that the rPEDV carrying EΔaa23-aa29 mutation in the E protein induced significantly higher IFN responses than the parental virus, partially attenuated, and remained immunogenic in piglets. For the first time, PEDV E was verified as an IFN antagonist in the infection context and identified as a virulence factor of PEDV. Our data also suggested that EΔaa23-aa29 mutation can be a good target for the development of live attenuated vaccines for PEDV and also provide new perspectives for the attenuated mechanism of PEDV.

Host Cells Actively Resist Porcine Reproductive and Respiratory Syndrome Virus Infection via the IRF8-MicroRNA-10a-SRP14 Regulatory Pathway.

MicroRNAs (miRNAs) play an important role in the virus-host interaction. Our previous work has indicated that the expression level of miR-10a increased in porcine alveolar macrophages (PAMs) during porcine reproductive and respiratory syndrome virus (PRRSV) infection and further inhibited viral replication through downregulates the expression of host molecule signal-recognition particle 14 (SRP14) protein. However, the molecular mechanism of miR-10a increased after PRRSV infection remains unknown. In the present study, transcription factor interferon regulatory factor 8 (IRF8) was identified as a negative regulator of miR-10a. PRRSV infection decreases the expression level of IRF8 in PAMs, leading to upregulating miR-10a expression to play an anti-PRRSV role. Meanwhile, this work first proved that IRF8 promoted PRRSV replication in an miR-10a-dependent manner. Further, we explained that SRP14, the target gene of miR-10a, promotes the synthesis of the PRRSV genome by interacting with the viral components Nsp2, thus facilitating PRRSV replication. In conclusion, we identified a novel IRF8-miR-10a-SRP14 regulatory pathway against PRRSV infection, which provides new insights into virus-host interactions and suggests potential new antiviral strategies to control PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has rapidly spread to the global pig industry and caused incalculable economic damage since first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. Understanding the molecular mechanisms of host resistance to PRRSV infection is necessary to develop safe and effective strategies to control PRRSV. During viral infection, miRNAs play vital roles in regulating the expression of viral or host genes at the posttranscriptional level. The significance of our study is that we revealed the transcriptional regulation mechanism of the antiviral molecule miR-10a after PRRSV infection. Moreover, our research also explained the mechanism of host molecule SRP14, the target gene of miR-10a regulating PRRSV replication. Thus, we report a novel regulatory pathway of IRF8-miR-10a-SRP14 against PRRSV infection, which provides new insights into virus-host interactions and suggests potential new control measures for future PRRSV outbreaks.

Genetic characterization and pathogenicity of a novel recombinant PRRSV from lineage 1, 8 and 3 in China failed to infect MARC-145 cells.

The diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in China is increasing rapidly along with mutation and recombination. Recombination could occur between inter- and intra-lineage of PRRSV, which accelerated the complexity of pathogenicity and cell tropism of the recombinant strain. In the present study, a novel PRRSV strain named HN-YL1711 was isolated from a pig farm suffering from severe respiratory difficulty in Henan province, China. The whole genomic sequence analysis indicated that the genome of HN-YL1711 was 15018 nt. It shared 86%, 87.3%, 88.1%, 91.1%, 84.2%, and 84.1% nucleotide similarities with PRRSVs VR2332, CH1a, JXA1, NADC30, QYYZ, and GM2, respectively. Based on phylogenetic analysis of Nsp2, ORF5 and complete genomes, HN-YL1711 was classified into lineage 1 of PRRSV. However, seven genomic break points were detected in recombination analysis, which indicated that the HN-YL1711 originated from multiple recombination among NADC30-like (major parent, lineage 1), JXA1-like (minor parent, lineage 8), and QYYZ-like (minor parent, lineage 3) PRRSV. Porcine alveolar macrophages (PAMs), 3D4/21-CD163 and MARC-145 cells were used to explore the viral adaptation of HN-YL1711. The results indicated that it could infect the PAMs but failed to infect MARC-145 cells. Challenge experiments showed that HN-YL1711 exhibits intermediate virulence in pigs, compared with HP-PRRSV JXA1 and LP-PRRSV CH1a. Taken together, our findings suggest that recombination remains an important factor in PRRSV evolution and that recombination further complicates the cell tropism and pathogenicity of PRRSV.

Genomic characteristics and pathogenicity of a new recombinant strain of porcine reproductive and respiratory syndrome virus.

Recombination is an important phenomenon that accelerates evolution and enriches the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant PRRSV isolates sometimes have different genetic backgrounds. In this study, we report a recombinant PRRSV (SD-YL1712) isolated from a pig farm. The genome of SD-YL1712 is 15,014 nucleotides in length, and its nucleotide and amino acid sequence conservation is higher than that of PRRSV strain JXA1 except within the NSP2 region. The NSP2 region of SDYL1712 shares the highest nucleotide (85.9%) and amino acid (84.1%) sequence identity with PRRSV strain NADC30. SD-YL1712 was found to contain a characteristic 131-amino-acid deletion in the NSP2 region. Two recombination breakpoints were detected at nt 2134 and nt 3958 within the NSP2 region, which revealed that SD-YL1712 originated from a recombination event between NADC30-like and HP-PRRSV-derived MLV-like strains. Interestingly, SD-YL1712 had an additional deletion at position 586, similar to that found in strain TJnh1501. Moreover, the pathogenicity of strain SD-YL1712 was found to be similar to that of HP-PRRSV JXA1, which was higher than that of the CH1a strain. Further analysis indicated that SD-YL1712 might be a transitional intermediate in the evolution of TJbd1401 to TJnh1501.

Porcine epidemic diarrhea virus: Molecular mechanisms of attenuation and vaccines.

Porcine epidemic diarrhea virus (PEDV) causes an emerging and re-emerging coronavirus disease characterized by vomiting, acute diarrhea, dehydration, and up to 100% mortality in neonatal suckling piglets, leading to huge economic losses in the global swine industry. Vaccination remains the most promising and effective way to prevent and control PEDV. However, effective vaccines for PEDV are still under development. Understanding the genomic structure and function of PEDV and the influence of the viral components on innate immunity is essential for developing effective vaccines. In the current review, we systematically describe the recent developments in vaccine against PEDV and the roles of structural proteins, non-structural proteins and accessory proteins of PEDV in affecting viral virulence and regulating innate immunity, which will provide insight into the rational design of effective and safe vaccines for PEDV or other coronaviruses.

A novel biotinylated nanobody-based blocking ELISA for the rapid and sensitive clinical detection of porcine epidemic diarrhea virus.

BACKGROUND: Porcine epidemic diarrhea virus (PEDV), which is characterized by severe watery diarrhea, vomiting, dehydration and a high mortality rate in piglets, leads to enormous economic losses to the pork industry and remains a large challenge worldwide. Thus, a rapid and reliable method is required for epidemiological investigations and to evaluate the effect of immunization. However, the current diagnostic methods for PEDV are time-consuming and very expensive and rarely meet the requirements for clinical application. Nanobodies have been used in the clinic to overcome these problems because of the advantages of their easy expression and high level of stability. In the present work, a novel biotinylated nanobody-based blocking ELISA (bELISA) was developed to detect anti-PEDV antibodies in clinical pig serum. RESULTS: Using phage display technology and periplasmic extraction ELISA (PE-ELISA), anti-PEDV N protein nanobodies from three strains of PEDV were successfully isolated after three consecutive rounds of bio-panning from a high quality phage display VHH library. Then, purified Nb2-Avi-tag fusion protein was biotinylated in vitro. A novel bELISA was subsequently developed for the first time with biotinylated Nb2. The cutoff value for bELISA was 29.27%. One hundred and fifty clinical serum samples were tested by both newly developed bELISA and commercial kits. The sensitivity and specificity of bELISA were 100% and 93.18%, respectively, and the coincidence rate between the two methods was 94%. CONCLUSIONS: In brief, bELISA is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PEDV vaccines efficacy and indirect diagnosis of PEDV infection.

Carbon monoxide and biliverdin suppress bovine viral diarrhoea virus replication.

Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.

Host cells reprogram lipid droplet synthesis through YY1 to resist PRRSV infection.

Metabolism in host cells can be modulated after viral infection, favoring viral survival or clearance. Here, we report that lipid droplet (LD) synthesis in host cells can be modulated by yin yang 1 (YY1) after porcine reproductive and respiratory syndrome virus (PRRSV) infection, resulting in active antiviral activity. As a ubiquitously distributed transcription factor, there was increased expression of YY1 upon PRRSV infection both in vitro and in vivo. YY1 silencing promoted the replication of PRRSV, whereas YY1 overexpression inhibited PRRSV replication. PRRSV infection led to a marked increase in LDs, while YY1 knockout inhibited LD synthesis, and YY1 overexpression enhanced LD accumulation, indicating that YY1 reprograms PRRSV infection-induced intracellular LD synthesis. We also showed that the viral components do not colocalize with LDs during PRRSV infection, and the effect of exogenously induced LD synthesis on PRRSV replication is nearly lethal. Moreover, we demonstrated that YY1 affects the synthesis of LDs by regulating the expression of lipid metabolism genes. YY1 negatively regulates the expression of fatty acid synthase (FASN) to weaken the fatty acid synthesis pathway and positively regulates the expression of peroxisome proliferator-activated receptor gamma (PPARγ) to promote the synthesis of LDs, thus inhibiting PRRSV replication. These novel findings indicate that YY1 plays a crucial role in regulating PRRSV replication by reprogramming LD synthesis. Therefore, our study provides a novel mechanism of host resistance to PRRSV and suggests potential new antiviral strategies against PRRSV infection.IMPORTANCEPorcine reproductive and respiratory virus (PRRSV) has caused incalculable economic damage to the global pig industry since it was first discovered in the 1980s. However, conventional vaccines do not provide satisfactory protection. It is well known that viruses are parasitic pathogens, and the completion of their replication life cycle is highly dependent on host cells. A better understanding of host resistance to PRRSV infection is essential for developing safe and effective strategies to control PRRSV. Here, we report a crucial host antiviral molecule, yin yang 1 (YY1), which is induced to be expressed upon PRRSV infection and subsequently inhibits virus replication by reprogramming lipid droplet (LD) synthesis through transcriptional regulation. Our work provides a novel antiviral mechanism against PRRSV infection and suggests that targeting YY1 could be a new strategy for controlling PRRSV.

Genomic similarity and antibody-dependent enhancement of immune serum potentially affect the protective efficacy of commercial MLV vaccines against NADC30-like PRRSV.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant diseases affecting the pig industry worldwide. The PRRSV mutation rate is the highest among the RNA viruses. To date, NADC30-like PRRSV and highly pathogenic PRRSV (HP-PRRSV) are the dominant epidemic strains in China; however, commercial vaccines do not always provide sufficient cross-protection, and the reasons for insufficient protection are unclear. This study isolated a wild-type NADC30-like PRRSV, SX-YL1806, from Shaanxi Province. Vaccination challenge experiments in piglets showed that commercial modified live virus (MLV) vaccines provided good protection against HP-PRRSV. However, it could not provide sufficient protection against the novel strain SX-YL1806. To explore the reasons for this phenomenon, we compared the genomic homology between the MLV strain and HP-PRRSV or NADC30-like PRRSV and found that the MLV strain had a lower genome similarity with NADC30-like PRRSV. Serum neutralization assay showed that MLV-immune serum slightly promoted the homologous HP-PRRSV replication and significantly promoted the heterologous NADC30-like PRRSV strain replication in vitro, suggesting that antibody-dependent enhancement (ADE) might also play a role in decreasing MLV protective efficacy. These findings expand our understanding of the potential factors affecting the protective effect of PRRSV MLV vaccines against the NADC30-like strains.

Hydrogen Sulfide Treatment Alleviates Chilling Injury in Cucumber Fruit by Regulating Antioxidant Capacity, Energy Metabolism and Proline Metabolism.

Although low-temperature storage could maintain the quality of fruits and vegetables, it may also result in chilling injury (CI) in cold-sensitive produce, such as cucumbers. This can seriously affect their quality." The antioxidant capacity, energy metabolism and proline metabolism of cucumbers treated with hydrogen sulfide (H2S) were studied in this assay. The outcomes displayed that H2S treatment effectively reduced CI and delayed the increase in electrolyte leakage (EL) and malondialdehyde (MDA) content. In addition, the H2S-treated cucumber fruit exhibited higher L* and hue angle values, as well as nutrients such as ascorbic acid (AsA). The H2S-treated fruit showed lower levels of reactive oxygen species (ROS) and higher antioxidant enzyme activities. Meanwhile, H2S treatment also increased the activities of the essential enzymes involved in energy metabolism, including cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), H+-ATPase and Ca2+-ATPase, which improved the energy supply. H2S induced higher ornithine δ-aminotransferase (OAT) and Δ-1-pyrroline-5-carboxylate synthetase (P5CS) activities, and reduced proline dehydrogenase (PDH) activity, promoting the accumulation of proline. These results indicated that H2S could alleviate CI in the cucumber fruit by modulating antioxidant capacity, energy metabolism and proline metabolism, thereby extending the shelf life of postharvest cucumbers.

Molecular Mechanism of Porcine Epidemic Diarrhea Virus Cell Tropism.

In the 21st century, several human and swine coronaviruses (CoVs) have emerged suddenly and caused great damage to people's lives and property. The porcine epidemic diarrhea virus (PEDV), leading to enormous economic losses to the pork industry and remains a large challenge. PEDV showed extensive cell tropism, and we cannot ignore the potential risk of cross-species transmission. However, the mechanism of adaptation and cell tropism of PEDV remains largely unknown and in vitro isolation of PEDV remains a huge challenge, which seriously impedes the development of vaccines. In this study, we confirmed that the spike (S) protein determines the adaptability of PEDV to monkey Vero cells and LLC-PK1 porcine cells, and isolated exchange of S1 and S2 subunits of adaptive strains did not make PEDV adapt to cells. Further, we found that the cellular adaptability of rCH/SX/2016-SHNXP depends on S1 and the first half of S2 (S3), and the 803L and 976H of the S2 subunit are critical for rCH/SX/2016-S1HNXP+S3HNXP adaptation to Vero cells. These findings highlight the decisive role of PEDV S protein in cell tropism and the potential role of coronaviruses S protein in cross-species transmissibility. Besides, our work also provides some different insight into finding PEDV receptors and developing PEDV and other coronaviruses vaccines. IMPORTANCE CoVs can spill from an animal reservoir into a naive host to cause diseases in humans or domestic animals. PEDV results in high mortality in piglets, which has caused immense economic losses in the pork industry. Virus isolation is the first step in studying viral pathogenesis and developing effective vaccines. However, the molecular mechanism of PEDV cell tropism is largely unknown, and isolation of endemic PEDV strains remains a major challenge. This study confirmed that the S gene is the decisive gene of PEDV adaptability to monkey Vero cells and porcine LLC-PK1 cells by the PEDV reverse genetics system. Isolated exchange of S1 and S2 of adaptive strains did not make PEDV adapt to cells, and the 803L and 976H of S2 subunit are critical for rCH/SX/2016-S1HNXP+S3HNXP adaptation to Vero cells. These results illustrate the decisive role of PEDV S protein in cell tropism and highlight the potential role of coronaviruses S protein in cross-species transmissibility. Besides, our finding also provides some unique insight into identifying PEDV functional receptors and has guiding significance for developing PEDV and other coronavirus vaccines.

Antibody dependent enhancement: Unavoidable problems in vaccine development.

In some cases, antibodies can enhance virus entry and replication in cells. This phenomenon is called antibody-dependent infection enhancement (ADE). ADE not only promotes the virus to be recognized by the target cell and enters the target cell, but also affects the signal transmission in the target cell. Early formalin-inactivated virus vaccines such as aluminum adjuvants (RSV and measles) have been shown to induce ADE. Although there is no direct evidence that there is ADE in COVID-19, this potential risk is a huge challenge for prevention and vaccine development. This article focuses on the virus-induced ADE phenomenon and its molecular mechanism. It also summarizes various attempts in vaccine research and development to eliminate the ADE phenomenon, and proposes to avoid ADE in vaccine development from the perspective of antigens and adjuvants.

[Development of a blocking ELISA based on a single-domain antibody target the S1 protein of porcine epidemic diarrhea virus].

The aim of this study was to develop a blocking enzyme-linked immunosorbent assay (bELISA) based on a biotinylated nanobody target the S1 protein of porcine epidemic diarrhea virus (PEDV) for detecting the anti-PEDV antibodies and evaluating the immune effect of the vaccine. The gene encoding the single-domain antibody sdAb3 target the PEDV S1 protein was amplified and the Avitag sequence was fused at its 3'-end. The PCR product was cloned into the expression vector pET-21b for expression and purification of the sdAb3-Avitag protein. The purified sdAb3-Avitag fusion protein was biotinylated and its activity was determined. Using the recombinant S1 protein as a coating antigen, a bELISA was established and optimized. Serum samples were tested in parallel by the bELISA and a commercial kit. The recombinant vector pET21b-sdAb3-Avitag was constructed to express the tagged sdAb3. After induction for expression, the biotin-labeled sdAb3 (sdAb3-Biotin) with high purity and good activity was obtained. For the optimized bELISA, the coating concentration of the S1 protein was 200 ng/well, the serum dilution was 1:2 and incubated for 2 h, the dilution ratio of the biotinylated sdAb3 was 1:8 000 and incubated for 30 min, the dilution of the enzyme-labeled antibody was 1:5 000 and incubated for 30 min. The bELISA had no cross reaction with the sera of major porcine viruses including transmissible gastroenteritis virus, porcine reproductive and respiratory syndrome virus and showed good specificity and reproducibility. For a total of 54 porcine serum samples tested, the overall compliance rate of the bELISA with a commercial kit was 92.56%. This study developed a rapid and reliable bELISA method, which can be used for serosurveillance and vaccine evaluation for PEDV.

Reverse genetic systems: Rational design of coronavirus live attenuated vaccines with immune sequelae.

Since the end of 2019, the global COVID-19 outbreak has once again made coronaviruses a hot topic. Vaccines are hoped to be an effective way to stop the spread of the virus. However, there are no clinically approved vaccines available for coronavirus infections. Reverse genetics technology can realize the operation of RNA virus genomes at the DNA level and provide new ideas and strategies for the development of new vaccines. In this review, we systematically describe the role of reverse genetics technology in studying the effects of coronavirus proteins on viral virulence and innate immunity, cell and tissue tropism and antiviral drug screening. An efficient reverse genetics platform is useful for obtaining the ideal attenuated strain to prepare an attenuated live vaccine.

A novel intracellularly expressed NS5B-specific nanobody suppresses bovine viral diarrhea virus replication.

Bovine viral diarrhea virus (BVDV) infection causes significant economic losses to the cattle industry worldwide and still represents a huge pressure on agricultural production. Thus, the development of novel anti-BVDV strategies are urgently needed. The nonstructural protein 5 (NS5B) of BVDV is essential for viral replication. Further, the camel single-domain antibody (nanobody) represents a promising antiviral approach with the advantages of small size, stable structure, high specificity and solubility, and the recognition of specific epitopes. However, no NS5B-specific nanobodies against BVDV have been reported. In this study, NS5B-specific nanobodies were isolated from a phage display library of variable domains of Camellidae heavy chain-only antibodies (VHHs). Further, an MDBK cell line stably expressing Nb1 was established to explore antiviral activity. Results showed that Nb1 could markedly suppress BVDV replication and interact with the BVDV NS5B protein. This suggests that nanobodies have potential for the development of novel antiviral drugs against BVDV infection.

Analysis of different fractionations of three-dimensional conformable radiotherapy for esophageal cancer.

This study aims to observe and discuss the curative and side effects of three different fractionation regimen of three-dimensional conformable radiotherapy (3DCRT) for esophageal cancer. A total of 169 untreated patients of esophageal cancer were randomized into three groups: groups A (conventional group, 2.0 Gy per time), B (2.5 Gy group, 2 Gy per time), and C (3.0 Gy group, 3.0 Gy per time), respectively. Groups A, B, and C are similar in terms of partial response (P = 0.35). However, the three groups had no significant differences in terms of the complete response (P = 0.63). The three-year survival rate of group B was higher than those of the other two groups, and the difference was significant (P = 0.047). For the three-year local control rate, that of group B was also higher than those of groups A and C, but the difference was not significant (P = 0.067). The incidence rate of 3 level esophagitis and bronchitis was highest in group C (P = 0.023 and P = 0.064). The 3 level tardive radioactive esophagitis in group C was higher than those in other two groups (P = 0.037 and P = 0.04). The incidence rate of the 3 level advanced lung reaction was also the highest in the three groups (P = 0.041). The effect is better and the side effect is tolerable for the 2.5 Gy per fraction, 5 times per week; thus, it can be used clinically for 3DCRT for esophageal carcinoma.

Protective effect of melatonin against Adriamycin-induced cardiotoxicity.

The aim of this in vivo study was to explore the protective properties of melatonin against Adriamycin-induced myocardial toxicity. A rat model of breast cancer was established and the rats were randomly divided into the blank group (Blank), the solvent group [Diss; dehydrated alcohol: physiological saline (1:9)], the Adriamycin group (ADM), the melatonin group (MLT) and the melatonin + Adriamycin group (M+A). The concentrations of lipid peroxide (LPO), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in myocardial tissues were detected, the changes in myocardial tissues were observed using light microscopy and electron microscopy, and the 1-month survival rates of each group of rats were compared. Breast cancer was established in 116 rats. In the ADM group, the concentration of LPO was higher and the concentrations of SOD and GSH-Px were significantly lower than those in the blank group. In the M+A group, compared with the ADM group, the concentration of LPO was lower (P<0.05) and the concentrations of SOD and GSH-Px were higher (P<0.05). It was observed using light and electron microscopy that the myocardial injuries to the M+A group were significantly alleviated in comparison with those in the ADM group; the 1-month survival rate in the M+A group was higher than that in the ADM group. Melatonin may have a protective role in the myocardium by reducing Adriamycin-induced myocardial oxidative damage.