Porcine epidemic diarrhea virus: A review of detection, inhibition of host gene expression and evasion of host innate immune.

As one of the most important swine enteropathogenic coronavirus, porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute and devastating enteric disease that causes lethal watery diarrhea in suckling piglets. Recent progress in studying PEDV has revealed many intriguing findings on its prevalence and genetic evolution, rapid diagnosis, suppression of host gene expression, and suppression of the host innate immune system. Due to the continuous mutation of the PEDV genome, viral evasions from innate immune defenses and mixed infection with other coronaviruses, the spread of the virus is becoming wider and faster, making it even more necessary to prevent the infections caused by wild-type PEDV variants. It has also been reported that PEDV nsp1 is an essential virulence determinant and is critical for inhibiting host gene expression by structural and biochemical analyses. The inhibition of host protein synthesis employed by PEDV nsp1 may contribute to the regulation of host cell proliferation and immune evasion-related biological functions. In this review, we critically evaluate the recent studies on these aspects of PEDV and assess prospects in understanding the function of PEDV proteins in regulating host innate immune response and viral virulence.

Synthesis and pharmacodynamic evaluation of Dihydropteridone derivatives against PDCoV in vivo and in vitro.

Porcine Delta Coronavirus (PDCoV) infection can induce serious dehydration, diarrhea and even death of piglets, which has caused huge losses to the breeding industry. PDCoV has been reported to have the potential for cross species transmission, and even reports of infecting humans have emerged. At present, there are still no effective prevention and control measures for PDCoV. In this study, we have designed and synthesized a series of unreported Dihydropteridone derivatives. All of these compounds were evaluated for the against PDCoV in vivo and in vitro for the first time. In this study, antiviral activity (17.34 ± 7.20 µM) and low cytotoxicity (>800 µM) was found in compound W8. Compound W8 exerts antiviral effect on PDCoV by inhibiting cell apoptosis and inflammatory factors caused by virus infection in vitro. In addition, lung and small intestinal lesions caused by PDCoV infection in mice could be significantly reduced by compound W8. These findings highlight the potential of compound W8 as a valuable therapeutic option against PDCoV infection, and lay a foundation for further research and development in this field.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. RESULTS: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. CONCLUSION: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Susceptibility to mice and potential evolutionary characteristics of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in suckling piglets and has the potential for cross-species transmission, posing a threat to animal and human health. However, the susceptibility profile of different species of mice to PDCoV infection and its evolutionary characteristics are still unclear. In the current study, we found that BALB/c and Kunming mice are susceptible to PDCoV. Our results showed that there were obvious lesions in intestinal and lung tissues from the infected mice. PDCoV RNAs were detected in the lung, kidney, and intestinal tissues from the infected mice of both strains, and there existed wider tissue tropism in the PDCoV-infected BALB/c mice. The RNA and protein levels of aminopeptidase N from mice were relatively high in the kidney and intestinal tissues and obviously increased after PDCoV infection. The viral-specific IgG and neutralizing antibodies against PDCoV were detected in the serum of infected mice. An interesting finding was that two key amino acid mutations, D138H and Q641K, in the S protein were identified in the PDCoV-infected mice. The essential roles of these two mutations for PDCoV-adaptive evolution were confirmed by cryo-electron microscope structure model analysis. The evolutionary characteristics of PDCoV among Deltacoronaviruses (δ-CoVs) were further analyzed. δ-CoVs from multiple mammals are closely related based on the phylogenetic analysis. The codon usage analysis demonstrated that similar codon usage patterns were used by most of the mammalian δ-CoVs at the global codon, synonymous codon, and amino acid usage levels. These results may provide more insights into the evolution, host ranges, and cross-species potential of PDCoV.

Discovery and pharmacodynamic evaluation of the novel butene lactone derivative M355 against influenza A virus in vitro and in vivo.

A new series of butene lactone derivatives were designed according to an influenza neuraminidase target and their antiviral activities against H1N1 infection of Madin-Darby canine kidney cells were evaluated. Among them, a compound that was given the name M355 was identified as the most potent against H1N1 (EC50 = 14.7 µM) with low toxicity (CC50 = 538.13 µM). It also visibly reduced the virus-induced cytopathic effect. Time-of-addition analysis indicated that H1N1 was mostly suppressed by M355 at the late stage of its infectious cycle. M355 inhibited neuraminidase in a dose-dependent fashion to a similar extent as oseltamivir, which was also indicated by a computer modeling experiment. In a mouse model, lung lesions and virus load were reduced and the expression of nucleoprotein was moderated by M355. The enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction analyses revealed that the levels of interferon-γ, interferon regulatory factor-3, Toll-like receptor-3, tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-8 were downregulated in the M355-treated groups, whereas the levels of IL-10 and IL-13 were upregulated. Similarly, IgG was found to be increased in infected mice plasma. These results demonstrate that M355 inhibit the expression of H1N1 in both cellular and animal models. Thus, M355 has the potential to be effective in the treatment of influenza A virus infection.

Expression of porcine interferon-α and its bioactivity analysis in vitro and in vivo.

Interferon α (IFN-α) plays a crucial role in the host's immune response. In this study, the amino acid sequence of porcine interferon α (PoIFN-α) was analyzed. Seven substitutions, S38F, H40Q, F43L, N78D, Y86C, S151A, and R156T, were mutated and obtained by aligning the sequences of PoIFN-α subtypes. The PoIFN-α mutants were designed, expressed, and purified in E. coli. The antiviral activities of these PoIFN-αs were measured in Vero and swine testis cells against vesicular stomatitis virus (VSV). Their inhibitory abilities on pseudorabies virus (PRV) were also examined. Commercial PoIFN-α was used as a control. We found the ideal inducer concentration of isopropyl ß-D-thiogalactoside was 1 mM, and the best time-point for induction was 8 h. The PoIFN-α mutant named PoIFN-α-156s had the highest antiviral activity, which was about 200-fold more than that of PoIFN-α. PoIFN-α-156s could inhibit VSV and PRV replication in a dose-dependent manner in vitro. The half-life of PoIFN-α-156s was longer than that of PoIFN-α in mice, and the effective antiviral action was higher than PoIFN-α. Animal experiments showed that PoIFN-α-156s could decrease the viral load after infection with VSV. Overall, these results suggest that recombinant PoIFN-α-156s has the ability of antivirus, and is feasible for veterinary clinical applications and fundamental research.

Assessments of different inactivating reagents in formulating transmissible gastroenteritis virus vaccine.

BACKGROUND: Transmissible gastroenteritis virus (TGEV) causes enteric infection in piglets, characterized by vomiting, severe diarrhea and dehydration, and the mortality in suckling piglets is often high up to 100%. Vaccination is an effective measure to control the disease caused by TGEV. METHODS: In this study, cell-cultured TGEV HN-2012 strain was inactivated by formaldehyde (FA), ß-propiolactone (BPL) or binaryethylenimine (BEI), respectively. Then the inactivated TGEV vaccine was prepared with freund's adjuvant, and the immunization effects were evaluated in mice. The TGEV-specific IgG level was detected by ELISA. The positive rates of CD4+, CD8+, CD4+IFN-γ+, CD4+IL-4+ T lymphocytes were detected by flow cytometry assay. Lymphocyte proliferation assay and gross pathology and histopathology examination were also performed to assess the three different inactivating reagents in formulating TGEV vaccine. RESULTS: The results showed that the TGEV-specific IgG level in FA group (n = 17) was earlier and stronger, while the BEI group produced much longer-term IgG level. The lymphocyte proliferation test demonstrated that the BEI group had a stronger ability to induce spleen lymphocyte proliferation. The positive rates of CD4+ and CD8+ T lymphocyte subsets of peripheral blood lymphocyte in BEI group was higher than that in FA group and BPL groups by flow cytometry assay. The positive rate of CD4+IFN-γ+ T lymphocyte subset was the highest in the BPL group, and the positive rate of CD4+IL-4+ T lymphocyte subset was the highest in the FA group. There were no obvious pathological changes in the vaccinated mice and the control group after the macroscopic and histopathological examination. CONCLUSIONS: These results indicated that all the three experimental groups could induce cellular and humoral immunity, and the FA group had the best humoral immunity effect, while the BEI group showed its excellent cellular immunity effect.

The role of different SIRT1-mediated signaling pathways in toxic injury.

Common environmental pollutants and drugs encountered in everyday life can cause toxic damage to the body through oxidative stress, inflammatory stimulation, induction of apoptosis, and inhibition of energy metabolism. Silent information regulator 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent deacetylase, is a member of the evolutionarily highly conserved Sir2 (silent information regulator 2) superprotein family, which is located in the nucleus and cytoplasm. It can deacetylate protein substrates in various signal transduction pathways to regulate gene expression, cell apoptosis and senescence, participate in the process of neuroprotection, energy metabolism, inflammation and the oxidative stress response in living organisms, and plays an important role in toxic damage caused by toxicants and in the process of SIRT1 activator/inhibitor antagonized toxic damage. This review summarizes the role that SIRT1 plays in toxic damage caused by toxicants via its interactions with protein substrates in certain signaling pathways.

A nano silicon adjuvant enhances inactivated transmissible gastroenteritis vaccine through activation the Toll-like receptors and promotes humoral and cellular immune responses.

Inactivated transmissible gastroenteritis virus (TGEV) vaccines are widely used in swine herds in China. These are limited, however, by the need to elicit both humoral and cellular immunity, as well as the efficiency of adjuvants. In this study, a 70-nm nano silicon particle was applied with inactivated TGEV vaccine in mice, and its immune-enhancing effects and mechanism of action investigated. We found that nano silicon applied with inactivated TGEV vaccine induced high antibody titers, increase IL-6, TNF-α and IFN-γ expression, and stimulate CD3+ T cell proliferation with a high CD4+/CD8+ T lymphocyte ratio. Nano silicon could quickly activate innate and adaptive immunity by stimulating Toll-like receptor signaling pathways, indicating that the nano silicon adjuvant enhanced long-term humoral and early cellular immune responses when combined with inactivated TGEV vaccine. Nano silicon could be considered for use as an antigen- carrier and adjuvant for veterinary vaccines.

Proteomics analysis of PK-15 cells infected with porcine parvovirus and the effect of PCBP1 on PPV replication.

Porcine parvovirus (PPV) is one of the most important pathogens that cause reproductive failure in pigs. However, the pathogenesis of PPV infection remains unclear. Proteomics is a powerful tool to understand the interaction between virus and host cells. In the present study, we analyzed the proteomics of PPV-infected PK-15 cells. A total of 32 and 345 proteins were differentially expressed at the early and replication stages, respectively. Subsequent gene ontology annotation and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed these differentially expressed proteins were significantly enriched in pathways including toll-like receptor signaling pathway, tumor necrosis factor signaling pathway, and viral carcinogenesis. The expression of poly (rC) binding protein 1 (PCBP1) was observed to decrease after PPV infection. Overexpressed or silenced PCBP1 expression inhibited or promoted PPV infection. Our studies established a foundation for further exploration of the multiplication mechanism of PPV. IMPORTANCE: Porcine parvovirus (PPV) is a cause of reproductive failure in the swine industry. Our knowledge of PPV remains limited, and there is no effective treatment for PPV infection. Proteomics of PPV-infected PK-15 cells was conducted to identify differentially expressed proteins at 6 hours post-infection (hpi) and 36 hpi. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that various pathways participate in PPV infection. Poly (rC) binding protein 1 was confirmed to inhibit PPV replication, which provided potential targets for anti-PPV infection. Our findings improve the understanding of PPV infection and pave the way for future research in this area.

Antiviral effect of baicalein on Porcine Deltacoronavirus infection by regulating the inflammatory responses through PI3K-Akt-NF-κB signaling pathway in cultured cells.

Porcine deltacoronavirus (PDCoV) is an newly emerged enteropathogenic coronavirus, mainly causing diarrhea in suckling piglets, and also has the potential for cross-species transmission. However, there are no effective vaccines or specific therapeutic agents for PDCoV. This study investigates the antiviral properties of baicalein against PDCoV infection in swine testicle cells (ST). It reveals that baicalein exerts a dose-dependent inhibitory effect on PDCoV replication, primarily targeting the replication stage of the viral infection by impeding viral RNA and protein synthesis. Furthermore, treatment with baicalein leads to reduced phosphorylation of PI3K, AKT, and NF-κB p65 proteins, along with decreased mRNA levels of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-α). These results signify that PDCoV replication is inhibited through the inhibition of the PI3K-Akt-NF-κB protein signaling pathway, thereby suppressing the inflammatory response. In conclusion, it underscores the potential of baicalein as a therapeutic candidate for treating PDCoV infection.

Molecular detection and genomic characterization of Torque teno sus virus 1 and 2 from domestic pigs in central China.

In the present study, Torque teno sus viruses (TTSuVs) were detected in tissue and blood samples obtained from domestic pigs in central China, and complete genomes of TTSuVs were characterized. A total of three tissue samples (3/20, 15 %) from post-weaning multisystemic wasting syndrome-affected pigs and 30 blood samples (30/40, 75 %) from healthy pigs were positive for Torque teno sus virus 1 (TTSuV1) and/or 2 (TTSuV2). Two TTSuV strains (TTV1Hn54 and TTV2Hn93) comprising 2,794 and 2,875 nucleotides, respectively, each had four open reading frames (ORFs) and the untranslated region with TATA box and GC-rich region. Genomic sequence of TTV2Hn93 strain was unique in length compared with other TTSuV2 genomic sequences. Interestingly, three rolling-circle replication (RCR) motif-IIIs (YXXK) which were located at amino acid (aa) position 166-169, 328-331, and 379-382, respectively, were found in the ORF1 of TTV1Hn54. Two RCR motif-IIIs (YXXK) at the aa position 105-108 and 480-483 respectively, were also identified in the ORF1 of TTV2Hn93. Phylogenetic tree based on complete genomes showed that TTV1Hn54 strain was designated into type TTSuV1b and had a slight high sequence identity of 91 % with the Canada strain (JQ120664). TTV2Hn93 strain was classified into subtype TTSuV2d and shared the highest identity (97 %) with the Spain strain (GU570207).

Expression of codon-optimized PDCoV-RBD protein in baculovirus expression system and immunogenicity evaluation in mice.

Porcine deltacoronavirus (PDCoV) is a global epidemic enteropathogenic coronavirus that mainly infects piglets, and causes huge losses to the pig industry. However, there are still no commercial vaccines available for PDCoV prevention and controlment. Receptor-binding domain (RBD) is located at the S1 subunit of PDCoV and is the major target for developing viral inhibitor and vaccine. In this study, the characteristics of the RBD were analyzed by bioinformatic tools, and codon optimization was performed to efficiently express the PDCoV-RBD protein in the insect baculovirus expression system. The purified PDCoV-RBD protein was obtained and fully emulsified with CPG2395 adjuvant, aqueous adjuvant and Al(OH)3 adjuvant, respectively, to develop vaccines. The humoral and cellular immune responses were assessed on mice. The results showed that both the RBD/CPG2395 and RBD/aqueous adjuvant could induce stronger immune responses in mice than that of RBD/Al(OH)3. In addition, the PDCoV challenge infection was conducted and the RBD/CPG2395 could provide better protection against PDCoV in mice. Our study showed that the RBD protein has good antigenicity and can be used as a protective antigen, which provided a basis for the development of the PDCoV vaccine.

Polydopamine-based nanomedicines for efficient antiviral and secondary injury protection therapy.

Viral infections continue to threaten human health. It remains a major challenge to efficiently inhibit viral infection while avoiding secondary injury. Here, we designed a multifunctional nanoplatform (termed as ODCM), prepared by oseltamivir phosphate (OP)-loaded polydopamine (PDA) nanoparticles camouflaged by the macrophage cell membrane (CM). OP can be efficiently loaded onto the PDA nanoparticles through the π-π stacking and hydrogen bonding interactions with a high drug-loading rate of 37.6%. In particular, the biomimetic nanoparticles can accumulate actively in the damaged lung model of viral infection. At the infection site, PDA nanoparticles can consume excess reactive oxygen species and be simultaneously oxidized and degraded to achieve controlled release of OP. This system exhibits enhanced delivery efficiency, inflammatory storm suppression, and viral replication inhibition. Therefore, the system exerts outstanding therapeutic effects while improving pulmonary edema and protecting lung injury in a mouse model of influenza A virus infection.

RPA-CRISPR/Cas12a-Based Detection of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis, HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for H. parasuis that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/µL of DNA template, p < 0.05), which is 104 -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of H. parasuis did not react with other common pig pathogens, including Streptococcus suis type II and IX, Actinobacillus pleuropneumoniae, Escherichia coli, Salmonella, Streptococcus suis, and Staphylococcus aureus (p < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of H. parasuis clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for H. parasuis at the field level.

Serological Investigation and Genetic Characteristics of Pseudorabies Virus between 2019 and 2021 in Henan Province of China.

In late 2011, severe pseudorabies (PR) outbreaks occurred among swine herds vaccinated with the Bartha-K61 vaccine in many provinces of China, causing enormous economic losses for the pork industry. To understand the epidemic profile and genetic characteristics of the pseudorabies virus (PRV), a total of 35,796 serum samples were collected from 1090 pig farms of different breeding scales between 2019 and 2021 in the Henan province where swine had been immunized with the Bartha-K61 vaccine, and PRV glycoprotein E (gE)-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA). The results reveal that the overall positive rate for PRV gE antibodies was 20.33% (7276/35,796), which decreased from 25.00% (2596/10,385) in 2019 to 16.69% (2222/13,315) in 2021, demonstrating that PR still existed widely in pig herds in the Henan province but displayed a decreasing trend. Further analysis suggested that the PRV-seropositive rate may be associated with farm size, farm category, quarter, region and the cross-regional transportation of livestock. Moreover, the gE gene complete sequences of 18 PRV isolates were obtained, and they shared a high identity (97.1-100.0%) with reference strains at the nucleotide level. Interestingly, the phylogenetic analysis based on the gE complete sequences found that there were both classical strains and variant strains in pig herds. The deduced amino acid sequence analysis of the gE gene showed that there were unique amino acids in the classical strains, the variant strains and genotype Ⅱ strains. This study provides epidemiological data that could be useful in the prevention of pseudorabies in Henan, China, and this finding contributed to our understanding of the epidemiology and evolution of PRV.

Co-infection of porcine deltacoronavirus and porcine epidemic diarrhoea virus alters gut microbiota diversity and composition in the colon of piglets.

Porcine deltacoronavirus (PDCoV) and porcine epidemic diarrhoea virus (PEDV) are the main porcine enteric coronaviruses that cause severe diarrhoea in piglets, posing huge threat to the swine industry. Our previous study verified that the co-infection of PDCoV and PEDV is common in natural swine infections and obviously enhances the disease severity in piglets. However, the effects of co-infection of PDCoV and PEDV on intestinal microbial community are unknown. In current study, the microbial composition and diversity in the colon of piglets were analyzed. Our results showed that both of PDCoV and PEDV were mainly distributed in the small intestines and caused severe damage of ileum but not colon in the co-inoculated piglets. Furthermore, we observed that PDCoV and PEDV co-infection alters the gut microbiota composition at the phylum, family and genus levels. The abundance of Mitsuokella and Collinsella at genus level were significantly increased in PDCoV-PEDV co-infection piglets. Spearman's correlation analysis further suggested that there existed strong positive correlation between Mitsuokella and TNF-α, IL-6 and IL-8 secretion, these two factors may together aggravating the small intestine pathological lesions. These results proved there existed obvious correlation between the disease severity caused by PDCoV-PEDV co-infection and intestinal microbial community.

Mechanism of selenomethionine inhibiting of PDCoV replication in LLC-PK1 cells based on STAT3/miR-125b-5p-1/HK2 signaling.

There are no licensed therapeutics or vaccines available against porcine delta coronavirus (PDCoV) to eliminate its potential for congenital disease. In the absence of effective treatments, it has led to significant economic losses in the swine industry worldwide. Similar to the current coronavirus disease 2019 (COVID-19) pandemic, PDCoV is trans-species transmissible and there is still a large desert for scientific exploration. We have reported that selenomethionine (SeMet) has potent antiviral activity against PDCoV. Here, we systematically investigated the endogenous immune mechanism of SeMet and found that STAT3/miR-125b-5p-1/HK2 signalling is essential for the exertion of SeMet anti-PDCoV replication function. Meanwhile, HK2, a key rate-limiting enzyme of the glycolytic pathway, was able to control PDCoV replication in LLC-PK1 cells, suggesting a strategy for viruses to evade innate immunity using glucose metabolism pathways. Overall, based on the ability of selenomethionine to control PDCoV infection and transmission, we provide a molecular basis for the development of new therapeutic approaches.

Preparation of a Single-Chain Antibody against Nucleocapsid Protein of Porcine Deltacoronavirus by Phage Display Technology.

Porcine deltacoronavirus (PDCoV) mainly causes severe diarrhea and intestinal pathological damage in piglets and poses a serious threat to pig farms. Currently, no effective reagents or vaccines are available to control PDCoV infection. Single-chain fragment variable (scFv) antibodies can effectively inhibit virus infection and may be a potential therapeutic reagent for PDCoV treatment. In this study, a porcine phage display antibody library from the peripheral blood lymphocytes of piglets infected with PDCoV was constructed and used to select PDCoV-specific scFv. The library was screened with four rounds of biopanning using the PDCoV N protein, and the colony with the highest affinity to the PDCoV N protein was obtained (namely, N53). Then, the N53-scFv gene fragment was cloned into plasmid pFUSE-hIgG-Fc2 and expressed in HEK-293T cells. The scFv-Fc antibody N53 (namely, scFv N53) was purified using Protein A-sepharose. The reactive activity of the purified antibody with the PDCoV N protein was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), western blot and indirect immunofluorescence assay (IFA). Finally, the antigenic epitopes that the scFv N53 recognized were identified by a series of truncated PDCoV N proteins. The amino acid residues 82GELPPNDTPATTRVT96 of the PDCoV N protein were verified as the minimal epitope that can be recognized by the scFv-Fc antibody N53. In addition, the interaction between the scFv-Fc antibody N53 and the PDCoV N protein was further analyzed by molecule docking. In conclusion, our research provides some references for the treatment and prevention of PDCoV.

Influenza-existing drugs and treatment prospects.

Influenza is a century-old disease that continues to baffle humans by its frequently changing nature, seasonal epidemics, and occasional pandemics. Approximately 9% of the world's population is infected by the influenza virus annually. The emergence of novel strains because of rapid mutations as well as interspecies disease contamination, limits the efficiency of strain-specific vaccines. Anti-influenza drugs such as neuraminidase inhibitors, M2 ion channel inhibitors, etc. have become the first line of defense in prophylaxis and early containment of the disease. But the growing drug resistance due to drug-induced selective pressure has also limited the efficacy of those drugs. Because we can't predict the next strain types, their virulence, or the severity of the next epidemic/pandemic caused by influenza virus, we ought to gear up for the development of novel anti-influenza drugs with a broad spectrum of reactivity against all strains and subtypes, better bioavailability, easier administrative pathways, and lesser adverse effects. Various new compounds with each having significantly different target molecules and pharmacologic activity have shown potential against influenza virus strains in laboratory situations as well as clinical trials. We should also consider combination therapy to boost the efficacy of existing drugs. This review is aiming to succinctly document the recent signs of progress regarding anti-influenza drugs both in the market and under investigation.