Evaluation of the immunogenicity of auxotrophic Lactobacillus with CRISPR-Cas9D10A system-mediated chromosomal editing to express porcine rotavirus capsid protein VP4.

Porcine rotavirus (PoRV) is an important pathogen, leading to the occurrence of viral diarrhoea . As the infection displays obvious enterotropism, intestinal mucosal immunity is the significant line of defence against pathogen invasion. Moreover, as lactic acid bacteria (LAB) show acid resistance, bile salt resistance and immune regulation, it is of great significance to develop an oral vaccine. Most traditional plasmid delivery vectors use antibiotic genes as selective markers, easily leading to antibiotic accumulation. Therefore, to select a food-grade marker in genetically engineering food-grade microorganisms is vital. Based on the CRISPR-Cas9D10A system, we constructed a stable auxotrophic Lactobacillus paracasei HLJ-27 (Lactobacillus △Alr HLJ-27) strain. In addition, as many plasmids replicate in the host bacteria, resulting in internal gene deletions. In this study,we used a temperature-sensitive gene editing plasmidto insert the VP4 gene into the genome, yielding the insertion mutant strains VP4/△Alr HLJ-27, VP4/△Alr W56, and VP4/W56. This recombinant bacterium efficiently induced secretory immunoglobulin A (SIgA)-based mucosal and immunoglobulin G (IgG)-based humoral immune responses. These oral mucosal vaccines have the potential to act as an alternative to the application of antibiotics in the future and induce efficient immune responses against PEDV infection.

Characteristics of patients with SARS-COV-2 PCR re-positivity after recovering from COVID-19.

The purpose of this study was to analyse the clinical characteristics of patients with severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) PCR re-positivity after recovering from coronavirus disease 2019 (COVID-19). Patients (n = 1391) from Guangzhou, China, who had recovered from COVID-19 were recruited between 7 September 2021 and 11 March 2022. Data on epidemiology, symptoms, laboratory test results and treatment were analysed. In this study, 42.7% of recovered patients had re-positive result. Most re-positive patients were asymptomatic, did not have severe comorbidities, and were not contagious. The re-positivity rate was 39%, 46%, 11% and 25% in patients who had received inactivated, mRNA, adenovirus vector and recombinant subunit vaccines, respectively. Seven independent risk factors for testing re-positive were identified, and a predictive model was constructed using these variables. The predictors of re-positivity were COVID-19 vaccination status, previous SARs-CoV-12 infection prior to the most recent episode, renal function, SARS-CoV-2 IgG and IgM antibody levels and white blood cell count. The predictive model could benefit the control of the spread of COVID-19.

Delivery of antigen to porcine dendritic cells by fusing antigen with porcine dendritic cells targeting peptide.

Dendritic cells (DCs) are professional antigen-presenting cells that can recognize, capture, and process antigens. Fusing molecules targeting DCs with antigens can effectively improve the efficiency with which antigens are recognized and captured by DCs. This targeting strategy can be used for vaccine development to effectively improve the efficiency of antigen recognition and capture by DCs. The targeting sequence of porcine cytotoxic T-lymphocyte associated protein 4 (CTLA4), which binds porcine DCs, was identified in this study. Recombinant Lactobacillus reuteri (L. reuteri) expressing CTLA4-6aa (LYPPPY) and CTLA4-87aa fused to the porcine epidemic diarrhea virus (PEDV) protective antigen core neutralizing epitope (COE) were used to evaluate the ability of the two targeting motifs to bind the B7 molecule on DCs. Our results demonstrate that CTLA4-6aa could bind porcine DCs, and recombinant Lactobacillus expressing the CTLA4-6aa captured by porcine DCs was more efficient than those expressing CTLA4-87aa. In addition, the expression of DC markers, toll-like receptors, and cytokines was significantly higher in the 6aa-COE/L. reuteri-stimulated porcine DCs compared to DCs treated with 87aa-COE/L. reuteri (p<0.01) and recombinant Lactobacillus expressing CTLA4-6aa enhanced the ability of porcine DCs to activate T-cell proliferation. Our analysis of the protein structure revealed that CTLA4-87aa contains intramolecular hydrogen bonds, which may have weakened the intermolecular force between the residues on porcine CTLA4 and that on B7. In conclusion, recombinant Lactobacillus expressing CTLA4-6aa were more efficiently captured by porcine DCs and had a stronger ability to promote DC maturation and enhance T-cell proliferation. The LYPPPY motif is the optimal sequence for binding to porcine DCs. Piglets immunized with recombinant Lactobacillus showed that recombinant Lactobacillus expressing CTLA4-6aa induced significant levels of anti-PEDV-specific IgG and IgA antibody responses. Our study may promote research on DC-targeting strategies to enhance the effectiveness of porcine vaccines.

Evaluation of the Immunogenicity in Mice Orally Immunized with Recombinant Lactobacillus casei Expressing Porcine Epidemic Diarrhea Virus S1 Protein.

Porcine epidemic diarrhea (PED), characterized by diarrhea, vomiting, and dehydration, is an acute enteric infectious disease of pigs. The disease is caused by porcine epidemic diarrhea virus (PEDV), which infects the intestinal mucosal surface. Therefore, mucosal immunization through the oral route is an effective method of immunization. Lactic acid bacteria, which are acid resistant and bile-salt resistant and improve mucosal immunity, are ideal carriers for oral vaccines. The S1 glycoprotein of PEDV mediates binding of the virus with cell receptors and induces neutralizing antibodies against the virus. Therefore, we reversely screened the recombinant strain pPG-SD-S1/Δupp ATCC 393 expressing PEDV S1 glycoprotein by Lactobacillus casei deficient in upp genotype (Δupp ATCC 393). Mice were orally immunized three times with the recombinant bacteria that had been identified for expression, and the changes of anti-PEDV IgG and secreted immunoglobulin A levels were observed over 70 days. The results indicated that the antibody levels notably increased after oral administration of recombinant bacteria. The detection of extracellular cytokines on the 42nd day after immunization indicated high levels of humoral and cellular immune responses in mice. The above results demonstrate that pPG-SD-S1/Δupp ATCC 393 has great potential as an oral vaccine against PEDV.

Effects of recombinant porcine Lactobacillus reuteri secreting expression of bovine lactoferrin peptides on growth performance of newborn piglets and protective effect against diarrhea virus infection

This experiment was conducted to investigate the effects of recombinant porcine Lactobacillus reuteri secreting bovine lactoferrin peptide (LFCA) on growth performance of newborn piglets and the protective effect on porcine transmissible gastroenteritis virus (TGEV)infection which caused piglet diarrhea. Experiment 1:thirty-six one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 3 groups, which were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group and control group, each group with 12 piglets. Piglets in each group were orally administered recombinant porcine Lactobacillus reuteri expressing LFCA pPG-LFCA/LR-CO21, containing empty vector plasmid PPG/LR-CO21 and equal volume phosphate buffer (PBS);oral administration continued for 3 days, and the observation time after oral administration was 14 d. During the period, piglets were fed freely, and the changes of body weight and diarrhea were recorded. Experiment 2:thirty one-day-old newborn piglets with an average body weight of about 1.5 kg were randomly divided into 5 groups and given TGEV with a half tissue culture infection dose (TCID50) of 10-7.50/mL by oral administration of 1, 3, 6, 9 and 12 mL, respectively. The observation period of 7 d was set to analyze the conditions of half lethal dose. Experiment 3:another thirty-two newborn piglets with an average body weight of about 1.5 kg were selected as experimental animals and randomly divided into 4 groups, with 8 piglets in each group. The groups were pPG-LFCA/LR-CO21 group, pPG/LR-CO21 group, control group and TGEV infect group. There were 8 replicates in each group and 1 piglet in each replicate. Each head of the experimental group was orally fed ppG-LFCA/LR-CO21, pPG/LR-CO21 and equal volume of PBS at a dose of 2..1010 CFU per day for 1 consecutive week. At 8 days of age, TGEV was infected by oral administration at half lethal dose, and samples were collected after 7 days of infection. The weight change and diarrhea of each group of piglets were recorded;hematoxylin-eosin staining was used to detect the length of intestinal villi and the depth of crypts;enzyme linked immunosorbent assay (ELISA) was used to determine total serum total immunoglobulin G (IgG) and total secretory immunoglobulin A (sIgA) antibody contents. RT-qPCR was used to detect the mRNA relative expression levels of Claudin-1, Occludin, tight junction protein-1 (ZO-1), inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8), interferon-P (IFN-P), tumor necrosis factor-a (TNF-a) and Toll-like receptor 2 (TLR2). The flora structure of the contents of the piglet's cecum was analyzed. After oral recombinant porcine Lactobacillusreuteri, compared with the control group, the average daily gain of newborn piglets in the pPG-LFCA/LR-CO21 group was significantly increased (P < 0.01), while the diarrhea rate was significantly decreased (P < 0.01). Compared with TGEV infection group, the average daily gain of piglets in pPG-LFCA/LR-CO21 group was increased and diarrhea rate was decreased, and the differences were significant (P < 0.05). Villus height and the ratio of villus height to crypt depth in jejunum and ileum were significantly increased (P < 0.05). The contents of total IgG and intestinal mucosal total sIgA antibody in serum of piglets were significantly increased (P < 0.05);the mRNA relative expression levels of tight junction protein-related genes Claudin-1, Occludin and ZO-1 in intestinal mucosal tissue were extremely significantly increased (P < 0.01), and the serum TNF-a content was extremely significantly decreased (P < 0.01). Serum IFN-P, IL-6, IL-8 and TLR2 contents were significantly increased (P < 0.01), and the survival rate of piglets was improved. The analysis of the bacterial diversity in the contents of the piglets' cecum showed that the proportion of normal intestinal flora of piglets decreased after TGEV infection. Compared with the TGEV infect group, the proportion of pathogenic bacteria Bacteroides in piglet's intestinal flora decreased by o

Public Health Measures During the COVID-19 Pandemic Reduce the Spread of Other Respiratory Infectious Diseases.

Background: Public health measures (such as wearing masks, physical distancing, and isolation) have significantly reduced the spread of the coronavirus disease-2019 (COVID-19), but the impact of public health measures on other respiratory infectious diseases is unclear. Objective: To assess the correlation between public health measures and the incidence of respiratory infectious diseases in China during the COVID-19 pandemic. Methods: We collected the data from the National Health and Construction Commission in China on the number of patients with six respiratory infectious diseases (measles, tuberculosis, pertussis, scarlet fever, influenza, and mumps) from 2017 to 2020 and assessed the correlation between public health measures and the incidence of respiratory infectious diseases. Finally, we used the data of the six respiratory infectious diseases in 2021 to verify our results. Results: We found public health measures significantly reduced the incidence of measles (p = 0.002), tuberculosis (p = 0.002), pertussis (p = 0.004), scarlet fever (p = 0.002), influenza (p = 0.034), and mumps (p = 0.002) in 2020, and prevented seasonal peaks. Moreover, the effects of public health measures were most marked during the peak seasons for these infections. Of the six respiratory infectious diseases considered, tuberculosis was least affected by public health measures. Conclusion: Public health measures were very effective in reducing the incidence of respiratory infectious diseases, especially when the respiratory infectious diseases would normally have been at their peak.

Discrimination and isolation of the virus from free RNA fragments for the highly sensitive measurement of SARS-CoV-2 abundance on surfaces using a graphene oxide nano surface.

It is highly important to sensitively measure the abundance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on various surfaces. Here, we present a nucleic acid-based detection method consisting of a new sample preparation protocol that isolates only viruses, not the free RNA fragments already present on the surfaces of indoor human-inhabited environments, using a graphene oxide-coated microbead filter. Wet wipes (100 cm2), not cotton swabs, were used to collect viruses from environmental surfaces with large areas, and viruses were concentrated and separated with a graphene oxide-coated microbead filter. Viral RNA from virus was recovered 88.10 ± 8.03% from the surface and free RNA fragment was removed by 99.75 ± 0.19% from the final eluted solution. When we tested the developed method under laboratory conditions, a 10-fold higher viral detection sensitivity (Detection limit: 1 pfu/100 cm2) than the current commercial protocol was observed. Using our new sample preparation protocol, we also confirmed that the virus was effectively removed from surfaces after chemical disinfection; we were unable to measure the disinfection efficiency using the current commercial protocol because it cannot distinguish between viral RNA and free RNA fragments. Finally, we investigated the presence of SARS-CoV-2 and bacteria in 12 individual negative pressure wards in which patients with SARS-CoV-2 infection had been hospitalized. Bacteria (based on 16 S DNA) were found in all samples collected from patient rooms; however, SARS-CoV-2 was mainly detected in rooms shared by two patients.

Recent Progress in the Drug Development Targeting SARS-CoV-2 Main Protease as Treatment for COVID-19.

The sudden outbreak of 2019 novel coronavirus (2019-nCoV, later named SARS-CoV-2) rapidly turned into an unprecedented pandemic of coronavirus disease 2019 (COVID-19). This global healthcare emergency marked the third occurrence of a deadly coronavirus (CoV) into the human society after entering the new millennium, which overwhelmed the worldwide healthcare system and affected the global economy. However, therapeutic options for COVID-19 are still very limited. Developing drugs targeting vital proteins in viral life cycle is a feasible approach to overcome this dilemma. Main protease (Mpro) plays a dominant role in processing CoV-encoded polyproteins which mediate the assembly of replication-transcription machinery and is thus recognized as an ideal antiviral target. Here we summarize the recent progress in the discovery of anti-SARS-CoV-2 agents against Mpro. Combining structural study, virtual screen, and experimental screen, numerous therapeutic candidates including repurposed drugs and ab initio designed compounds have been proposed. Such collaborative effort from the scientific community would accelerate the pace of developing efficacious treatment for COVID-19.

Preparation of monoclonal antibodies against VP2 protein of Bovine parvovirus and development of a double antibodies sandwich ELISA method

In order to establish a rapid method for the detection of bovine parvovirus (BPV), primers were designed according to the VP2 sequence of BPV from NCBI. The VP2 gene was amplified from the viral DNA by PCR and cloned into a recombinant expression vector pProHTa. The recombinant plasmid pProHTa-BPV-VP2 was transformed into Escherichia coli Rosetta cells, which were induced to express the VP2 protein. The purified VP2 protein was immunized to mice (100 g/mouse). After cell fusion, screening, and subcloning, five positive hybridoma cell lines were obtained. They were designated as 5G9, 2B5, 6A3, 7E8, and 2B6, respectively. The subtypes of the five monoclonal antibodies (McAbs) were identified as IgG2a, IgG2b, IgM, IgM, and IgA, respectively. Indirect immunofluorescence assay showed that all McAbs reacted with BPV specifically. In the meanwhile, the purified VP2 protein was immunized to New Zealand rabbits to prepare anti-VP2 poly-antibodies (PcAbs). Subsequently, a double antibody sandwich ELISA was established for the detection of BPV using the PcAbs as capture antibody and 2B5 as detecting antibody. The specificity detection showed that only the BPV was positive, and there was no reaction with BRV, PRV, TGEV, PEDV, or BVDV. The sensitivity test showed that the minimum detection amount of this method was 3.125 x 102.8TCID50/mL, which showed high sensitivity. The results of the repetitive test showed that the intra-and inter-batch coefficients of variation were less than 10%. This method was used to detect 269 clinical diarrhea samples, in which 14 samples were positive for BPV. The coincidence rate was 100% between this method and PCR. In summary, a double antibody sandwich ELISA method was established using anti-BPV VP2 protein McAbs and anti-VP2 PcAbs. This method has good specificity, sensitivity, and reproducibility, which can provide an effective detection method for the rapid diagnosis of BPV infection and a reliable means for epidemiological investigation as well as disease prevention and control.

Epidemiological investigation of feline infectious peritonitis in cats living in Harbin, Northeast China from 2017 to 2019 using a combination of an EvaGreen-based real-time RT-PCR and serum chemistry assays.

Feline infectious peritonitis (FIP) is caused by the FIP virus (FIPV), a highly virulent mutant form of feline coronavirus (FCoV). This disease is one of the most important infectious diseases in cats, and it is associated with high mortality, particularly among younger cats. In this study, we isolated a wild-type FIPV HRB-17 epidemic strain from the blood sample of household pet cat exhibiting the characteristic wet-form FIP symptoms, which has been confirmed further by animal infection. Further, we developed an EvaGreen-based real-time RT-PCR assay for the accurate detection of FCoV based on the amplification of the highly conserved FIPV N gene. Then, using a combination of the real-time RT-PCR approach and a serum chemistry assay, we performed an epidemiological survey of FIPV infection in cats living in Harbin City, Northeast China. The results indicated that the EvaGreen-based real-time RT-PCR assay can be used for screening FCoV infection in the affected cats at an analytical detection limit of 8.2 × 101 viral genome copies/µL, but could not effectively distinguish FIPVs from FECVs. Additionally, the results of the epidemiological survey investigating feline blood samples (n = 1523) collected between July 2017 to July 2019 revealed an FIPV prevalence of approximately 12% (189/1523). Maybe, the prevalence would be less than 12% due to the real-time RT-PCR assay could not accurately differentiate FIPV and FECV. Nevertheless, it still highlighted the severity of the FIP epidemic in cats and reiterated the urgent need to develop effective anti-FIP therapeutic agents and anti-FIPV vaccines. As pet cats are household animals, risk communication and continuous region-extended surveillance cat programs are recommended.

Cloning, Prokaryotic Soluble Expression, and Analysis of Antiviral Activity of Two Novel Feline IFN-ω Proteins

Cats are becoming more popular as household companions and pets, forming close relationships with humans. Although feline viral diseases can pose serious health hazards to pet cats, commercialized preventative vaccines are lacking. Interferons (IFNs), especially type I IFNs (IFN-α, IFN-ß, and interferon omega (IFN-ω)), have been explored as effective therapeutic drugs against viral diseases in cats. Nevertheless, there is limited knowledge regarding feline IFN-ω (feIFN-ω), compared to IFN-α and IFN-ß. In this study, we cloned the genes encoding feIFN-ωa and feIFN-ωb from cat spleen lymphocytes. Homology and phylogenetic tree analysis revealed that these two genes belonged to new subtypes of feIFN-ω. The recombinant feIFN-ωa and feIFN-ωb proteins were expressed in their soluble forms in Escherichia coli, followed by purification. Both proteins exhibited effective anti-vesicular stomatitis virus (VSV) activity in Vero, F81 (feline kidney cell), Madin-Darby bovine kidney (MDBK), Madin-Darby canine kidney (MDCK), and porcine kidney (PK-15) cells, showing broader cross-species antiviral activity than the INTERCAT IFN antiviral drug. Furthermore, the recombinant feIFN-ωa and feIFN-ωb proteins demonstrated antiviral activity against VSV, feline coronavirus (FCoV), canine parvovirus (CPV), bovine viral diarrhea virus (BVDV), and porcine epidemic diarrhea virus (PEDV), indicating better broad-spectrum antiviral activity than the INTERCAT IFN. The two novel feIFN-ω proteins (feIFN-ωa and feIFN-ωb) described in this study show promising potential to serve as effective therapeutic agents for treating viral infections in pet cats.