Establishment and application of triple PCR method for CPV, CCoV and CRV

Canine parvovirus (CPV), canine coronavirus (CCoV) and canine rotavirus (CRV) are the three main causative viruses of diarrhea in dogs with similar clinical symptoms;thereby it is necessary to establish a high effective molecular detection method for differentiating the above pathogens. By optimizing the primer concentration and annealing temperature, a triple PCR method was established for simultaneous detection of CPV, CCoV and CRV, and then the specificity, sensitivity and repeatability of the method were tested. The results showed that the target fragments of CPV VP2 gene (253 bp), CCoV ORF-1b gene (379 bp) and CRV VP6 gene (852 bp) could be accurately amplified by the triple PCR method with high specificity, the detection limits of CPV, CCOV and CRV were 6.44x10-1 pg/L, 8.72x10-1 pg/L and 8.35x10-1 pg/L respectively with high sensitivity, and the method had good stability. Using this triple PCR method, 135 canine diarrhea fecal samples collected in Chengdu region from 2019 to 2020 were detected, and compared with those of single PCR method. The detection rates of CPV, CCoV and CRV were 16.30%, 20.74% and 4.44%, respectively, and the total infection rate was 51.11% (65/135) with 20.00% (13/65) co-infection rate. The detection results were consistent with three single PCR methods. In conclusion, CPV/CCoV/CRV triple PCR method successfully established in this paper can be applied as an effective molecular method to detection of related pathogens and to the epidemiological investigation.

Establishment of a duplex fluorescent quantitative PCR assay for detection of Porcine circovirus type 2 and type 3

To establish a method for simultaneous detection of porcine circovirus type 2 (PCV2) and porcine circovirus type 3 (PCV3), specific primers and TaqMan probes were designed after sequence alignment according to the specific sequences of PCV2 Cap gene and PCV3 Cap gene on GenBank. By optimizing the reaction conditions, a duplex fluorescence quantitative PCR detection method for simultaneous detection of porcine circovirus type 2 and 3 was established, and the specificity, sensitivity, and reproducibility were tested. Specificity test results showed that in addition to the positive test results for PCV2 and PCV3, tests for PRRSV, CSFV, PPV, PRV, PEDV, and TGEV were all negative with no cross-reaction, indicating its good specificity. Sensitivity test results showed that the minimum detection limit for detection of PCV2 and PCV3 can both reach 10 copies.L-1, indicating its high sensitivity. The coefficient of variation within and between groups of this method was less than 2%, indicating its good stability. A total of 181 pork and whole blood samples collected from Zhejiang Province were tested using the detection method established in this article and the standard common fluorescent PCR detection method. The results showed that the positive rate of PCV2 was 50.83% (92/181), the positive rate of PCV3 was 37.57% (68/181), and the co-infection rate of PCV2 and PCV3 was 12.15% (22/181). The above detection results of ordinary fluorescent PCR were 50.28% (91/181), 36.46% (66/181), and the co-infection rate was 11.60% (21/181). The coincidence rates of the two methods for PCV2 and PCV3 can reach 98.91% and 97.06%, and the coincidence rate for PCV2 and PCV3 mixed infection were 95.45%. In summary, the duplex fluorescence quantitative PCR detection method established in this experiment can distinguish PCV2 and PCV3 rapidly, which can be used for pathogen detection and epidemiological investigation.

Silent superbug killers in a river near you how factory farms contaminate public water courses on three continents

Water downstream from factory farms harbours an invisible threat to people's health which could eclipse the COVID-19 crisis. The threat? Antibiotic Resistance Genes (ARGs) which are driving antimicrobial resistance the world's superbug crisis - projected to kill up to 10 million people annually by 2050. This publication reports the presence of ARGs in animal waste discharged from industrial farms into public waterways or onto soil (or crops) in four countries. Gauge community impact and sentiment regarding the issue was also highlighted. The water and sediment from public water courses connected to effluent discharges from 6-10 pig farms were tested in each of four countries (Canada, Spain, Thailand and the USA).

Effects of Porcine deltacoronavirus on the transduction pathway of the innate immune signal

Porcine deltacoronavirus (PDCOV) is a new type of pig intestinal coronavirus, which targets pig small intestinal epithelial cells to cause severe enteritis. After infecting the host, PDCoV finishes its proliferation in the host cell by antagonism or escape the innate immune signaling transduction pathway. In order to understand the action mechanism of PDCOV 0n the congenital immune signal transduction pathways, this paper reviews the effects of PDCOV on RLR, Jak-STAT, MAPK and mitochondrial signaling pathway to clarify the relationship between PDCOV and host innate immune signaling transduction pathways in order to provide help for the prevention and treatment of PDCOV infection.

Effect of Drebrin protein on the proliferation of PEDV in Vero cells

Previous studies have revealed that developmental regulated brain protein (Drebrin) is involved in cell- to-cell communication, nerve transmission, tumor metastasis, spermatogenesis and other life activities, but there are few studies on viruses. The aim of the current research was therefore, to study the function of Drebrin and its effect on the proliferation of porcine epidemic diarrhea virus (PEDV). The Drebrin gene was cloned according to the Drebrin gene sequence (XM_008015438.2) of Chlorocebus sabaeus registered by GenBank, and the phylogenetic tree was constructed to analyze its homology. The results showed that the CDS region of Vero cells Drebrin gene was 2088 bp long, encoding 695 amino acids, and was relatively conserved and had high homology with all species. To investigate the effect of Drebrin on the proliferation of PEDV in Vero cells, the eukaryotic expression vector pcDNA3.1-Drebrin-Flag was constructed. After transfection of Vero cells with different concentrations of pcDNA3.1-Drebrin-Flag, cells were infected with PEDV. Our results showed that overexpression of Drebrin in Vero cells could significantly inhibit the intracellular PEDV mRNA level and N protein expression, reduce the extracellular virus titer and inhibit the proliferation of PEDV. Further study on the interaction between Drebrin and PEDV S proteins by laser confocal technique was also performed. The results showed that Drebrin and S protein were co-located in the cytoplasm, suggesting that the two proteins may interact with each other. This study demonstrated for the first time that Drebrin can inhibit PEDV proliferation in Vero cells, laying a foundation for further research in to Drebrin function and provides a valuable information for anti-PEDV research.

Cloning, expression and identification of truncated spike gene of IBV (Sf200) and chicken granulocyte- macrophage colony stimulating factor (GM-CSF)

The aim of this study was to clone, express and identify the truncated S1 gene of nephrotropic infectious bronchitis virus (IBV) and granulocyte-monocyte colony stimulating factor (GM-CSF) of chicken. Firstly, two genes were amplified by polymerase chain reaction (PCR) and cloned into pMD18-T vector. The truncated S1 gene designated as Sf200 containing five antigenic sites of S1 glycoprotein on amino acid residues (aa) 24-61, (aa) 291-398 and (aa) 497-543 and GM-CSF were then amplified from the respective recombinant pMD18-T plasmids and cloned into pET-32a (+) vector resulting pET-Sf200, pET-GM which were identified by restriction enzyme digestion and sequencing analysis. The in vitro expression of truncated Sf200 and GM-CSF constructs were later expressed in E. coli BL21 with a molecular mass of approximately 38 kDa and 29 kDa respectively as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polyclonal antibodies were developed by injecting E. coli expressed Sf200 and GM-CSF into the SPF mice and were used to identify the recombinant proteins by Western blot analysis. These findings indicated that the polyclonal antibodies produced in mice could be used to detect the recombinant truncated Sf200 and GM-CSF and vice versa.

Glycyrrhizin inhibits swine acute diarrhea syndrome coronavirus infection via the HMGB1/TLR4/JNK signal pathway

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a newly discovered enteric coronavirus, is the etiological agent that causes severe clinical diarrhea and intestinal pathological damage in piglets. In this study, Vero E6 and IPI-2I cells were pretreated with different concentrations of glycyrrhizin (GLY) for 2 hours, and then infected with different concentrations of SADSCoV, aiming to investigate the inhibitory effect of GLY on SADS-CoV. Western blot and TCID50 results revealed a significantly decreased N protein expression and viral titer, indicating that GLY can inhibit the infection of SADS-CoV. Vero E6 and IPI-2I cells were pretreated with different concentrations of GLY for 2 hours and infected with SADS-CoV. Western blot results showed that when the concentration of GLY was 0.8 mmol/L, the expression of N protein decreased significantly, indicating that GLY inhibited the invasion of the virus. At first, cells were treated with 0.4 mmol/L GLY, and cell samples were collected at 2 hours, 6 hours and 12 hours after being infected with SADS-CoV for analysis, and the expression of N protein were found to be significantly reduced at all points, indicating that GLY had a significant inhibitory effect on the replication of the virus. GLY is a competitive inhibitor of high mobility group box 1 (HMGB1), and the receptors of HMGB1 mainly include TLR4 and RAGE. Based on this fact, the mutant plasmid at the key sites of HMGB1 (C45S, C106S, C45/106S) and the siRNA of the RAGE receptor were transfected to Vero E6 cells and infected with SADS-CoV, and the cell supernatant and samples were harvested. The western blot and TCID50 results showed that the expression of N protein and the virus titer were decreased, suggesting that GLY exerts its function by affecting the binding of HMGB1/TLR4/RAGE during SADS-CoV infection. To further explore the signaling pathway through which GLY functions, Vero E6 and IPI-2I cells were inoculated with SADS-CoV, and cell samples were harvested, western blot was used to detect the changes of MAPK proteins. The results showed that the protein expression levels of p-p38, p-JNK and p-ERK were up-regulated in the early and late stages, indicating that the MAPK pathway was activated by SADS-CoV infection. Vero E6 and IPI-2I were pretreated with different concentrations of GLY and TLR4 inhibitor TAK for 2 hours and infected with SADS-CoV. Protein samples were harvested and analysed by western blot which showed a decreased p-JNK and N proteins, while other proteins showed no significant changes. These results indicated that GLY and TAK regulated the phosphorylation of JNK but did not regulate the phosphorylation of p38 and ERK. Also, Vero E6 cells were treated with HMGB1 antibody, the siRNA of HMGB1 and HMGB1 mutants plasmid, and infected with SADS-CoV. Protein samples were harvested, western blot results showed that phosphorylation of JNK decreased, indicating that HMGB1 affected JNK phosphorylation. Finally, Vero E6 and IPI-2I cells were pretreated with different concentrations of JNK inhibitor SP600125 to infect SADS-CoV, western blot, TCID50 and IFA results showed that the expression of N protein and virus titer, as well as virus replication were reduced, indicating that SP600125 inhibited virus replication. In conclusion, our results revealed that GLY can inhibit in vitro replication of SADS- CoV, mainly through the HMGB1/TLR4/JNK signaling pathway. The discovery of this pathway provides theoretical support for the research of novel anti-SADS-CoV drugs.

Fructus aurantii extracts ameliorate intestinal inflammation and regulate the intestinal barrier through Fc epsilon RI signaling pathway

Fructus Aurantii (FA) is the dry and immature fruit of Citrus aurantium L. and its rutaceous cultivars. FA has been widely used to treat digestive system diseases since ancient China, and it promotes gastrointestinal (GI) motility in functional dyspepsia (FD), but its potential therapeutic mechanisms remain unclear. We examined the effects of FA ethanol extracts in an iodoacetamide (IA)-induced FD rat model. Firstly, key FA therapy targets for FD were gathered using systematic pharmacology. Combined with systemic pharmacological analyses, plasma metabolomics based on UPLC-QTOF-MS were conducted. Then, MetaboAnalyst was used to jointly analyze systemic pharmacology targets and metabolomic metabolites to select key metabolic pathways. Finally, the key path is verified by experiments. FA exerted distinct therapeutic effects in anti-inflammation and promoting gastrointestinal motility in our IA-induced FD rat model. When compared with the model group, FA down-regulated the inflammatory factors interleukin 1beta and tumor necrosis factor-a. At the same time, FA up-regulated tight junction proteins in the intestinal epithelial barrier. Through the integrated analysis of metabolomics and systemic pharmacology, we conducted experimental verification on Fc epsilon RI signaling pathway. When compared with the model group, FA down-regulatedphospho-mitogen activated protein kinase, phospho-extracellular signal regulated kinase1/2, myosin light chain kinase, and phospho-myosin regulatory light chain protein levels. Thus, FA ameliorated FD by regulating the Fc epsilon RI signaling pathway. Our integrated strategy identified underlying FA mechanisms toward FD treatment and provided a foundation for FA development as a clinical agent for FD.

Isolation, identification, and S1 gene sequencing of two strains of Avian Infectious bronchitis virus

Objective: Epidemiology and genetic variations of the infectious bronchitis virus(IBV) in Fujian province were studied. Method: Two strains of virus isolated from the diseased chickens in Fujian in 2021 were identified by chicken embryo pathogenicity test, electron microscope observation, and RT-PCR. S1 genes of the isolates were cloned, sequenced, and analyzed using biological software. Result: The two IBV strains were code named FJ-NP01 and FJ-FZ01. The full length of S1 of FJ-NP01 was 1 629 nt encoding 543 amino acids, and that of FJ-FZ01, 1 620 nt encoding 540 amino acids. The S1 gene cleavage site of FJ-FZ01 was HRRRR, same as all reference strains of genotype I branch;while that of FJ-NP01 HRRKR differed from the reported site of IBV isolated from genotype IV but same as that of TC07-2 reference strain of genotype VI. The homology of nucleotide and amino acid between the two isolates was 83.2% and 79.6%, respectively, but merely 75.7%-76.3%and 77.1%-83.5% with the Mass-type conventional vaccines H120 and H52, respectively. Further analysis showed that FJ-NP01was from a recombination event between CK CH GD LZ12-4 and L-1148, the homology of nucleotide acid between 1438-1506 nt of FJ-NP01 with CK CH GD LZ12-4 was 97%, and 95.9% between the other nucleotide acid of S1 gene with L-1148. Conclusion: It appeared that the IBV epidemic experienced in the province was complex in nature and that the existing Mass vaccines would not provide sufficient immune protection to deter the spread.

Interaction between target peptide and complementary peptide by bimolecular fluorescence complementary technology

[Objective] Using the bimolecular fluorescence complementation (BiFC) technology, the present experiment aimed to study the interaction relationship and localization of the target peptide and the complementary peptide based on the porcine epidemic diarrhea virus (PEDV) S protein receptor binding site peptide in living cells, so as to provide the foundation and theoretical support for the further use of the peptide in the detection of porcine epidemic diarrhea virus. [Method] The target peptide was designed according to the physical and chemical characteristics of the target protein, such as the amino acid composition, the type of charge, the ability to form intennolecular hydrogen bonds, the strength of polarity, and hydrophobicity;According to the amino acid composition of the target protein, a complementary peptide that interacted with it in theory was designed, and the target peptide and complementary peptide were predicted and analyzed by using bioinfonnatics tools;The target peptide and complementary peptide were inserted into the pBiFC-VC155 and pBiFC-VN173 vector, which was double digested by the EcoRI/XhoI and NotI/SalI, respectively, verified by enzyme digestion and sequencing, and then transfected into Vero cells to study the interaction between the target peptide and the complementary peptide, and the precise localization of BiFC complex in cells. [Result] Bioinfonnatics analysis showed that the target peptide and complementary peptide had hydrophilic and hydrophobic domains, respectively, and the hydrophilic domains were both positively and negatively charged, which could generate electrostatic attraction. The results of enzyme digestion and sequencing showed that the pBiFC-VC155-target peptide and pBiFC-VNI73-complementary peptide plasmids were successfully constructed;Cell transfection experiments showed that the target peptide and complementary peptide could form BiFC complexes in Vcro cells after co-transfection of recombinant plasmids, indicating that they could interact with each other;Indirect immuttolluorescence assay confirmed that the BiFC complex was mainly distributed in the nucleus. [Conclusion] It was confirmed that the peptide designed based on the PEW/ S protein receptor binding site can interact with each other in living cells, demonstrating the feasibility of the peptide for detection.

Antiviral activitiy of curcumin, demethoxycurcumin, bisdemethoxycurcumin and cyclocurcumin compounds of Curcuma longa against NSP3 on SARS-CoV-2

SARS-CoV-2 genome encodes two large polyproteins (pp), pp1a and pp1ab which are cleaved and transformed into a mature form by a protease, non-structural protein 3 (NSP3). NSP3 is encoded by open reading frame (ORF) 1a/b. Curcuma longa (C. longa) or turmeric has been documented to have antiviral effects. The aim of this study was to assess the viral activities of C. longa against SARS-CoV-2 focusing on its potency to inhibit viral replication by targeting NSP3. PubChem databases were used to obtain the metabolic profile of C. longa. The compound's interaction with nucleocapsid was analyzed using molecular docking with Molegro Virtual Docker. Bioinformatics analysis based on rerank score presents all compounds of C. longa have higher binding affinity than the native ligand with cyclocurcumin as the lowest score (-128.38 kcal/mol). This anti-viral activity was hypothesized from the similarity of hydrogen bonds with amino acid residues Ser 128 and Asn 40 as key residues present in Ribavirin. This study reveals that C. longa is the potential to be developed as an antiviral agent through replication inhibition in SARS-CoV-2 targeting its replication mediated by NSP3.

A review of bioinformatics studies on the function of structural and nonstructural proteins and the level of glycoprotein inhibiting heme metabolism by SARS-CoV-2 virus

Coronavirus disease 2019 (COVID-19) is an acute respiratory infection. Its virus called SARS-COV-2 which is an RNA virus with high homology to the bat coronavirus. In this review study, first the molecular and cellular characteristics and the proliferation and replication of SARS-COV-2 are investigated. Then, by reviewing bioinformatics studies regarding protected domain analysis, homology modeling, and molecular docking, the biological role of some specific SARS-COV-2 proteins are examined. The results showed that the open reading frame 8 (ORF8) and surface glycoprotein could bind to porphyrin. At the same time, ORF1ab, ORF10, and ORF3a can attack the heme part of hemoglobin to dissociate iron and form porphyrin. This attack reduces hemoglobin ability to carry oxygen and carbon dioxide. As a result, lung cells become severely inflamed due to their inability to exchange carbon dioxide and oxygen, which leads to large ground-glass opacities on CT scan images. Based on the bioinformatics results, chloroquine can prevent ORF1ab, ORF3a, and ORF10 from attacking hemoglobin to form porphyrin and avoid the binding of ORF8 and surface glycoprotein to porphyrin, which effectively relieves the symptoms of acute respiratory syndrome. In the current pandemic, bioinformatics studies are of great importance for preventing the spread of COVID-19, developing drugs and vaccines, and clinical practice.

Pathogenicity analysis of one avian infectious bronchitis virus stain isolated from Shanxi

In order to perform the isolation of avian infectious bronchitis virus (IBV) and study the pathogenicity of IBV isolate, the RT-PCR was used to detect nucleic acid extracted from a clinical sample of chickens, which were suspected to be infected with infectious bronchitis virus (IBV) and provided by a farmer in Yuncheng, Shanxi province. And the sample was detected as IBV positive by RT-PCR. Then 9-11-day-old SPF chicken embryonated eggs were inoculated with the sample filtered from the grinding fluid, and the obtained allantoic fluid was blindly passed by three generations (F3) and was also tested as IBV positive;The F11 generation passaged in embryonated eggs caused typical "dwarf embryo" lesions to SPF chicken embryonated eggs, and induced the loss of cilia in tracheal rings. The results showed that an IBV strain was isolated and named as YC181031. The S1 gene amplification and sequencing analysis showed that YC181031 strain belonged to IBV GI-22 genotype, which is also nephropathogenic type IBV. Seven-day-old SPF chicks were used to test the pathogenicity of the isolate. The results showed that several clinical symptoms were showed in chicks infected with YC181031, such as breathing with difficulty, depression, excreting watery droppings and death. The mortality of infected chicks was 20%. Typical pathological changes such as enlargement of kidney and urate deposition in the kidney were observed in infected chicks. The immunohistochemical assay and viral load detection were performed for the tissue samples from infected and dead chicks. The tissue lesions and distribution of virus were observed in the kidney, trachea, lung, glandular stomach, spleen and liver samples of infected chicks. RT-PCR detection of pharyngeal anal swabs showed that the virus shedding by infected chicks could be continuously detected within 14 days of the test period;The viral loads of various tissues were detected by RT-qPCR and the results showed that the viral load from high to low was kidney, trachea, lung, stomach, spleen and liver. The viral load of kidney was significantly higher than that of other tissues (P < 0.05).In this study, the pathogenicity characteristics of GI-22 genotype strain were systematically studied for the first time, providing a reference for the prevention and treatment of the disease.

Using molecular tools to compare various concentration protocols prior to detecting SARS-CoV-2 RNA in wastewater. (Special Issue: Chemistry and global challenges (part A).)

Background: Reports showed presence of SARS-CoV-2 genetic material in wastewater. Wastewater concentration methods are optimized for detection of non-enveloped viruses so need to be adopted for enveloped viruses and their genetic material. Methods: Conventional (cRT-PCR) and quantitative real time RT-PCR (qRT-PCR) were used as readouts to compare 4 water concentration methods namely, (A) filtration on negatively charged membrane followed by extracting RNA from it, (B) adsorbtion-elution method, (C) flocculation with skimmed milk and (D) polyethylene glycol precipitation, to detect SARS-CoV-2 RNA and 229E human coronavirus (229E-HCoV) as a model for spike-containing enveloped virus from fresh and wastewater. Results: On using cRT-PCR: recovery rate of SARS-CoV-2 RNA was better using method A then B for fresh water and method B then D for wastewater. 229E-HCoV recovery from fresh water was better using method C then A and methods B then D for wastewater. On using qRT-PCR, both methods A and B were better for SARS-CoV-2 RNA recovery from both fresh and wastewater. For the 229E-HCoV methods A was the most efficient for fresh water and method B for wastewater. Conclusion: Method B is recommended for SARS-CoV-2 RNA or whole 229E-HCoV recovery from wastewater.

Assessment of SARS-CoV-2 infection in astrocytes

Discovered in late 2019 in a market in the city of Wuhan, Hubei Province, China, SARS-CoV-2 is an important member of the Coronaviridae family, responsible for bringing the whole world into a state of alert causing a global pandemic. The virus has been identified as causing a characteristic clinical condition known as "Corona-virus disease 2019" (COVID-19), causing an Acute Respiratory Syndrome. Being a respiratory virus, transmitted by direct contact with an infected person and by touching contaminated surfaces, SARS-CoV-2 quickly spread throughout the world, causing a pandemic, having today more than 535 million people infected and causing more than million deaths. In addition to the respiratory system, the virus is present in other cells in the body. Findings show the presence of SARS-CoV-2 in cerebrospinal fluid associated with changes in the expression of neuronal inflammation markers, as well as an increased expression of cytokines released by astrocytes, indicating an alteration in the Central Nervous System (CNS). In this project, we analyzed the effects of SARS-CoV-2 infection directly on astrocytes, glial cells that are extremely important for the maintenance of homeostasis and CNS defense. Therefore, we produced astrocytes from three human iPSC strains to verify aspects of cell morphology and physiology, as well as gene and protein expression, after infection with the virus. We found that SARS-CoV-2 is capable of infecting astrocytes, but some studies are still needed to better elucidate its role in the interaction with this cell type in the CNS.

Expression of Brucella abortus Omp25 protein in Lactococcus lactis probiotic bacteria

Background and purpose: The sequence of Omp25 is conserved in all Brucella species. The high antigenicity of the product of this gene stimulates the host's immune system. Using engineered probiotic bacteria is an appropriate method for vaccine transport. The aim of this study was to express the Omp25 of the Brucella abortus pathogenic bacterium in Lactococcus lactis probiotic bacterium. Materials and methods: In this experimental study, the required vector was designed and synthesized to include the gene of interest and a signal peptide (pNZ8148-Usp45-Omp25). E. coli strain TOP10F was transformed using the pNZ8148-Usp45-Omp25 expression vector based on induction by nisin. The recombinant plasmid was extracted from the transformed bacteria using a plasmid extraction kit. The L. lactis was transformed by pNZ8148-Usp45-Omp25 vector using electroporation. Evaluation of the expression of Omp25 gene at the RNA level was assessed by reverse transcription method and confirming the presence of recombinant Omp25 protein in the engineered bacteria using SDS-PAGE method. Results: Successful expression of B. abortus Omp25 in L. lactis was verified by RT-PCR. Subsequently, the proteins were separated based on molecular weight using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). The protein expression analysis showed the expression of Omp25 as a 25 kDa extra band in transformed L. lactis compared to the L. lactis receiving the vector lacking the target gene. Conclusion: This study shows that Omp25 is expressed in L. lactis transformed via pNZ8148-Usp45-Omp25 by electroporation. Transformed L. lactis can be successfully used as a subunit oral vaccine in prevention of Brucellosis.

Role of initial and follow-up IL-6 (interleukin-6) titre in COVID-19 pneumonia: a single center experience

Background: Robust data of IL-6 is available in bacterial infection, and now it can be utilized in currently ongoing COVID-19 (corona virus disease-19) pneumonia pandemic to guide treatment strategy as marker of inflammation. Methods: Prospective, observational study included 1,000 COVID-19 cases confirmed with RT PCR (reverse transcription polymerase chain reaction). All cases were undergone categorized after clinical details, HRCT (high resolution computerized tomography) thorax, oxygen saturation, IL-6 (interleukin 6) at entry point and follow up. Age, gender, comorbidity and use BIPAP/NIV (bilevel positive airway pressure/non-invasive ventilation), and outcome as with or without lung fibrosis as per HRCT severity were key observations. Statistical analysis is done by using Chi-square test. Results: In study of 1,000 COVID-19 pneumonia cases, age (<50 and >50 years) and gender has significant association with IL-6. HRCT severity score at entry point has significant correlation with IL-6 level (p < 0.00001). IL-6 level has significant association with duration of illness (p < 0.00001). Comorbidities has significant association with IL-6 level (p < 0.00001). IL-6 level has significant association with oxygen saturation (p < 0.00001). BIPAP/NIV requirement has significant association with IL-6 level (p < 0.00001). Timing of BIPAP/NIV requirement during course of hospitalization has significant association with IL-6 level (p < 0.00001). Follow-up IL-6 titer during hospitalization as compared to entry point normal and abnormal IL-6 has significant association with post-COVID-19 lung fibrosis, respectively (p < 0.00001). Conclusion: IL-6 has very crucialrole in COVID-19 pneumonia in predicting severity of illness, progression of illness including 'cytokine storm' and assessing response to treatment during hospitalization and follow-up titers in analyzing post-COVID-19 lung fibrosis.

Isolation, identification and genetic evolution analysis of porcine epidemic diarrhea virus SNJ-P strain in Sichuan Province

Purpose: To investigate the epidemic variation of porcine epidemic diarrhea virus (PEDV) strains in Sichuan Province, and to analyze the causes of poor vaccination effect. Methods: Piglet intestinal samples were collected from a pig farm in Sichuan Province for PCR detection, virus purification, determination of virus titer and virus infection experiments. Whole genome sequencing of isolated strains was determined. The S gene sequence of the isolated strain was compared with the strains from other regions and vaccine strains, and the phylogenetic tree was established. The amino acid site variation of S protein between the isolated strain and the classical vaccine strain CV777 was compared. Results: A PEDV strain was successfully isolated and named as PEDV SNJ-P. The determination of virus titer was 1..107.5/100 L. Animal infection experiments showed that the isolated strain could cause diarrhea, dehydration and other symptoms and lead to death in piglets. Genome sequencing and phylogenetic tree analysis showed that the whole gene of PEDV SNJ-P strain was 28003 bp, and the genotype of the strain was S non-INDEL type. The strains were closely related to the strains of PEDV-WS, CH/JLDH/2016 and CH/HNLH/2015 isolated from China, and were relatively distant with the same type vaccine strain, and were far from the classical vaccine strain. Compared with the classical vaccine strain CV777, the S protein of SNJ-P strain had multiple amino acid mutations, deletions and insertions. Conclusion: Due to the continuous variation of strains, SNJ-P strain is far from the vaccine strain, and the current vaccines cannot provide effective protection. The results of this study are expected to provide reference for the study of PEDV strains and vaccine development in China.

Epidemiological characteristics of a local COVID-19 outbreak caused by omicron variant (BA.2) in Qinzhou, Guangxi

Objective: To analyze the epidemiological characteristics of a local COVID-19 outbreak in Qinzhou, Guangxi Zhuang autonomous region, and provide reference for the prevention and control of COVID-19 in the future. Methods The epidemiological and clinical information, analysis reports and laboratory test results of the COVID-19 cases were collected for a descriptive epidemiological analysis. Results A total of 97 COVID-19 cases, including 79 asymptomatic cases and 18 confirmed cases, were reported in Qinzhou during 12-24 March, 2022. Forty nine cases were males and 48 cases were females. The median of age of the cases was 32 (17.0, 44.5) years. The median of incubation period was 3 days. The median of latent period was 2 days. A total of 3841 close contacts were screened, in whom 61 were infected. The secondary attack rates in 3841 close contacts was 1.59%. The secondary attack rate in household contacts was 65% in 8 family clustering (95% CL: 20%-100%). Three mass gathering were identified in a local recreation center box, a wedding ceremony and a family feast for new home, in which the attack rates were 63.64%, 9.38% and 12.16%, respectively. Transmission firstly occurred in the people attending the activity in the recreation center box. At least 5 generations of transmission were identified in the outbreak. The results of genome second generation sequencing of the isolates from 20 infection cases revealed that the pathogen of the outbreak was SARS-CoV-2 Omicron variant (BA.2). Conclusion Analysis on the cases caused by Omicron variant (BA.2) indicated early prevention measures are important for the control of further spread of COVID-19.

Identification of feline corona- virus in breeding catteries by molecular biological methods and shedding persistency

Infectious peritonitis virus (FIPV) causes a fatal disease in cats. This virus occurs both in cats bred in households with optimal welfare and outdoor cats. Feline patients with the effusive form of disease usually survive a few days to weeks from the appearance of the first clinical signs. Cats with the non- effusive form survive for weeks to months. FIPV is caused by a mutation from feline enteric coronavirus (FECV). In our study, we diagnosed feline coronavirus from the feces of 82% of the tested cats. The persistence of the feline coronavirus in the organism is influenced by environmental factors, the genome of the host and the causative agent. Negative environmental conditions that increase the likelihood of FIPV disease are long-term stress, mainly more labile individuals and a high concentration of domesticated cats in one place. In the host, there are important factors such as immune system performance, age, breed and genetic background. In our study, we primarily verified the real time RT-PCR method for identifying the virus from the feces of 71 cats and subsequently gaine the valuable data on the dynamics of feline coronavirus excretion, primarily for epizootological purposes and for the purposes of genetic analyzes of susceptibility to infection.