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.

Small-angle polarimetry as a technique for identification of nucleotide sequences in bioinformatics

Background and Objectives: The method of identification of symbolic sequences associated with the genetic structure of biological objects using the principles of small-angle polarimetry is considered. This method of analyzing and visualizing symbolic sequences obtained by sequencing DNA fragments can be defined as small-angle polarimetry of phase-modulating structures associated with genetic information. Materials and Methods: The analyzed symbolic sequence is represented by a two-dimensional phase-modulating matrix, each element of which corresponds to one of the four basic nucleotides (adenine, cytosine, thymine, guanine), and the depth of modulation of the phase of the reading coherent linearly polarized beam is determined by the content of this nucleotide in the corresponding triplet in the nucleotide sequence. As a result of the diffraction of a reading coherent beam with a polarization plane oriented at an angle of 45° to the sides of the phase-modulating matrix, a spatial distribution of local polarization states of the reading field diffracted on the matrix is formed in the paraxial region of the far diffraction zone. Discrimination of local polarization states in accordance with the proposed algorithm makes it possible to synthesize a binary spatial distribution, which is a unique identifier of the analyzed symbol sequence. Results: Modeling of the processes of phase coding and subsequent analysis of local polarization states in the near-axial region using sequencing results for the strains "Wuhan”, "Delta” and "Omicron” of the SARS-CoV-2 virus has shown a high sensitivity of the method to local changes in the structure of nucleotide sequences. Conclusion: The results of the simulation allow us to conclude that binary distributions of local polarization states of light fields diffracted on DNA-associated phase-modulating structures recorded in the axial region are characterized by high sensitivity to local mutational changes in the structure of nucleotide sequences. The results obtained can be used as a basis for creating effective hybrid methods for analyzing genetic information using the principles of polarization coding and small-angle polarimetry. © 2023 Vestnik Novosibirskogo Gosudarstvennogo Universiteta, Seriya: Istoriya, Filologiya. All rights reserved.

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.

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.

Comparative analysis of SARS-CoV-2 variants with two high-throughput sequencing platforms

Whole-genome sequencing of upper respiratory tract specimens from patients with confirmed COVID-19 in Henan Province was performed to compare the performance of the Illumina and Oxford Nanopore sequencing platforms, thus providing a reference for whole-genome monitoring of the novel coronavirus (SARS-CoV-2). Ten samples from COVID-19 cases in Henan Province from June 2021 to January 2022 were collected and sequenced with Illumina and Nanopore high-through-put sequencing technology to obtain full genome sequences of the novel coronavirus, which were compared with the Wuhan reference sequence (Wuhan-Hu-1). Bioinformatics software (CLC) was used for sequence alignment analysis. Three of the ten samples were Omicron (BA.1) variants with 55,61 nucleotide variation sites. One sample was an Alpha (B.1.1.7) variant with 41 nucleotide variation sites. Six samples were Delta (8.1.617.2) variants with 35,42,47 nucleotide variation sites. The sequence identity of mutation sites in six samples was 100%, and the mutation sites in the S genome segment of seven samples were consistent. For samples with a Ct value < 33, both next-generation and third-generation sequencing achieved high genome coverage and sequencing depth. A significant difference in coverage was observed between second-generation sequencing and third-generation sequencing (t=-2.037, P < 0.06). However, the coverage at different time points of the third-generation sequencing did not significantly differ (F=2.498, P > 0.05). The needs for SARS-CoV-2 mutant detection could be met through use of either high-throughput sequencing platform. The identification of mutations in the novel coronavirus through Illumina high-throughput sequencing was more accurate, whereas Nanopore high-throughput sequencing technology could be used for rapid detection and typing of different novel coronaviruses.

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.

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.

Studying mutations of SARS-CoV-2 different variants (alpha, beta, delta, gamma, omicron)

Being pushed by natural selection, random genetic drift, gene editions, and receptor immunity response, viruses develop constantly through mutations affecting different genes and leading to genetic diversity and producing new variants. In order to know well how a mutation could have an impact on the possibility of being infected, on transmission, and on aggressivity of SARS-CoV-2 it would be important to study these mutations. To be able to carry out a comparative study between variants and undergone mutations over many countries in the world, we've dealt with many genomic sequences that have been rapidly accumulated in the GenBank since January 2020, and published by many laboratories over the world. These sequences allowed us to establish phylogenetical trees using a strong bioinformatic tool, just enhanced to study Covid which is MEGA version 11. Distribution of shifted sequences of different variants over the world within phylogenetical trees shows that the overwhelming majority of detected mutations are accumulated in the 5 known variants Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), Gamma (P.1) et Omicron (B.1.1.529), especially within their most variable genes, structural genes of which are N (Nucleocapsid protein) and S (Spike glycoprotein) added to functional ones ORF (Open Reading Frame : ORF1ab, ORF3a);hence, variants holding these mutations are the most dominant and the most infectious this time in the world.

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.

Source investigation of a COVID-19 outbreak in the sea-land border city

Objective: To analyze the epidemiological characteristic and tracing process of an outbreak of COVID-19 in a sea-land border city (D city) of Guangxi in December 2021, and provide scientific data for for the emergency management and scientific traceability of similar outbreaks in the future. Methods Epidemiological investigation of cases was carried out under the guideline of the Novel Coronavirus Epidemiological Investigation Programme on Cases of Pneumonia (Edition 8). RT-PCR method was used for samples testing. Furthermore, positive samples were analyzed by whole-genome sequence and phylogenetic analysis. R software 4.1.3 version was used for data analysis. Results There were 20 cases in this outbreak which related 6 families. The average incubation period was (4.6..2.2) d. Compared with the Wuhan reference strain (NC_045512), the genome sequence analysis showed that there were 35-36 nucleotide mutation sites in the novel Coronavirus genome sequence of 19 local cases, which belonged to VOC/Delta variant strain (AY.57 evolutionary branch). The 11 amino acid mutation sites were the same in all the novel Coronavirus spikes (S) proteins, which were highly homologous to the 2 COVID-19 genome sequences uploaded from a neighboring country in the GISAID genome database. Conclusion This outbreak was caused by fishermen who were infected by contacting with persons of neighboring country in the public sea area and causing located community transmission. The management of border villagers and the monitoring of epidemic strains should be strengthened to detect and deal with the outbreak as early as possible in the future.

Whole genome sequence analysis of SARS-CoV-2 from 40 imported cases of confirmed COVID-19 in Sichuan

Objective: To understand the genomic characteristics of SARS-CoV-2 from 40 imported cases with confirmed COVID-19 in Sichuan during January and March 2022. Methods: Total viral RNA was extracted from respiratory samples of 182 confirmed COVID-19 cases who entered China through Chendu International Airport from January to March 2022. Mutation nucleic acid detection kit was used to identify the mutant strains and Illumina sequencing platform was applied for whole genome sequence(WGS) of virus. SARS-CoV-2 reference sequences were downloaded from NCBI database for genetic evolution and antigen variation analysis. The Nextclade and Pangolin online virus analysis platform were used to determine the virus family and type, and to analyze the mutation loci of the virus. The phylogenetic tree was constructed, along with the epidemiological data of cases to analyze the source and correlation of viruses. Results: Among 182 imported COVID-19 cases,B.1.617.2 mutations were identified in 3 cases and B.1.1.529 mutations were detected in 57 cases.A total of 40 SARS-CoV-2 whole genome sequences with coverage>95% were obtained in this study. Nextclade typing analysis showed that 3 sequences belonged to 21J(Delta),5 sequences belonged to 21K(Omicron)and the remaining 32 sequences belonged to 21L(Omicron). Pangolin typing analysis showed that the 3 sequences of 21J(Delta)belonged to AY.4,AY.109and B.1.617.2, the 5sequences of 21K(Omicron)all belonged to BA.1.1, and the remaining 32 sequences of 21L(Omicron)belonged to BA.2. Our sequence results were99.7% consistency with the Omicron variants sequences in current GISAID database. Compared with the reference sequence strain Wuhan-Hu-1(NC_045512.2),45,47and 42nucleotide variation sites and 36,25 and 36amino acid variation sites were found in the 3 sequences of 21J(Delta). There were average 59(26-64)nucleotide mutation sites and 48(10-53)amino acid mutation sites in the 5sequences of 21K(Omicron). The median number of nucleotide mutation sites of 71(66-76)and amino acid mutation sites of 53(40-56)were identified in the 32sequences of 21L(Omicron). Phylogenetic tree analysis showed that 40SARS-CoV-2WGSs were all related to the current variants of concern(VOC). Conclusions Continuous: sequencing of SARS-CoV-2whole genome from imported cases with confirmed COVID-19is of great significance for the prevention and control of the outbreak and prevalence of local epidemic caused by imported viruses in Sichuan.

One-step multiple TaqMan real-time RT-PCR for simultaneous detection of swine diarrhea viruses

Objective: The aim of this study was to establish a one-step multiplex real-time RT-PCR method to simultaneously detect and quantify five swine diarrhea related viruses, PEDV, GARV, PDCoV, SADS-CoV and PTV, so as to provide an efficient and sensitive tool for rapid diagnosis and epidemiological investigation of porcine diarrhea. Method: The ORF3 gene sequences of several genotypes of PEDV were analyzed, and then the primers and probes were designed for detection of PEDV field strains by referring to the ORF3 genes, which contained deletion mutations in attenuated strains. The 5'-end conserved region of NSP5 genes of GARV G3, G4, G5 and G9 strains were analyzed for design of probes and primers. The specific primers and probes targeting to the conserved regions of PDCoV M, PTV 5'UTR and SADS-CoV N genes were designed for detection of the pathogens. The ROC curves were completed by referring to parameters that were set in RStudio. The specificity value, sensitivity value, and areas under the curves (AUC) and Youden value were calculated according to ROC curves to determine the cut-off CT value. The amplified fragments were cloned into pEASY-T1 vector. The standards prepared through in vitro transcription were named as cRNA-PEDV, cRNA-GARV, cRNA-PDCoV, cRNA-PTV and cRNA-SADS-CoV. The sensitivity, specificity and repeatability of one-step multiplex real-time RT-PCR were evaluated. Coincidence rate between this and another similar method were compared in the detection of clinical samples. Result: Both the annealing temperature and optimal concentrations of primers and probes were obtained for detection of the five pathogens. According to the ROC curve, the CT cut off values for detection of PEDV, GARV, PDCoV, PTV, and SADS-CoV were set as 35.78, 34.25, 34.98, 34.60, and 35.70, respectively. The detection sensitivity of this method for the five pathogens could reach 1..102 copies/L. The standard curves had a good linear relationship and the amplification efficiency was between 96.3% and 104%. The established method could not detect the PEDV vaccine strains and other swine infecting viruses and bacteria including TGEV, CSFV, PRV, PRRSV, S.choleraesuis, P.multocida, E.coli, S.suis and S.aureus. The repeatability test showed the range of intra-assay and inter-assay coefficients of variability: 0.22% to 3.08% and 0.89% to 4.0%, respectively. The detection coincidence rates of the established detection method and another similar method for the five pathogens in 242 clinical samples were 97.9%, 98.8%, 100%, 98.3% and 100% for PEDV, GARV, PDCoV, PTV and SADS-CoV, respectively. The Kappa values were all higher than 0.9. The method had advantage over a commercial diagnostic kit for detection of PEDV wild strains in accuracy. Detection results with clinical samples showed that positive rates of PEDV, GARV, PDCoV and PTV was 10.7% (26/242), 13.6% (33/242), 18.2% (44/242) and 14.5% (35/242), respectively, demonstrating the prevalence state of the four pathogens in Sichuan province in the years. SADS-CoV was not detectable in any areas, but the phenomenon of coinfection with different diarrhea causing viruses was common. Therefore, it was necessary to strengthen the surveillance of several porcine diarrhea viruses in Sichuan province for preventive control. Conclusion: In this study, a one-step multiplex real-time RT-PCR was established for simultaneous detection of PEDV wild strains, PDCoV, SADS-COV and GARV, PTV multiple genotypes, which provided an efficient and sensitive tool for the differential diagnosis and epidemiological investigation of swine diarrhea disease.

Complete genome sequencing and molecular genetic evolution analysis of Guangxi mutant of porcine epidemic diarrhea virus

In order to understand the genomic characteristics and molecular genetic diversity of porcine epidemic diarrhea virus(PEDV) in Guangxi in recent years, 11 pairs of specific primers were designed to detect the whole genome of PEDV GXNN isolated from porcine diarrhea in Nanning, Guangxi, China, and similarity comparison, genetic evolution, gene variation and S gene recombination were also analyzed. The results showed that full length of the GXNN strain was 28 035 bp, had similar genomic characteristics with other PEDV isolates, about 96.4%-98.7% nucleotide similarity with different reference strains, and the nucleotide similarity of S, ORF3, M and N genes was 93.7%-98.9%, 90.9%-99.4%, 97.4%-99.7% and 95.6%-99.2%;the amino acid similarity of them was 92.9%-99.5%, 91.3%-99.1%, 97.4%-99.1% and 96.4%-99.5%. GXNN is closely related to most domestic isolates in recent years. Phylogenetic tree showed that GXNN closely related to most strains isolated in China recent years, belonged to GII-b subtype. However, it was low relatedness to classic vaccine strains, domestic early epidemic strains, foreign epidemic strains and Guangxi CH-GX-2015-750 A, they belong to different subtypes. Compared with the 5 vaccine strains, the S gene of GXNN stain has a large variation, by inserting amino acid Q at positions 118 844 and 905 sizes, four unique amino acid mutations in the core neutralizing epitope(COE)region and the main epitope region, and 14 mutations in other regions. 126 T/A, 199 A/V and 103 T/A site mutations of ORF3, M and N genes were happened at position 126, 4 D4 region and PN-D4 region, respectively. Recombination analysis revealed that there was a potential recombination region in the hypervariable region of S gene at 826-3 142 nt. This study successfully obtained the complete genome sequence of a PEDV strain, and analyzed its genetic variation and provided a reference for PEDV molecular epidemiology research and new vaccine development.

STING gene knockout pig alveolar macrophage cell model constructed by CRISPR/Cas9 technology and its effect on type I interferon transcription. (Special Issue: Swine industry science.)

Objective: To elucidate the mechanism of interferon gene stimulating factor (STING) in the anti-pathogenic microbial infection of pigs, so as to further provide a reference for the scientific prevention and control of viral diseases such as porcine transmissible gastroenteritis, epidemic diarrhea and porcine pseudorabies. Method: High-scored targets were found in exons 4 and 8 of STING gene and corresponding sgRNA sequences were designed based on CRISPR/ Cas9 technology. The annealed sgRNAs were linked with the enzyme digested LentiCRISPRV2 carrier with T4 DNA ligase to obtain LentiCRISPRV2-STING-sgRNA lentivirus carrier(STING-sgRNA);Different combinations of STING sgRNA lentivirus carriers, packaging plasmid psPAX2 and envelope plasmid pMD2.G were transfected into 293T cells to obtain lentivirus containing sgRNA and then transduced into 3D4/21 cells. Monoclonal cell lines were obtained by puromycin screening and limited dilution method. The knockout efficiencies of the STING gene were identified by PCR amplification, Sequencing and Western blotting;The effect of STING gene knockout on the expression of type I interferon was verified by real-time fluorescent quantitative PCR. Result: When 293T cells were transfected with different combinations of STING-sgRNA lentivirus carrier and HA-STING over expression vector, the editing effect of STING eukaryotic expression carrier could be detected in cells, and the combination of STING-sgRNA(1+5)lentivirus carrier showed the supreme editing efficiency. Thus, the STING-sgRNA(1+5)lentivirus carrier combined with the packaging plasmid psPAX2 and the envelope plasmid pMD2.G were transfected 293T cells to package lentivirus, and then infected 3D4/21 cells with lentivirus. The results showed that a 3D4/21 cell line with a large deletion of the STING gene(4989 bp)was obtained. The STING protein was not observed by Western blotting, indicating that the STING gene knockout 3D4/21 cells(3D4/ 21-STING-/-)were successfully constructed. The transcription level of IFN-beta in 3D4/21-STING-/- cells decreased significantly (P<0.05) compared with parental cells when stimulated by transfection of Haemophilusparasuis DNA. Conclusion : By applying CRISPR/Cas9 technology, STING gene is successfully knock out in 3D4/21 cells, resulting in loss of function of STING gene;STING knockout leads to the transcription disorder of type I interferon when cells are stimulated by DNA, which also suggests that STING gene may be a key factor in the anti-pathogenic microbial infection of pigs.

Quantification of the Diversity in Gene Structures Using the Principles of Polarization Mapping.

Results of computational analysis and visualization of differences in gene structures using polarization coding are presented. A two-dimensional phase screen, where each element of which corresponds to a specific basic nucleotide (adenine, cytosine, guanine, or thymine), displays the analyzed nucleotide sequence. Readout of the screen with a coherent beam characterized by a given polarization state forms a diffracted light field with a local polarization structure that is unique for the analyzed nucleotide sequence. This unique structure is described by spatial distributions of local values of the Stokes vector components. Analysis of these distributions allows the comparison of nucleotide sequences for different strains of pathogenic microorganisms and frequency analysis of the sequences. The possibilities of this polarization-based technique are illustrated by the model data obtained from a comparative analysis of the spike protein gene sequences for three different model variants (Wuhan, Delta, and Omicron) of the SARS-CoV-2 virus. Various modifications of polarization encoding and analysis of gene structures and a possibility for instrumental implementation of the proposed method are discussed.

Molecular dual actions of hsa-miRNA and v-miRNA in oncogenic EBV

Viruses are one of the main reasons that cause healthy cells to proliferate and become cancerous. Several viruses have been identified as causative factors for various forms of cancer. Tumor occurrence can be caused by viral oncoprotein activity, persistent infection or inflammation. The molecular process is still complicated to be understood. In recent decades, Homo sapiens cell microRNA (hsa-miRNA) has been discovered by small non-coding RNAs that affect post-transcriptional gene expression. hsa-miRNA is a key control factor for several key biological processes and has a much greater impact on the target gene group. even though they occupy a small part of the genome, they play a great role in the development of cancer. Several viruses produce this tiny RNA, which can regulate their gene expression or affect the host's gene expression. A new hypothesis is that Epstein-Barr virus (EBV) is the first cancer causing virus that is found to produce microRNAs (v-miRNAs). In addition, evidence shows that miRNA encoded by EBV contributes to the occurrence and progression of EBV-related malignancies. Generally, these compounds reduce messenger RNA (mRNA) instability, such as genes that regulate tumorigenesis mechanisms like inflammation, cell cycle control, stress response, differentiation, apoptosis, invasion, and immune pathways. Therefore, EBV-miRNAs are important in the complex interaction between host, virus and EBV tumorigenesis. In terms of malignant tumors, the combinatorial process behind EBV-miRNA still needs further study. In this article, we will introduce EBV-miRNA, including the cellular processes affected by the virus, and their ability to promote cancer.

Unsupervised outlier detection applied to SARS-CoV-2 nucleotide sequences can identify sequences of common variants and other variants of interest.

As of June 2022, the GISAID database contains more than 11 million SARS-CoV-2 genomes, including several thousand nucleotide sequences for the most common variants such as delta or omicron. These SARS-CoV-2 strains have been collected from patients around the world since the beginning of the pandemic. We start by assessing the similarity of all pairs of nucleotide sequences using the Jaccard index and principal component analysis. As shown previously in the literature, an unsupervised cluster analysis applied to the SARS-CoV-2 genomes results in clusters of sequences according to certain characteristics such as their strain or their clade. Importantly, we observe that nucleotide sequences of common variants are often outliers in clusters of sequences stemming from variants identified earlier on during the pandemic. Motivated by this finding, we are interested in applying outlier detection to nucleotide sequences. We demonstrate that nucleotide sequences of common variants (such as alpha, delta, or omicron) can be identified solely based on a statistical outlier criterion. We argue that outlier detection might be a useful surveillance tool to identify emerging variants in real time as the pandemic progresses.

Identification and Analysis the Polymorphism in FGA Gene among Covid-19 Patients in Babylon Province/Iraq

SARS-COV-2, a beta coronavirus, was discovered to be the virus that caused the coronavirus disease pandemic in 2019 (Covid-19). It's been proven that abnormal coagulation function has a role in COVID-19 illness progression. However, there is no apparent link between D-dimer levels and COVID-19 severity. The goal of the study was to see if there was a link between D-dimer levels and the severity of COVID-19 investigation of SNPs in FGA gene. Interactors have been found in a variety of proteins, and fibrinogen chains (FGA) are a type of fibrinogen, which is an anti-infective organ, the liver produces this glycoprotein. And serves as an important coagulation factor and an acute phase reactant;for its versatile role in coagulation, inflammation, blood viscosity, and the implications in the management of (COVID 19). Fibrinogen's role in acute COVID 19 patients and clot formation has been studied been investigated by researchers. There have been no research on the direct association of Fibrinogen alpha chain (FGA) with SARS-Cov-2 so far. The current study proved that FGA did not affect the vaccinated people, but effect of sars-cov2 infected patients. Sequencing study of the investigated section revealed 392 bp of the gene FGA. The presence of an insertion mutation in the fifth axon results in a shift in amino acid sequence from Histidine CAU (GTA) to Threonine ACA (TGT), resulting a change in the amino acid sequence. © 2022, ResearchTrentz Academy Publishing Education Services. All rights reserved.

A novel deep learning model for predicting severity of the disease using COVID-19 long non-coding RNA sequence

The Covid-19 is very critical situation throughout the world from December 2019. In this pandemic situation some of the patients are symptomless and others have many varying symptoms of the disease. The severe symptom cases are leading to damage of multi organ and death. The mechanism used to indicate severity of the disease is still unknown. Early detection of the infected patients of Covid-19 is very important to control the spreading of diseases and also severity level of the disease helps to decrease the mortality rate. So deep learning model is developed to predict the severity of the Covid-19 disease by using lncRNA sequence which is a new approach. To train this deep learning model 3363 lncRNA mutation sequences of Covid-19 patients is used. Severity is predicted by the risk score of this model which indicates whether the patient suffering with severe and non-severe disease level. The performance is evaluated by comparing this model with other three Deep Learning algorithms i.e., LSTMs, RNN and GANs. The performance of this proposed model with CNN algorithm is best among all other algorithms and also it is able to predict the severity of the disease with high accuracy prediction of 96%, which helps to reduce mortality rate.