Artificial Synthesis of Conserved Segment S Gene Fragment of Rift Valley Fever Virus and Preliminary Study of Its Reverse Transcription Loop-Mediated Isothermal Amplification Detection Method

Purpose: To develop a rapid detection method for Rift Valley fever virus (RVFV) diagnosis.Methods: According to the reference sequences of RVFV published in GenBank, nine overlapping polymerase chain reaction (PCR) primers and four specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) primers were designed using DNAStar and LAMP primer design software, respectively. Based on the synthesis of a conserved part of the RVFV S segment gene sequence using overlapping PCR, RT-LAMP assay was first established and evaluated after a series of tests, including, optimization of reaction conditions, and sensitivity and specificity tests.Result: A target RVFV S segment gene fragment of 288 bp was synthesised. The optimal reaction conditions for RT-LAMP assay were 63 °C for 45 min: the assay has a specific ladder-like pattern of amplification bands from about 120 bp. The lowest target gene copy number of RT-LAMP for RVFV detection was 70 copies. The assay showed good specificity as only the synthesised target RVFV gene was amplified with no amplification for the detection of Peste des petits ruminants virus, Epidemic encephalitis B virus, E. coli, Pasteurella multocida, or Salmonella.Conclusion: This study provides a rapid, sensitive, specific RT-LAMP method for RVFV detection.Keywords: Rift valley fever virus, Overlapping polymerase chain reaction, Reverse transcription loopmediated isothermal amplification, Rapid diagnosis tes

Design of a multi-epitope vaccine against Haemophilus parasuis based on pan-genome and immunoinformatics approaches

BackgroundGlässer's disease, caused by Haemophilus parasuis (HPS), is responsible for economic losses in the pig industry worldwide. However, the existing commercial vaccines offer poor protection and there are significant barriers to the development of effective vaccines.MethodsIn the current study, we aimed to identify potential vaccine candidates and design a multi-epitope vaccine against HPS by performing pan-genomic analysis of 121 strains and using a reverse vaccinology approach.ResultsThe designed vaccine constructs consist of predicted epitopes of B and T cells derived from the outer membrane proteins of the HPS core genome. The vaccine was found to be highly immunogenic, non-toxic, and non-allergenic as well as have stable physicochemical properties. It has a high binding affinity to Toll-like receptor 2. In addition, in silico immune simulation results showed that the vaccine elicited an effective immune response. Moreover, the mouse polyclonal antibody obtained by immunizing the vaccine protein can be combined with different serotypes and non-typable Haemophilus parasuis in vitro.ConclusionThe overall results of the study suggest that the designed multi-epitope vaccine is a promising candidate for pan-prophylaxis against different strains of HPS

Pan-genome wide association study of Glaesserella parasuis highlights genes associated with virulence and biofilm formation

Glaesserella parasuis is a gram-negative bacterium that causes fibrotic polyserositis and arthritis in pig, significantly affecting the pig industry. The pan-genome of G. parasuis is open. As the number of genes increases, the core and accessory genomes may show more pronounced differences. The genes associated with virulence and biofilm formation are also still unclear due to the diversity of G. parasuis. Therefore, we have applied a pan-genome-wide association study (Pan-GWAS) to 121 strains G. parasuis. Our analysis revealed that the core genome consists of 1,133 genes associated with the cytoskeleton, virulence, and basic biological processes. The accessory genome is highly variable and is a major cause of genetic diversity in G. parasuis. Furthermore, two biologically important traits (virulence, biofilm formation) of G. parasuis were studied via pan-GWAS to search for genes associated with the traits. A total of 142 genes were associated with strong virulence traits. By affecting metabolic pathways and capturing the host nutrients, these genes are involved in signal pathways and virulence factors, which are beneficial for bacterial survival and biofilm formation. This research lays the foundation for further studies on virulence and biofilm formation and provides potential new drug and vaccine targets against G. parasuis

Pan-genome analysis of Streptococcus suis serotype 2 highlights genes associated with virulence and antibiotic resistance

Streptococcus suis serotype 2 (SS2) is a Gram-positive bacterium. It is a common and significant pathogen in pigs and a common cause of zoonotic meningitis in humans. It can lead to sepsis, endocarditis, arthritis, and pneumonia. If not diagnosed and treated promptly, it has a high mortality rate. The pan-genome of SS2 is open, and with an increasing number of genes, the core genome and accessory genome may exhibit more pronounced differences. Due to the diversity of SS2, the genes related to its virulence and resistance are still unclear. In this study, a strain of SS2 was isolated from a pig farm in Sichuan Province, China, and subjected to whole-genome sequencing and characterization. Subsequently, we conducted a Pan-Genome-Wide Association Study (Pan-GWAS) on 230 strains of SS2. Our analysis indicates that the core genome is composed of 1,458 genes related to the basic life processes of the bacterium. The accessory genome, consisting of 4,337 genes, is highly variable and a major contributor to the genetic diversity of SS2. Furthermore, we identified important virulence and resistance genes in SS2 through pan-GWAS. The virulence genes of SS2 are mainly associated with bacterial adhesion. In addition, resistance genes in the core genome may confer natural resistance of SS2 to fluoroquinolone and glycopeptide antibiotics. This study lays the foundation for further research on the virulence and resistance of SS2, providing potential new drug and vaccine targets against SS2

Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B

Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single stranded RNA (ssRNA) viruses, including CSFV. Based on genetic variation, the CSFV RdRps could be clearly divided into two major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Different evolutionary patterns of classical swine fever virus envelope proteins

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious disease of the domestic pig as well as wild boar. The proteins Erns, E1, and E2 are components of the viral envelope membrane. They are also implicated in virus attachment and entry, replication, and/or anti-immune response. Here, we studied the genetic variations of these envelope proteins in the evolution of CSFV. The results reveal that the envelope proteins underwent different evolutionary fates. In Erns and E1, but not E2, a number of amino acid sites experienced functional divergence. Furthermore, the diversification in Erns and E1 were generally episodic because the divergence-related changes of E1 have only occurred with the split between two major groups of CSFV and that of Erns have taken place with the division of one major group. The major divergence-related sites of Erns are located on one of the substrate-binding regions of RNase domain and C-terminal extension. These functional domains have been reported to block activation of the innate immune system and attachment and entry into host cells, respectively. Our results might shed some light on the divergent roles of the envelope proteins.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author