Microbiology and Immunology最新文献

Reverse genetics enables the generation of recombinant viruses; however, conventional approaches using full-length cDNA cloned into bacterial artificial chromosomes or plasmids are time-consuming and technically challenging. While the circular polymerase extension reaction (CPER) has been applied to RNA viruses, it has not been used for DNA viruses. Here, we successfully applied CPER to generate infectious recombinant adenoviruses (AdVs) of two serotypes. The resulting viruses replicated comparably to parental strains, and replication was confirmed by immunostaining. These findings demonstrate that CPER is a rapid and efficient platform for reverse genetics of DNA viruses and may accelerate AdV research and therapeutic development.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious respiratory disease. It is classified into two biovars on the basis of NAD requirement for growth: biovar 1 (NAD-dependent) and biovar 2 (NAD-independent). Biovar 1 consists of 17 serovars (1−12 and 15−19) and biovar 2 has 8 serovars (2, 4, 7, 9, 11, 13, 14, and 17). Several virulence factors are responsible for the pathogenesis of A. pleuropneumoniae, particularly Apx toxins, capsular polysaccharides, lipopolysaccharides, and membrane proteins. In this review, the current knowledge of biovars, serovars, epidemiology, virulence factors of A. pleuropneumoniae as well as laboratory diagnosis and vaccines are summarized.

Dengue fever poses a grave threat to public health worldwide, claiming numerous fatalities each year with tropical regions being particularly hard hit by dengue outbreaks. The multiple-epitope construct is tailored to the dengue virus's geographical prevalence and the genetics of the Pakistani population. 14 experimentally validated MHC-I and MHC-II epitope sequences, were employed to generate the variants by taking into account the conservancy in all serotypes. Subsequently, the binding affinities of each derived variant against the human leukocyte antigen alleles most common among the Pakistani population were analyzed. A total of three epitopes, two Class-I (GTSGSPIINR and RSWNTGFDW), and one Class-II (ILAPTRVVAAEMEEA), with a combined Pakistani population coverage of more than 73%, together with five linear B cell epitopes were used to create six possible multi-epitope fusion constructs. The molecular docking analysis indicated that two constructs demonstrated notable binding affinities for the most abundant HLA-A*11:01 in Pakistan. Additionally, molecular dynamics (MD) simulations identified one of the constructs as a promising therapeutic candidate. The vaccine construct selected from this analysis could aid in future vaccine design for the dengue virus following further in vitro test validation and in vivo studies to investigate its immune protection capacity.

We investigated the receptor usage of the hepatitis C virus (HCV) genotype 3a S310 strain using cell culture–derived HCV (HCVcc) and trans-complemented HCV particles (HCVtcp). Infection by HCV strains of genotypes 1, 2, and 3 was inhibited by anti-CD81 antibody. By contrast, anti–claudin (CLDN)1 antibody reduced infection by the genotype 1b TH strain and genotype 2a JFH-1 strain but had no effect on the S310 strain (genotype 3a). Moreover, CLDN1-knockout cells remained permissive to infection with a chimeric HCVcc bearing the S310 envelope in a JFH-1 backbone. In CLDN1-deficient cells, infection by HCVtcp derived from the S310 strain was significantly reduced by treatment with anti–claudin-6 (CLDN6) antibody or knockdown of CLDN6 mRNA, suggesting that the S310 strain utilizes CLDN6 as an alternate entry factor. Further analyses revealed that HCVtcp of genotype 4a (ED43 strain) and genotype 6a (HK6a strain) also infected CLDN1-deficient cells. These findings provide new insights into CLDN usage by diverse HCV genotypes and raise the possibility that CLDN tropism may affect viral entry, infection efficiency, and pathogenesis.

Surveillance of SARS-CoV-2 in white-tailed deer (WTD, Odocoileus virginianus) has revealed its widespread and sustained transmission across North America, with evidence suggesting possible transmission from deer to humans. In the following surveillance studies in other deer species, however, little evidence of infection spread was found, including in sika deer (Cervus nippon) in our previous study. Differences in the structure of the virus entry receptor angiotensin-converting enzyme 2 (ACE2) are known to act as one of the functional barriers to SARS-CoV-2 infection. To investigate the molecular basis of the lack of SARS-CoV-2 transmission to sika deer, we performed structural and functional analyses of the sika deer ACE2 in comparison with WTD ACE2. Comparison of sika deer ACE2 sequence and those of cervid species with WTD ACE2, followed by in silico molecular dynamics analysis, revealed a substitution of lysine to asparagine in position 31 commonly found in cervid ACE2s can potentially alter binding to the SARS-CoV-2 spike (S) protein receptor-binding domain (RBD). Functional assays in cells expressing sika deer and WTD ACE2s showed minimal differences in viral binding and replication, demonstrating that SARS-CoV-2 can similarly utilize ACE2 from both species. These findings suggest that sika deer and possibly other cervids may be highly susceptible to SARS-CoV-2 and highlight the need to investigate other factors impacting virus spread in deer populations.