Virulence In Mice Research Articles

Effects of Sublethally Injured Campylobacter jejuni in Mice.

ABSTRACTGlobally, Campylobacter spp. are the most common food-associated bacterial cause of human gastrointestinal disease. Campylobacteriosis is primarily associated with the consumption of contaminated chicken meat. Chemical decontamination of chicken carcasses during processing is one of the most effective interventions to mitigate Campylobacter contamination. Following exposure to sanitizers, however, sublethally injured populations of bacteria may persist. The risk that sublethally injured Campylobacter pose for public health is unknown. Furthermore, the virulence potential of sublethally injured Campylobacter jejuni during prolonged storage in relation to host pathogenesis and the host immune response has not been well established. Therefore, we evaluated the effects of sublethally injured C. jejuni on the host, after storage in chicken meat juice. C57BL/6 mice were infected with two C. jejuni chicken meat isolates or the ATCC 33291 strain that had been stored in the chicken meat juice, after exposure to chlorine or acidified sodium chlorite (ASC). Although chlorine exposure was unable to reduce intestinal colonization by C. jejuni, exposure to ASC significantly reduced the intestinal colonization and tissue translocation in mice. The expression of pro- and anti-inflammatory cytokine genes for interleukin-6 (IL-6), IL23a, and IL-10, Toll-like receptor 2 (TLR2) and TLR4 genes, and host stress response genes (CRP, MBL1, and NF-κB1) were significantly reduced following the exposure to ASC. Our results demonstrated that sublethally injured C. jejuni has reduced virulence potential and colonization in mice. The data contribute toward clarification of the importance of chemical decontamination during processing to minimize human campylobacteriosis.IMPORTANCE Campylobacter is the most common cause of bacterial gastrointestinal disease, and consumption of contaminated poultry is frequently identified as the source of bacteria. The survivability and virulence potential of sublethally injured Campylobacter following exposure to chemicals which are commonly used to eliminate Campylobacter during the poultry meat processing are of concern to the food industry, government health officials, and consumers. Here, we demonstrate that sublethally injured Campylobacter jejuni has reduced bacterial virulence and colonization potential in mice.

The C-Terminal Repeat Units of SpaA Mediate Adhesion of Erysipelothrix rhusiopathiae to Host Cells and Regulate Its Virulence.

Simple SummaryErysipelothrix rhusiopathiae is an important zoonotic pathogen, which poses a serious harm to the pig industry. We aimed to evaluate the genomic differences between virulent and avirulent strains to study the pathogenic mechanism of Erysipelothrix rhusiopathiae. The results showed that the spaA gene of avirulent strain lacked 120bp, encoding repeat units at the C-terminal of SpaA, the virulence of the virulent strain with this 120 bp deletion was attenuated, and the mutant strain decreased adhesion to porcine iliac artery endothelial cells.Erysipelothrix rhusiopathiae is a causative agent of erysipelas in animals and erysipeloid in humans. However, current information regarding E. rhusiopathiae pathogenesis remains limited. Previously, we identified two E. rhusiopathiae strains, SE38 and G4T10, which were virulent and avirulent in pigs, respectively. Here, to further study the pathogenic mechanism of E. rhusiopathiae, we sequenced and assembled the genomes of strains SE38 and G4T10, and performed a comparative genomic analysis to identify differences or mutations in virulence-associated genes. Next, we comparatively analyzed 25 E. rhusiopathiae virulence-associated genes in SE38 and G4T10. Compared with that of SE38, the spaA gene of the G4T10 strain lacked 120 bp, encoding repeat units at the C-terminal of SpaA. To examine whether these deletions or splits influence E. rhusiopathiae virulence, these 120 bp were successfully deleted from the spaA gene in strain SE38 by homologous recombination. The mutant strain ΔspaA displayed attenuated virulence in mice and decreased adhesion to porcine iliac artery endothelial cells, which was also observed using the corresponding mutant protein SpaA’. Our results demonstrate that SpaA-mediated adhesion between E. rhusiopathiae and host cells is dependent on its C-terminal repeat units.

Recombinant vesicular stomatitis vaccine against Nipah virus has a favorable safety profile: Model for assessment of live vaccines with neurotropic potential.

Nipah virus (NiV) disease is a bat-borne zoonosis responsible for outbreaks with high lethality and is a priority for vaccine development. With funding from the Coalition of Epidemic Preparedness Innovations (CEPI), we are developing a chimeric vaccine (PHV02) composed of recombinant vesicular stomatitis virus (VSV) expressing the envelope glycoproteins of both Ebola virus (EBOV) and NiV. The EBOV glycoprotein (GP) mediates fusion and viral entry and the NiV attachment glycoprotein (G) is a ligand for cell receptors, and stimulates neutralizing antibody, the putative mediator of protection against NiV. PHV02 is identical in construction to the registered Ebola vaccine (Ervebo) with the addition of the NiV G gene. NiV ephrin B2 and B3 receptors are expressed on neural cells and the wild-type NiV is neurotropic and causes encephalitis in affected patients. It was therefore important to assess whether the NiV G alters tropism of the rVSV vector and serves as a virulence factor. PHV02 was fully attenuated in adult hamsters inoculated by the intramuscular (IM) route, whereas parental wild-type VSV was 100% lethal. Two rodent models (mice, hamsters) were infected by the intracerebral (IC) route with graded doses of PHV02. Comparator active controls in various experiments included rVSV-EBOV (representative of Ebola vaccine) and yellow fever (YF) 17DD commercial vaccine. These studies showed PHV02 to be more neurovirulent than both rVSV-EBOV and YF 17DD in infant animals. PHV02 was lethal for adult hamsters inoculated IC but not for adult mice. In contrast YF 17DD retained virulence for adult mice inoculated IC but was not virulent for adult hamsters. Because of the inconsistency of neurovirulence patterns in the rodent models, a monkey neurovirulence test (MNVT) was performed, using YF 17DD as the active comparator because it has a well-established profile of quantifiable microscopic changes in brain centers and a known reporting rate of neurotropic adverse events in humans. In the MNVT PHV02 was significantly less neurovirulent than the YF 17DD vaccine reference control, indicating that the vaccine will have an acceptable safety profile for humans. The findings are important because they illustrate the complexities of phenotypic assessment of novel viral vectors with tissue tropisms determined by transgenic proteins, and because it is unprecedented to use a heterologous comparator virus (YF vaccine) in a regulatory-enabling study. This approach may have value in future studies of other novel viral vectors.

Secretion of the siderophore rhizoferrin is regulated by the cAMP-PKA pathway and is involved in the virulence of Mucor lusitanicus

Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.

Deletion C-terminal thioesterase abolishes melanin biosynthesis, affects metabolism and reduces the pathogenesis of Fonsecaea monophora.

Dematiaceous Fonsecaea monophora is one of the major pathogens of chromoblastomycosis. It has been well established that melanization is catalyzed by the type I polyketide synthase (PKS) in F. monophora. Multidomain protein Type I PKS is encoded by six genes, in which the last enzyme thioesterase (TE) catalyzes the cyclization and releases polyketide. Two PKS genes AYO21_03016 (pks1) and AYO21_10638 have been found in F. monophora and both PKS loci have the same gene arrangement but the TE domain in AYO21_10638 is truncated at 3’- end. TE may be the key enzyme to maintain the function of pks1. To test this hypothesis, we constructed a 3’-end 500 bp deletion mutant of AYO21_03016 (Δpks1-TE-C500) and its complemented strain. We profiled metabolome of this mutant and analyzed the consequences of impaired metabolism in this mutant by fungal growth in vitro and by pathogenesis in vivo. Compared with wild-type strain, we found that the mutant repressed pks1 expression and other 5 genes expression levels were reduced by more than 50%, perhaps leading to a corresponding melanin loss. The mutant also reduced sporulation and delayed germination, became vulnerable to various environmental stresses and was less resistance to macrophage or neutrophil killings in vitro, and less virulence in mice footpad model. Metabolomic analysis indicated that many metabolites were remarkably affected in Δpks1-TE-C500, in particular, an increased nicotinamide and antioxidant glutathione. In conclusion, we confirmed the crucial role of C-terminal TE in maintaining fully function of pks1 in F. monophora. Deletion of TE negatively impacts on the synthesis of melanin and metabolites that eventually affect growth and virulence of F. monophora. Any potential inhibitor of TE then could be a novel antifungal target for drug development.

D2I and F9Y Mutations in the NS1 Protein of Influenza A Virus Affect Viral Replication via Regulating Host Innate Immune Responses.

Influenza A viruses (IAV) modulate host antiviral responses to promote viral growth and pathogenicity. The non-structural (NS1) protein of influenza A virus has played an indispensable role in the inhibition of host immune responses, especially in limiting interferon (IFN) production. In this study, random site mutations were introduced into the NS1 gene of A/WSN/1933 (WSN, H1N1) via an error prone PCR to construct a random mutant plasmid library. The NS1 random mutant virus library was generated by reverse genetics. To screen out the unidentified NS1 functional mutants, the library viruses were lung-to-lung passaged in mice and individual plaques were picked from the fourth passage in mice lungs. Sanger sequencing revealed that eight different kinds of mutations in the NS1 gene were obtained from the passaged library virus. We found that the NS1 F9Y mutation significantly enhanced viral growth in vitro (MDCK and A549 cells) and in vivo (BALB/c mice) as well as increased virulence in mice. The NS1 D2I mutation attenuated the viral replication and pathogenicity in both in vitro and in vivo models. Further studies demonstrated that the NS1 F9Y mutant virus exhibited systematic and selective inhibition of cytokine responses as well as inhibited the expression of IFN. In addition, the expression levels of innate immunity-related cytokines were significantly up-regulated after the rNS1 D2I virus infected A549 cells. Collectively, our results revealed that the two mutations in the N-terminal of the NS1 protein could alter the viral properties of IAV and provide additional evidence that the NS1 protein is a critical virulence factor. The two characterized NS1 mutations may serve as potential targets for antiviral drugs as well as attenuated vaccine development.

Pat- and Pta-mediated protein acetylation is required for horizontally-acquired virulence gene expression in Salmonella Typhimurium.

Salmonella Typhimurium is a Gram-negative facultative pathogen that causes a range of diseases, from mild gastroenteritis to severe systemic infection in a variety of animal hosts. S. Typhimurium regulates virulence gene expression by a silencing mechanism using nucleoid-associated proteins such as Histone-like Nucleoid Structuring protein (H-NS) silencing. We hypothesize that the posttranslational modification, specifically protein acetylation, of proteins in gene silencing systems could affect the pathogenic gene expression of S. Typhimurium. Therefore, we created acetylation-deficient mutant by deleting two genes, pat and pta, which are involved in the protein acetylation pathway. We observed that the pat and pta deletion attenuates mouse virulence and also decreases Salmonella's replication within macrophages. In addition, the Δpat Δpta strain showed a decreased expression of the horizontally-acquired virulence genes, mgtC, pagC, and ugtL, which are highly expressed in low Mg2+. The decreased virulence gene expression is possibly due to higher H-NS occupancy to those promoters because the pat and pta deletion increases H-NS occupancy whereas the same mutation decreases occupancy of RNA polymerase. Our results suggest that Pat- and Pta-mediated protein acetylation system promotes the expression of virulence genes by regulating the binding affinity of H-NS in S. Typhimurium.

Evidence for Water-Borne Transmission of Highly Pathogenic Avian Influenza H5N1 Viruses.

In this study, we isolated 10 H5N1 strains from water samples in Dongting Lake and 4 H5N1 strains from lakeside backyard poultry. These isolates belonged to three distinct clades (clade 2.3.2, 2.3.4, and 7). Phylogenetic analysis showed a diversified genome constellation. The genetic characteristics of some viruses isolated from water samples were extremely similar to those from lakeside poultry. Pathogenic experiments showed that selected represented isolates in this study were highly pathogenic for SPF chickens but had a diversified virulence in mice. The results of our study suggested the potential transmission of avian influenza (H5N1) between the poultry and wild waterfowls and water body around the habitat may play an important role.

Comparative study of the phenotype and virulence of recent serovar 1a, 1b, and 2a isolates of Erysipelothrix rhusiopathiae in Japan

Erysipelothrix rhusiopathiae causes swine erysipelas (SE) and is classified –into 16 serovars based on cell surface antigens. Our previous study suggested that recent SE outbreaks were mostly caused by serovar 1a of E. rhusiopathiae with the surface protective antigen (Spa)A protein characterized by methionine and isoleucine at positions 203 and 257 (M203/I257 SpaA). In this study, four recent E. rhusiopathiae isolates comprising two serovar 1a with M203/I257 SpaA strains (2012 Miyazaki and 2012 Chiba), one serovar 1b strain (2015 Miyazaki), and one serovar 2a strain (2012 Nagano) were compared with each other and with the serovar 1a Fujisawa reference strain regarding in vitro phenotypes and in vivo virulence in mice and pigs. The serovar 1b and 2a strains, which are the less prevalent strains in the field in Japan, showed lower growth in liquid culture and lower virulence in animals than the serovar 1a variants. Adhesion of the serovar 2a strain to porcine endothelial cells was weaker than that of the serovar 1a and 1b strains. Several advantages of serovar 1a strains were found, but no plausible cause of the M203/I257 SpaA type variants to be selected for the most prevalent strains among serovar 1a strains was identified in this study.

A Macroporous Magnesium Oxide-Templated Carbon Adsorbs Shiga Toxins and Type III Secretory Proteins in Enterohemorrhagic Escherichia coli, Which Attenuates Virulence.

Enterohemorrhagic Escherichia coli (EHEC) is one of the most common foodborne pathogens. However, no drug that prevents the severe complications caused by this bacterium has been approved yet. This study showed that a macroporous magnesium oxide (MgO)-templated carbon material (MgOC150) adsorbs Shiga toxins, and Type III secretory EspA/EspB proteins responsible for EHEC pathogenesis, and decreases the extracellular levels of these proteins. On the other hand, this material did not affect the growth of EHEC. Citrobacter rodentium traditionally used to estimate Type III secretion system-associated virulence in mice is highly virulent. The survival period of infected mice was prolonged when MgOC150 was administered. This adsorbent disturbed neither mammalian cells nor normal intestinal bacteria, such as Enterococcus hirae, Lactobacillus acidophilus, and Lactobacillus casei. In contrast, MgOC150 adsorbed antimicrobial agents, including β-lactams, quinolones, tetracyclines, and trimethoprim/sulfamethoxazole. However, fosfomycin and amikacin were not adsorbed. Thus, MgOC150 can be used with fosfomycin and amikacin to treat infections. MgOC150 is used for industrial purposes, such as an electrode catalyst, a bioelectrode, and enzyme immobilization. The study proposed another potential application of MgOC150, assisting anti-EHEC chemotherapy.

Pathological study of Pasteurella Multocida Recombinant Clone ABA392

Objective: In current study Molecular analysis of the recombinant clone ABA392 was carried out Methodology. In this study, detailed molecular analysis of the recombinant clone ABA392 were carried out by examining plasmid stability, plasmid profiling, determination of the plasmid molecular weight, and restriction endonuclease analysis. Results: From the experiments above, the plasmid size of ABA392 was estimated as 3.5 kb, while the size of the insert gene of ABA392 was estimated as 0.8 kb (double digested with PstI and SmaI restriction enzymes). A Pathogenicity study of recombinant clone ABA392 was carried out via a mouse virulence test and histopathological analysis, where it was found that mice, which were challenged with ABA392 and the parent strain of Pasteurella multocida serotype B strain PMB202, were lethal within 24 to 72 hours upon inoculation. Based on microscopic examination, the virulence effect was observed by marked haemorrhage in lung, liver, spleen, and kidney tissues. Conclusion: This study has also proven that the plasmid of recombinant clone ABA392 was stably maintained in the Escherichia coli JM101 host system as reported previously. A preliminary protein analysis via SDS-PAGE was performed and a distinct protein band was most likely to be detected, which was approximately 16kD. Nonetheless, further characterization of the recombinant clone ABA392 proteins needs to be done to determine the nature of these proteins, which are involved in the virulence mechanism of hemorrhagic septicemia. Keywords: Plasmid profiling; Pathogenicity; recombinant clone; Pasteurella multocida

A bacterial endosymbiont of the fungus Rhizopus microsporus drives phagocyte evasion and opportunistic virulence

SummaryOpportunistic infections by environmental fungi are a growing clinical problem, driven by an increasing population of people with immunocompromising conditions. Spores of the Mucorales order are ubiquitous in the environment but can also cause acute invasive infections in humans through germination and evasion of the mammalian host immune system. How they achieve this and the evolutionary drivers underlying the acquisition of virulence mechanisms are poorly understood. Here, we show that a clinical isolate of Rhizopus microsporus contains a Ralstonia pickettii bacterial endosymbiont required for virulence in both zebrafish and mice and that this endosymbiosis enables the secretion of factors that potently suppress growth of the soil amoeba Dictyostelium discoideum, as well as their ability to engulf and kill other microbes. As amoebas are natural environmental predators of both bacteria and fungi, we propose that this tri-kingdom interaction contributes to establishing endosymbiosis and the acquisition of anti-phagocyte activity. Importantly, we show that this activity also protects fungal spores from phagocytosis and clearance by human macrophages, and endosymbiont removal renders the fungal spores avirulent in vivo. Together, these findings describe a new role for a bacterial endosymbiont in Rhizopus microsporus pathogenesis in animals and suggest a mechanism of virulence acquisition through environmental interactions with amoebas.

Diagnostic Value of Circulating Antigens in the Serum of Piglets with Experimental Acute Toxoplasmosis.

Toxoplasmosis, caused by Toxoplasma gondii, an apicomplexan parasite, infects all warm-blooded animals, including a third of the human population. Laboratory diagnosis of acute toxoplasmosis is based on the detection of anti-T. gondii IgM and IgG and T. gondii nucleic acid; however, these assays have certain limitations. Circulating Ags (CAgs) are reliable diagnostic indicators of acute infection. In this study, we established a model of acute T. gondii infection in Large White pigs. CAg levels peaked between 3 and 5 d after inoculation, and 28 CAgs were identified using an immunoprecipitation-shotgun approach, among which dolichol-phosphate-mannose synthase family protein (TgDPM), C3HC zinc finger-like protein (TgZFLP3), and ribosomal protein RPL7 (TgRPL7) were selected to further investigate their value in the diagnosis of acute toxoplasmosis. Immunofluorescence assays revealed that TgDPM and TgRPL7 were localized in the membrane surface, while TgZFLP3 was localized in the apical end. Western blotting revealed the presence of the three proteins in the serum during acute infection. Indirect ELISA results indicate that TgZFLP3 is likely to be a novel candidate for the diagnosis of acute toxoplasmosis. However, these three proteins may not be useful as candidate vaccines against toxoplasmosis owing to their low protective ability. In addition, deletion of the zflp3 gene partially attenuated virulence in Kunming mice. Collectively, we identified 28 CAgs in the serum of piglets with experimental acute toxoplasmosis and confirmed that TgZFLP3 is a potential biomarker for acute T. gondii infection. The results of this study provide data to improve the detection efficiency of acute toxoplasmosis.

The PB1 gene from H9N2 avian influenza virus showed high compatibility and increased mutation rate after reassorting with a human H1N1 influenza virus

BackgroundReassortment between human and avian influenza viruses (AIV) may result in novel viruses with new characteristics that may threaten human health when causing the next flu pandemic. A particular risk may be posed by avian influenza viruses of subtype H9N2 that are currently massively circulating in domestic poultry in Asia and have been shown to infect humans. In this study, we investigate the characteristics and compatibility of a human H1N1 virus with avian H9N2 derived genes.MethodsThe polymerase activity of the viral ribonucleoprotein (RNP) complex as combinations of polymerase-related gene segments derived from different reassortment events was tested in luciferase reporter assays. Reassortant viruses were generated by reverse genetics. Gene segments of the human WSN-H1N1 virus (A/WSN/1933) were replaced by gene segments of the avian A2093-H9N2 virus (A/chicken/Jiangsu/A2093/2011), which were both the Hemagglutinin (HA) and Neuraminidase (NA) gene segments in combination with one of the genes involved in the RNP complex (either PB2, PB1, PA or NP). The growth kinetics and virulence of reassortant viruses were tested on cell lines and mice. The reassortant viruses were then passaged for five generations in MDCK cells and mice lungs. The HA gene of progeny viruses from different passaging paths was analyzed using Next-Generation Sequencing (NGS).ResultsWe discovered that the avian PB1 gene of H9N2 increased the polymerase activity of the RNP complex in backbone of H1N1. Reassortant viruses were able to replicate in MDCK and DF1 cells and mice. Analysis of the NGS data showed a higher substitution rate for the PB1-reassortant virus. In particular, for the PB1-reassortant virus, increased virulence for mice was measured by increased body weight loss after infection in mice.ConclusionsThe higher polymerase activity and increased mutation frequency measured for the PB1-reassortant virus suggests that the avian PB1 gene of H9N2 may drive the evolution and adaptation of reassortant viruses to the human host. This study provides novel insights in the characteristics of viruses that may arise by reassortment of human and avian influenza viruses. Surveillance for infections with H9N2 viruses and the emergence of the reassortant viruses in humans is important for pandemic preparedness.

Combined insertion of basic and non-basic amino acids at hemagglutinin cleavage site of highly pathogenic H7N9 virus promotes replication and pathogenicity in chickens and mice.

Since mid-2016, the low pathogenic H7N9 influenza virus has evolved into a highly pathogenic (HP) phenotype in China, raising many concerns about public health and poultry industry. The insertion of a “KRTA” motif at hemagglutinin cleavage site (HACS) occurred in the early stage of HP H7N9 variants. During the co-circulation, the HACS of HP-H7N9 variants were more polymorphic in birds and humans. Although HP-H7N9 variants, unlike the H5 subtype virus, exhibited the insertions of basic and non-basic amino acids, the underlying function of those insertions and substitutions remains unclear. The results of bioinformatics analysis indicated that the PEVPKRKRTAR/G motif of HACS had become the dominant motif in China. Then, we generated six H7N9 viruses bearing the PEIPKGR/G, PEVPKGR/G, PEVPKRKRTAR/G, PEVPKGKRTAR/G, PEVPKGKRIAR/G, and PEVPKRKRR/G motifs. Interestingly, after the deletion of threonine and alanine (TA) at HACS, the H7N9 viruses manifested decreased thermostability and virulence in mice, and the PEVPKRKRTAR/G-motif virus is prevalent in birds and humans probably due to its increased transmissibility and moderate virulence. By contrast, the insertion of non-basic amino acid isoleucine and alanine (IA) decreased the transmissibility in chickens and virulence in mice. Remarkably, the I335V substitution of H7N9 virus enhanced infectivity and transmission in chickens, suggesting that the combination of mutations and insertions of amino acids at the HACS promoted replication and pathogenicity in chickens and mice. The ongoing evolution of H7N9 increasingly threatens public health and poultry industry, so, its comprehensive surveillance and prevention of H7N9 viruses should be pursued.

Functional Characterization of 17 Protein Serine/Threonine Phosphatases in Toxoplasma gondii Using CRISPR-Cas9 System.

Protein serine/threonine phosphatases (PSPs), found in various plants and protozoa, are involved in the regulation of various biological processes. However, very little is known about the role of PSPs in the pathogenicity of the apicomplexan protozoan Toxoplasma gondii. Herein, the subcellular localization of 17 PSPs (PP5, PP7, EFPP, SLP, PPM3F, PPM4, PPM5A, PPM5B, PPM6, PPM8, PPM9, PPM12, PPM14, PPM18, CTD1, CTD2, and CTD3) was examined by 6× HA tagging of endogenous genes in C-terminal. The PSPs were detected in the cytoplasm (PP5, EFPP, PPM8, and CTD2), dense granules (SLP), nucleus (PPM4 and PPM9), inner membrane complex (PPM12), basal complex (CTD3), and apical pole (PP7). The remaining PSPs exhibited low or undetectable level of expression. To characterize the contribution of these genes to the infectivity of T. gondii, knock-out (KO) strains of type I RH strain deficient in the 17 psp genes and KO type II Pru strain deficient in pp7 and slp genes were constructed. The pathogenicity of individual RHΔpsp mutants was characterized in vitro using plaque, egress, and intracellular replication assays, and mouse infection, while pathogenicity of PruΔpp7 and PruΔslp mutant strains was evaluated by examining the parasite lytic cycle in vitro and assessment of brain cyst burden in mice. No significant differences were observed between 16 RHΔpsp strains and wild-type (WT) RH strain. However, RHΔpp7 exhibited significantly lower invasion efficiency and parasitophorous vacuole formation in vitro, and less virulence in mice compared with other RHΔpsp and WT strains. In addition, PruΔpp7 exhibited marked attenuation of virulence and significant reduction in the brain cyst burden in mice compared with PruΔslp and WT strains, suggesting the key role of PP7 in the virulence of T. gondii. Comparative transcriptomic profiling of the 17 psp genes showed that they may play different roles in the pathogenesis of different genotypes or life cycle stages of T. gondii. These findings provide new insight into the role of PSPs in the pathogenesis of T. gondii.

Pseudorabies virus UL16 protein influences the inhibition of LRPPRC for the viral proliferation

Pseudorabies is caused by pseudorabies virus (PRV), a member of the Herpesvirus family, and has caused tremendous damage to the pig industry. Protein unique lone 16 (pUL16) is a conserved envelope protein in all herpesviruses, that is known to play an important role in several aspects, including virus diffusion in cells and virulence in mice. It has been shown that the pUL16 can interact with the virus proteins UL11, UL49, UL21, gD, and gE. However, the research to date on pUL16 has only focused on etiology, without discussing the possible cellular pathways involved in PRV infection. Leucine-rich PPR motif-containing protein (LRPPRC) is a multifunctional cellular protein that participates in various cellular processes, such as RNA processing, splicing, stabilization, editing, translation, and energy metabolism. This was the first caspase-independent apoptosis protein to be identified. In this study, immune precipitation and mass spectrometry was performed to define the function of the pUL16 in PRV infection to study the possible cellular pathways in which pUL16 may participate. It was found that LRPRRC could interact with PRV pUL16, which may indicate that UL16 is involved in a redox reaction or cellular apoptosis. This is the first study of the interaction between pUL16 and host proteins, which has positive significance to gain a further understanding of the pUL16.

Single dose of chimeric dengue-2/Zika vaccine candidate protects mice and non-human primates against Zika virus

The development of a safe and effective Zika virus (ZIKV) vaccine has become a global health priority since the widespread epidemic in 2015-2016. Based on previous experience in using the well-characterized and clinically proven dengue virus serotype-2 (DENV-2) PDK-53 vaccine backbone for live-attenuated chimeric flavivirus vaccine development, we developed chimeric DENV-2/ZIKV vaccine candidates optimized for growth and genetic stability in Vero cells. These vaccine candidates retain all previously characterized attenuation phenotypes of the PDK-53 vaccine virus, including attenuation of neurovirulence for 1-day-old CD-1 mice, absence of virulence in interferon receptor-deficient mice, and lack of transmissibility in the main mosquito vectors. A single DENV-2/ZIKV dose provides protection against ZIKV challenge in mice and rhesus macaques. Overall, these data indicate that the ZIKV live-attenuated vaccine candidates are safe, immunogenic and effective at preventing ZIKV infection in multiple animal models, warranting continued development.

Emergence, Evolution, and Pathogenicity of Influenza A(H7N4) Virus in Shorebirds in China.

ABSTRACTA 2-year surveillance study of influenza A viruses in migratory birds was conducted to understand the subsequent risk during the migratory seasons in Dandong Yalu River Estuary Coastal Wetland National Nature Reserve, Liaoning Province, China, a major stopover site on the East Asian-Australasian flyway. Overall, we isolated 27 influenza A viruses with multiple subtypes, including H3N8 (n = 2), H4N6 (n = 2), H4N7 (n = 2), H7N4 (n = 9), H7N7 (n = 1), H10N7 (n = 7), and H13N6 (n = 4). Particularly, a novel reassortant influenza A(H7N4) virus was first identified in a woman and her backyard poultry flock in Jiangsu Province, China, posing a serious threat to public health. Here, we describe the genetic characterization and pathogenicity of the nine influenza A(H7N4) isolates. Phylogenetic analysis indicated that complex viral gene flow occurred among Asian countries. We also demonstrated a similar evolutionary trajectory of the surface genes of the A(H7N4) isolates and Jiangsu human-related A(H7N4) viruses. Our A(H7N4) isolates exhibited differing degrees of virulence in mice, suggesting a potential risk to other mammalian species, including humans. We revealed multiple mutations that might affect viral virulence in mice. Our report highlights the importance and need for the long-term surveillance of avian influenza virus in migratory birds combined with domestic poultry surveillance along migratory routes and flyways and, thereby, the development of measures to manage potential health threats.IMPORTANCE The H7 subtype avian influenza viruses, such as H7N2, H7N3, H7N4, H7N7, and H7N9, were documented as being capable of infecting humans, and the H7 subtype low pathogenicity avian influenza viruses are capable of mutating into highly pathogenic avian influenza; therefore, they pose a serious threat to public health. Here, we investigated the evolutionary history, molecular characteristics, and pathogenicity of shorebird-origin influenza A(H7N4) viruses, showing a similar evolutionary trajectory with Jiangsu human A(H7N4) viruses in HA and NA genes. Moreover, our isolates exhibited variable virulence (including moderate virulence) in mice, suggesting a potential risk to other mammalian species, including humans.

Roles of OmpA in Type III Secretion System-Mediated Virulence of Enterohemorrhagic Escherichia coli.

Outer membrane proteins are commonly produced by gram-negative bacteria, and they have diverse functions. A subgroup of proteins, which includes OmpA, OmpW and OmpX, is often involved in bacterial pathogenesis. Here we show that OmpA, rather than OmpW or OmpX, contributes to the virulence of enterohemorrhagic Escherichia coli (EHEC) through its type III secretion system (T3SS). Deletion of ompA decreased secretion of the T3SS proteins EspA and EspB; however, the expression level of the LEE genes that encode a set of T3SS proteins did not decrease. The ompA mutant had less abilities to form A/E lesions in host epithelial cells and lyse human red blood cells than the parent strain. Moreover, the virulence of an ompA mutant of Citrobacter rodentium (traditionally used to estimate T3SS-associated virulence in mice) was attenuated. Mice infected with the ompA mutant survived longer than those infected with the parent strain. Furthermore, mice infected with ompA developed symptoms of diarrhea more slowly than mice infected with the parent strain. Altogether, these results suggest that OmpA sustains the activity of the T3SS and is required for optimal virulence in EHEC. This work expands the roles of outer membrane proteins in bacterial pathogenesis.