Wild-type Infection Research Articles

Burden of post–acute COVID-19 sequelae in healthcare workers and its course over a 30-month period–results from a prospective multicentre cohort

Abstract Purpose As healthcare workers (HCW) have been disproportionally affected by COVID-19, its post-acute sequelae (PASC) in HCW can impact healthcare systems. We assessed the burden and course of PASC in HCW over a 30-month period. Methods In a prospective multicentre HCW cohort in Switzerland, PASC surveys were conducted in 03/2021, 09/2021, 06/2022, 04/2023, and 10/2023. Stratified by viral variant at first infection, the prevalence of PASC symptoms, self-experienced PASC and the Post-COVID Functional Status (PCFS) were analysed cross-sectionally in 10/2023, self-perceived success of therapeutic measures used was assessed. The evolution of PASC symptoms and PCFS in Wild-type and non-Wild-type infected HCW compared to uninfected controls was analysed longitudinally across all surveys. Results In cross-sectional analysis, 1704 HCW (median age 47 years, 82.2% female) were included. Thereof, 30.7% reported ≥ 1 PASC symptom in 10/2023, with 115 (6.7%) stating to have or have had PASC. Both were most common after Wild-type infection compared to other variants. Overall, 17/115 (15%) indicated relevant/severe restrictions in their daily activities and of 85 (74%) that tried ≥ 1 measure against their symptoms, 69 (81%) reported having benefitted. Longitudinal analysis (n = 653) showed a significantly higher proportion of Wild-type infected HCW to report PASC symptoms compared to controls in 03/2021 (+ 21%, 95% CI 4–39), with decreasing trend (+ 7%, 95%CI -10–25 in 10/2023). This effect was not evident for non-Wild-type infected HCW. Conclusions Over a 30 month period, overall PASC burden in our HCW cohort decreased, although 1% still experience relevant restrictions in their daily life; Wild-type infected individuals show the highest disease burden.

Host species-specific activity of the poxvirus PKR inhibitors E3 and K3 mediate host range function.

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. The expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix αG was resistant to wild-type VACV infection and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA.IMPORTANCEThe molecular mechanisms that govern the host range of viruses are incompletely understood. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. The current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters double-stranded (ds) RNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete and that dsRNA sequestration is not the primary mechanism for E3 activity.

Agrobacterium virulence factors induce the expression of host DNA repair-related genes without promoting major genomic damage

This study aimed to investigate whether the plant DNA damage levels and DNA damage response (DDR) are regulated during Agrobacterium infection and potentially manipulated by Agrobacterium to facilitate T-DNA integration. We investigated the plant genomic response to Agrobacterium infection by measuring gamma H2AX levels, which reflect the levels of double-strand DNA breaks (DSBs), and by characterizing transcription of three major DNA repair marker genes NAC82, KU70, and AGO2. These experiments revealed that, globally, Agrobacterium infection did not result in a major increase in DSB content in the host genome. The transcription of the DNA damage repair genes, on the other hand, was elevated upon the wild-type Agrobacterium infection. This transcriptional outcome was largely negated by a mutation in the bacterial virB5 gene which encodes the virulence (Vir) protein B5, a minor component of Agrobacterium pilus necessary for the translocation of Vir effector proteins into the host cell, suggesting that the transcriptional activation of the cellular DNA damage repair machinery requires the transport into the host cell of the Agrobacterium effectors, i.e., the VirD2, VirD5, VirE2, VirE3, and VirF proteins. Most likely, a combination of several of these Vir effectors is required to activate the host DNA repair as their individual loss- or gain-of-function mutants did not significantly affect this process.

Pseudorabies Virus UL4 protein promotes the ASC-dependent inflammasome activation and pyroptosis to exacerbate inflammation.

Pseudorabies virus (PRV) infection causes systemic inflammatory responses and inflammatory damages in infected animals, which are associated with the activation of inflammasome and pyroptosis in infected tissues. Here, we identified a critical function of PRV non-structural protein UL4 that enhanced ASC-dependent inflammasome activation to promote pyroptosis. Whereas, the deficiency of viral UL4 was able to reduce ASC-dependent inflammasome activation and the occurrences of pyroptosis. Mechanistically, the 132-145 aa of UL4 permitted its translocation from the nucleus to the cytoplasm to interact with cytoplasmic ASC to promote the activation of NLRP3 and AIM2 inflammasome. Further research showed that UL4 promoted the phosphorylation levels of SYK and JNK to enhance the ASC phosphorylation, which led to the increase of ASC oligomerization, thus promoting the activation of NLRP3 and AIM2 inflammasome and enhanced GSDMD-mediated pyroptosis. In vivo experiments further showed that PRV UL4 (132DVAADAAAEAAAAE145) mutated strain (PRV-UL4mut) infection did not lead to a significant decrease in viral titers at 12 h. p. i, but it induced lower levels of IL-1β, IL-18, and GSDMD-NT, which led to an alleviated inflammatory infiltration and pathological damage in the lungs and brains, and a lower death rate compared with wild-type PRV strain infection. Taken together, our findings unravel that UL4 is an important viral regulator to manipulate the inflammasome signaling and pyroptosis of host cells to promote the pathogenicity of PRV, which might be further exploited as a new target for live attenuated vaccines or therapeutic strategies against pseudorabies in the future.

Vaccine-associated measles in an immunocompromised host: Hospital infection prevention and control and public health response

Introduction: Vaccine-associated measles is generally not considered to be transmissible, as opposed to wild-type measles, which is one of the most highly contagious diseases. Data on contact and exposure management of vaccine-associated measles is limited, with varied approaches to such cases described in the literature. Methods: We report the case of a 2-year-old immunosuppressed child who developed a febrile exanthem with mild conjunctivitis 18 days after receiving the measles-mumps-rubella-varicella vaccine. Results: Given the patient's recent measles-containing vaccination while on immunosuppressive medications, consistent clinical findings and the lack of epidemiological risk factors for wild-type infection, the decision was made to treat this as a presumptive case of vaccine-associated measles virus prior to return of confirmatory genotyping results. After consultation with public health experts, contact tracing was not considered necessary. No secondary measles cases were identified, despite a large exposure potential due to lack of consistent airborne precautions during hospital admission. Discussion: This case highlights the lack of transmissibility of vaccine-associated measles in immunocompromised hosts, adding to the scant body of literature on this topic, with the potential to inform hospital infection prevention and control as well as public health management in similar situations.

Long-Term Protective Immunity against Ehrlichia chaffeensis Infection Induced by a Genetically Modified Live Vaccine.

Human monocytic ehrlichiosis, an emerging tick-borne disease, is caused by Ehrlichia chaffeensis. Infections with the pathogen are also common in the canine host. Our previous studies demonstrated that functional disruption within the E. chaffeensis phage head-to-tail connector protein gene results in bacterial attenuation, creating a modified live attenuated vaccine (MLAV). The MLAV confers protective immunity against intravenous and tick transmission challenges one month following vaccination. In this study, we evaluated the duration of MLAV protection. Dogs vaccinated with the MLAV were challenged with wild-type E. chaffeensis via intravenous infection at 4-, 8-, and 12-months post-vaccination. Immunized dogs rapidly cleared the wild-type pathogen infection and tested positive for bacteremia less frequently than unvaccinated controls. While immune responses varied among dogs, vaccinees consistently mounted IgG and CD4+ T-cell responses specific to E. chaffeensis throughout the assessment period. Our findings demonstrate that MLAV-mediated immune protection persists for at least one year against wild-type bacterial infection, marking a major advancement in combating this serious tick-borne disease. The data presented here serve as the foundation for further studies, elucidating the molecular mechanisms underlying virulence and vaccine development and aiding in preventing the diseases caused by E. chaffeensis and other tick-borne rickettsial pathogens.

Both middle and large envelope proteins can mediate neutralization of hepatitis B virus infectivity by anti-preS2 antibodies: escape by naturally occurring preS2 deletions.

Hepatitis B virus (HBV) expresses co-terminal large (L), middle (M), and small (S) envelope proteins containing preS1/preS2/S, preS2/S, and S domain alone, respectively. S and preS1 domains mediate sequential virion attachment to heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP), respectively, which can be blocked by anti-S and anti-preS1 antibodies. How anti-preS2 antibodies neutralize HBV infectivity remains enigmatic. The late stage of chronic HBV infection often selects for mutated preS2 translation initiation codon to prevent M protein expression, or in-frame preS2 deletions to shorten both L and M proteins. When introduced to infectious clone of genotype C or D, both M-minus mutations and most 5' preS2 deletions sustained virion production. Such mutant progeny viral particles were infectious in NTCP-reconstituted HepG2 cells. Neutralization experiments were performed on the genotype D clone. Although remaining susceptible to anti-preS1 and anti-S neutralizing antibodies, M-minus mutants were only partially neutralized by two anti-preS2 antibodies tested while preS2 deletion mutants were resistant. By infection experiments using viral particles with lost versus increased M protein expression, or a neutralization escaping preS2 deletion only present on L or M protein, we found that both full-length L and M proteins contributed to virus neutralization by the two anti-preS2 antibodies. Thus, immune escape could be a driving force for the selection of M-minus mutations, and especially preS2 deletions. The fact that both L and M proteins could mediate neutralization by anti-preS2 antibodies may shed light on the underlying molecular mechanism.IMPORTANCEThe large (L), middle (M), and small (S) envelope proteins of hepatitis B virus (HBV) contain preS1/preS2/S, preS2/S, and S domain alone, respectively. The discovery of heparan sulfate proteoglycans and sodium taurocholate cotransporting polypeptide (NTCP) as the low- and high-affinity HBV receptors could explain neutralizing potential of anti-S and anti-preS1 antibodies, respectively, but how anti-preS2 neutralizing antibodies work remains enigmatic. In this study, we found two M-minus mutants in the context of genotype D partially escaped two anti-preS2 neutralizing antibodies in NTCP-reconstituted HepG2 cells, while several naturally occurring preS2 deletion mutants escaped both antibodies. By point mutations to eliminate or enhance M protein expression, and by introducing preS2 deletion selectively to L or M protein, we found binding of anti-preS2 antibodies to both L and M proteins contributed to neutralization of wild-type HBV infectivity. Our finding may shed light on the possible mechanism(s) whereby anti-preS2 antibodies neutralize HBV infectivity.

A phosphodiesterase CpdB in Yersinia pseudotuberculosis degrades CDNs to inhibit innate immune response

Yersinia pseudotuberculosis (Yptb) is a pathogenic gram-negative bacterium that can colonize the intestines of different animals. Its infection leads to the activation of the host’s innate immunity. Both host and bacterial-derived cyclic dinucleotides (CDNs) could activate the innate immune response of host cells. In bacteria, CDNs like c-di-AMP, c-di-GMP, or 3′3'-cGAMP can be hydrolyzed by different hydrolases. Recent studies showed that the degradation of those second messengers helps the pathogen evade immune detection. In this study, we identified a hydrolase, YPK_3776, namely CpdB in Yptb. CpdB is predicted to bind bacterial-derived c-di-AMP, c-di-GMP, 3′3'-cGAMP and host-derived 2′3'-cGAMP. Surprisingly, by using high-performance liquid chromatography (HPLC), we found that CpdB could only degrade bacterial-derived CDNs but not host-derived 2′3'-cGAMP. In addition, CpdB has 2′3'-cNMP activity. Consistently, the Yptb mutant lacking the cpdB gene exhibited a higher level of intracellular c-di-GMP. Furthermore, the ∆cpdB mutant elicited stronger innate immune responses during Yptb infection in macrophages, suggesting CpdB enables Yptb to evade host immune surveillance. Furthermore, CpdB inhibited the Yptb-induced innate immune response in a STING-dependent manner. Finally, we showed the ∆cpdB infection in mice model exhibited in lower bacterial burden, as compared to wild-type strain infection, indicating CpdB is important for bacterial survival in the host. Together, we identified a cyclic dinucleotide hydrolase CpdB in Yptb that could degrade bacterial-derived CDNs which help the pathogen to evade immune detection via the STING pathway.

Glutamate is a key nutrient for Porphyromonas gingivalis growth and survival during intracellular autophagic life under nutritionally limited conditions.

This study reveals that P. gingivalis heavily depends on preferential utilization of Glutamate (Glu) for autophagic vacuolar growth and survival in human GECs. Several novel observations are made to support this: (i) GdhA of P. gingivalis is highly enriched in these vacuoles, (ii) P. gingivalis induces a large conversion of intracellular glutamine to Glu, (iii) size of vacuoles are significantly increased in the presence of Glu-Glu in P. gingivalis wild-type strain infection which is opposite in a Δ gdhA strain, (iv) P. gingivalis uptakes 14 C-Glu and preferentially utilizes Glu-Glu dipeptide, (v) similarly, wild-type strain shows growth increase in a nutritionally reduced bacterial culture media, and (vi) finally, Glu-Glu supplementation increases bacterial cell-volume of P. gingivalis wild-type but not Δ gdhA strain, an indicator of higher metabolism and growth. Taken together, this study highlights the pathophysiological importance of Glu for P. gingivalis growth-rate, biomass induction and survival in nutritionally limited host subcellular environments.

Measles Infection Dose Responses: Insights from Mathematical Modeling

How viral infections develop can change based on the number of viruses initially entering the body. The understanding of the impacts of infection doses remains incomplete, in part due to challenging constraints, and a lack of research. Gaining more insights is crucial regarding the measles virus (MV). The higher the MV infection dose, the earlier the peak of acute viremia, but the magnitude of the peak viremia remains almost constant. Measles is highly contagious, causes immunosuppression such as lymphopenia, and contributes substantially to childhood morbidity and mortality. This work investigated mechanisms underlying the observed wild-type measles infection dose responses in cynomolgus monkeys. We fitted longitudinal data on viremia using maximum likelihood estimation, and used the Akaike Information Criterion (AIC) to evaluate relevant biological hypotheses and their respective model parameterizations. The lowest AIC indicates a linear relationship between the infection dose, the initial viral load, and the initial number of activated MV-specific T cells. Early peak viremia is associated with high initial number of activated MV-specific T cells. Thus, when MV infection dose increases, the initial viremia and associated immune cell stimulation increase, and reduce the time it takes for T cell killing to be sufficient, thereby allowing dose-independent peaks for viremia, MV-specific T cells, and lymphocyte depletion. Together, these results suggest that the development of measles depends on virus-host interactions at the start and the efficiency of viral control by cellular immunity. These relationships are additional motivations for prevention, vaccination, and early treatment for measles.Graphical abstractMeasles infection dose responses: insights from mathematical modeling. Top: Model-data fits for acute viremia in response to changes in measles infection doses. 104\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$10^4$$\\end{document}, 103\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$10^3$$\\end{document}, 102\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$10^2$$\\end{document}, 10 and 1 TCID50\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$_{50}$$\\end{document} correspond to red diamonds, blue stars, orange triangles, magenta dots, and green squares respectively. The solid lines represent the trajectories generated by the proposed model parameterization. The shapes represent data. The dark grey dotted dashed line represents the limit of detection < 0.3. Bottom: Cartoon illustrating that when the measles infection dose increases, the stimulation of the measles-specific cellular immune responses increases early on post-infection. This enhanced immune response reduces the time required to clear the infectious viral load and helps maintain similar levels of viral loads and lymphocyte depletion irrespective of the initial dose.

Engineering of the complementary mutation site in tobacco mosaic virus p126 to develop a stable attenuated mutant for cross-protection

Tobacco mosaic virus (TMV; genus Tobamovirus) is one of the most prevailing pathogens that seriously affects the quality and yield of tobacco (Nicotiana tabacum) leaves. Cross-protection using mild strains is a potential strategy for the biological prevention of plant viral diseases. Complementary mutations in attenuated strains may cause attenuated ones to suddenly evolve into virulent strains, which limits the application of cross-protection in practice. To data there has been no study on engineering the complementary mutation sites to generate stable attenuated mutants for cross-protection. In this study, we found that the substitution of the conserved arginine at position 88 (R88) in p126 protein with alanine (A) abolished the cell-to-cell movement and reduced the replication of TMV. However, a spontaneous complementary mutation of serine at position 114 (S114) to lysine (K) in p126 restored TMV virulence. Substitution of S114 with R in p126 restored the systemic infection but not the virulence of TMV, therefore, the mutant TMV-R88A/S114R was an attenuated one. Furthermore, our results showed that TMV-R88A/S114R was a stable attenuated mutant, and could effectively protect tobacco plants against the wild-type TMV infection. This study reports a promising TMV mild mutant for cross-protection in tobacco plants by modifying the complementary mutation site in p126.

Role of core lipopolysaccharide biosynthetic genes in the infection and adsorption of broad-host-range bacteriophages of Rhizobium etli.

In this study, we examined the role of the lipopolysaccharide (LPS) core of Rhizobium etli in facilitating the adsorption and infection of phages with broad host range. When the plasmid-encoded LPS biosynthesis genes, wreU and wreV, were disrupted, distinct and contrasting effects on phage infection were observed. The wreU mutant strains exhibited wild-type adsorption and infection properties, whereas the wreV mutant demonstrated resistance to phage infection, but retained the capacity to adsorb phages. Complementation of the wreV mutant strains with a recombinant plasmid containing the wreU and wreV, restored the susceptibility to the phages. However, the presence of this recombinant plasmid in a strain devoid of the native lps-encoding plasmid was insufficient to restore phage susceptibility. These results suggest that the absence of wreV impedes the proper assembly of the complete LPS core, potentially affecting the formation of UDP-KdgNAg or KDO precursors for the O-antigen. In addition, a protein not yet identified, but residing in the native lps-encoding plasmid, may be necessary for complete phage infection.

Shedding of measles vaccine RNA in children after receiving measles, mumps and rubella vaccination

BackgroundMeasles, mumps, and rubella(MMR) vaccination is critical to measles outbreak responses. However, vaccine reactions and detection of measles vaccine RNA in recently immunized persons may complicate case classification especially in those presenting with another respiratory viral illness. We aim to characterize cases of measles vaccine shedding in recently vaccinated children presenting with respiratory viral symptoms. MethodsChildren who were tested with a multiplex respiratory panel <30 days after receiving MMR were identified. Remnant nasopharyngeal(NP) samples were tested for measles vaccine by PCR. Medical records were reviewed for demographics, presenting symptoms, and test results. ResultsFrom January 2022 to March 2023, 127 NP from children who received MMR were tested. Ninety-six NP were collected after the first dose, of which 33(34.4 %) were positive for vaccine RNA. The median interval between MMR and detection was 11.0 days. Thirty-one NP were collected after the second MMR and 1(3.2 %) was positive; time between the vaccination and detection was 18.9 days. Median cycle threshold(Ct) value of the measles PCR for vaccine shedding was significantly higher than median Ct in children with wild-type infection. ConclusionShedding of measles vaccine RNA is not uncommon and vaccine RNA can be detected up to 29 days post MMR; the amount of vaccine RNA shedding is low indicated by high Ct values. Clinicians and public health officials should consider performing measles vaccine testing on those testing positive for measles within one month of MMR vaccination, especially if the Ct value is high and definitive epidemiological links are absent.

Alveolar macrophage (AM) Toll-like Receptors (TLRs) signaling drives inflammation and pathogenic neutrophilic infiltrate during neonatal murine Influenza virus infection

Abstract Like human neonates, murine neonates are highly susceptible to influenza viral (IV) infection. AMs are tissue-resident immune cells which are activated through TLRs to initiate immune cell recruitment to the site of infection, critical for viral control. However, bystander damage can also occur in the delicate alveoli. Therefore, we hypothesized that AM TLRs contribute to neonatal murine IV pathogenesis via neutrophil recruitment. To determine the relative contribution of AM TLR signaling, we utilized the Cre-flox system to generate AM-specific TLR deletions (LysMCre-MyD88flox). Three-day-old transgenic mice were infected with influenza type A virus strain PR/8/34 (H1N1) and tracked for survival. LysMCre-MyD88flox neonates demonstrated improved survival (70%) compared to wild type (WT) (33%). Moreover, LysMCre-MyD88flox neonates had decreased inflammatory cytokine concentrations in the broncho-alveolar lavage (BAL) compared to WT, specifically IL-18, IL-6, TNF-α and myeloid recruiting chemokines MCP-1 and CXCL1. Flow analysis of the BAL revealed lower neutrophils in the LysMCre-MyD88flox group, consistent with the cytokine concentrations. To test the pathogenic role of neutrophils during IV infection, infected WT neonates were neutrophil-depleted and had improved survival (60% vs. 24%, isotype group). These findings suggest AM TLRs increase IV pathogenesis in neonatal mice and that TLR-blockade is a potential therapeutic target in this vulnerable population.

A Review on the Methodology and Use of the Pregnant Mouse Model in the Study of Brucella Reproductive Pathogenesis and Its Abortifacient Effect.

Brucellosis is one of the most common and widespread bacterial zoonoses and is caused by Gram-negative bacteria belonging to the genus Brucella. These organisms are able to infect and replicate within the placenta, resulting in abortion, one of the main clinical signs of brucellosis. Although the mouse model is widely used to study Brucella virulence and, more recently, to evaluate the protection of new vaccines, there is no clear consensus on the experimental conditions (e.g., mouse strains, doses, routes of inoculation, infection/pregnancy time) and the natural host reproducibility of the pregnant mouse model for reproductive brucellosis. This lack of consensus calls for a review that integrates the major findings regarding the effect of Brucella wild-type and vaccine strains infections on mouse pregnancy. We found sufficient evidence on the utility of the pregnant mouse model to study Brucella-induced placentitis and abortion and propose suitable experimental conditions (dose, time of infection) and pregnancy outcome readouts for B. abortus and B. melitensis studies. Finally, we discuss the utility and limitations of the pregnant mouse as a predictive model for the abortifacient effect of live Brucella vaccines.

Nanobodies against African swine fever virus p72 and CD2v proteins as reagents for developing two cELISAs to detect viral antibodies

African swine fever virus (ASFV) poses a significant threat to the global swine industry. Currently, there are no effective vaccines or treatments available to combat ASFV infection in pigs. The primary means of controlling the spread of the disease is through rapid detection and subsequent elimination of infected pig. Recently, a lower virulent ASFV isolate with a deleted EP402R gene (CD2v-deleted) has been reported in China, which further complicates the control of ASFV infection in pig farms. Furthermore, an EP402R-deleted ASFV variant has been developed as a potential live attenuated vaccine candidate strain. Therefore, it is crucial to develop detection methods that can distinguish wild-type and EP402R-deleted ASFV infections. In this study, two recombinant ASFV-p72 and -CD2v proteins were expressed using a prokaryotic system and used to immunize Bactrian camels. Subsequently, eight nanobodies against ASFV-p72 and ten nanobodies against ASFV-CD2v were screened. Following the production of these nanobodies with horse radish peroxidase (HRP) fusion proteins, the ASFV-p72-Nb2-HRP and ASFV-CD2v-Nb22-HRP fusions were selected for the development of two competitive ELISAs (cELISAs) to detect anti-ASFV antibodies. The two cELISAs exhibited high sensitivity, good specificity, repeatability, and stability. The coincidence rate between the two cELISAs and commercial ELISA kits was 98.6% and 97.6%, respectively. Collectively, the two cELISA for detecting antibodies against ASFV demonstrated ease of operation, a low cost, and a simple production process. The two cELISAs could determine whether pigs were infected with wild-type or CD2v-deleted ASFV, and could play an important role in monitoring ASFV infections in pig farms.

Gasdermin D promotes influenza virus-induced mortality through neutrophil amplification of inflammation

Influenza virus activates cellular inflammasome pathways, which can be both beneficial and detrimental to infection outcomes. Here, we investigate the function of the inflammasome-activated, pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice (Gsdmd−/−) significantly attenuates influenza virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected Gsdmd−/− mice exhibit decreased inflammatory gene signatures shown by lung transcriptomics. Among these, diminished neutrophil gene activation signatures are corroborated by decreased detection of neutrophil elastase and myeloperoxidase in KO mouse lungs. Indeed, directly infected neutrophils are observed in vivo and infection of neutrophils in vitro induces release of DNA and tissue-damaging enzymes that is largely dependent on GSDMD. Neutrophil depletion in infected WT mice recapitulates the reductions in mortality, lung inflammation, and lung dysfunction observed in Gsdmd−/− animals, while depletion does not have additive protective effects in Gsdmd−/− mice. These findings implicate a function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a therapeutic avenue for treating severe influenza.

Abstract 2798: Oral fungal infection tweaks the innate immune tumor microenvironment promoting

Abstract It is yet unknown how a fungus and tumor interplay impairs innate immunity, modulating tumorigenesis. Here, we found that oral infection with Cladosporium cladosporioides of mice with Ikka deficiency in epithelial stem cells enhanced STAT3-dependent oral squamous cell carcinoma (SCC) development and fungal colonization. In wild-type mice infection was cleared up, suggesting a synergic tumor-fungus loop involved in the accelerated carcinogenesis. C. cladosporioides-mediated EGFR activation stimulated intratumor STAT3-led pathways to generate an acidic oral milieu, which disrupted bacterial compositions and suppressed neutrophil’s ROS but increased Il1b expression, dampening neutrophil’s fungitoxicity and allowing oral fungal and bacterial overgrowth. In addition, fungus-induced IL-1β/IL-17A augmented STAT3 activity in SCC cells, contributing to local and distal carcinogenesis. Significantly, fungal signals were greater in human head and neck SCCs (HNSCCs) and SCC-adjacent tissues than in healthy controls and were co-localized with IL1B-expressing neutrophils in HNSCCs. These similar traits of oncogenic pathways, microbiota dysbiosis, and defective neutrophils in HNSCC patients with poor survival highlight the importance of increased fungal infection for human malignant development and therapy. Citation Format: Xin Li, Na-Young Song, Amit Kumar Singh, Jonathan Badger, Chengfei Jiang, Feng Zhu, Debra Tross, Yongmei Zhao, Bao Tran, Yinling Hu. Oral fungal infection tweaks the innate immune tumor microenvironment promoting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2798.

Difference in mortality risk predicted by leukocyte and lymphocyte levels in COVID-19 patients infected with the Wild-type, Delta, and Omicron strains.

This study aimed to investigate the changing trends, level differences, and prognostic performance of the leukocyte and lymphocyte levels of patients infected with the Wild strains, Delta strains and Omicron strains to provide a reference for prognostic assessment. In the current study, we conducted a retrospective cross-sectional study to evaluate the changing trends, level differences, and prognostic performance of leukocyte and lymphocyte of different strains at admission and discharge may already exist in patients with coronavirus disease-2019 (COVID-19) infected with the Wild type, Delta, and Omicron strains. A retrospective cross-sectional study was conducted. We recruited and screened the 243 cases infected with the Wild-type strains in Wuhan, the 629 cases infected with the Delta and 116 cases infected strains with the Omicron strains in Xi'an. The leukocyte and lymphocyte levels were compared the cohort of Wild-type infection with the cohort of Delta and the Omicron. The changes in the levels of leukocytes and lymphocytes exhibit a completely opposite trend in patients with COVID-19 infected with the different strains. The lymphocyte level at admission and discharge in patients with COVID-19 infected with Omicron strains (area under curve [AUC] receiver operating characteristic curve [ROC] 72.8-90.2%, 82.8-97.2%) presented better performance compared patients with COVID-19 infected with Wild type strains (AUC ROC 60.9-80.7%, 82.3-97.2%) and Delta strains (AUC ROC 56.1-84.7%, 40.3-93.3%). Kaplan-Meier curves showed that the leukocyte levels above newly established cutoff values and the lymphocyte levels below newly established cutoff values had a significantly higher risk of in-hospital mortality in COVID-19 patients with Wild-type and Omicron strains (P < .01). The levels of leukocyte and lymphocyte at admission and discharge in patients with COVID-19 infected with the Wild type, Delta, and Omicron strains may be differences among strains, which indicates different death risks. Our research may help clinicians identify patients with a poor prognosis for severe acute respiratory syndrome coronavirus 2 infection.

Rational attenuation of canine distemper virus (CDV) to develop a morbillivirus animal model that mimics measles in humans.

Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimental MeV infections in non-human primates are restricted by ethical and practical constraints, and animal morbillivirus infections in natural host species have been considered as alternatives. Inoculation of ferrets with wild-type canine distemper virus (CDV) has been used for this purpose, but in most cases, the virus overwhelms the immune system and causes highly lethal disease. Introduction of an additional transcription unit and an additional attenuating point mutation in the polymerase yielded a candidate virus that caused self-limiting disease with transient viremia and virus shedding. This rationally attenuated CDV strain can be used for experimental morbillivirus infections in ferrets that reflect measles in humans.