Lamin B1 curtails early human papillomavirus infection by safeguarding nuclear compartmentalization and autophagic capacity.

Human papillomavirus (HPV) infection is a primary cause of cervical and head-and-neck cancers. The HPV genome enters the nucleus during mitosis when the nuclear envelope disassembles. Given that lamins maintain nuclear integrity during interphase, we asked to what extent their loss would affect early HPV infection. To address this question, we infected human cervical cancer cells and keratinocytes lacking the major lamins with a HPV16 pseudovirus (HP-PsV) encoding an EGFP reporter. We found that a sustained reduction or complete loss of lamin B1 significantly increased HP-PsV infection rate. A corresponding greater nuclear HP-PsV load in LMNB1 knockout cells was directly related to their prolonged mitotic window and extensive nuclear rupture propensity. Despite the increased HP-PsV presence, EGFP transcript levels remained virtually unchanged, indicating an additional defect in protein turnover. Further investigation revealed that LMNB1 knockout led to a substantial decrease in autophagic capacity, possibly linked to the persistent activation of cGAS by cytoplasmic chromatin exposure. Thus, the attrition of lamin B1 increases nuclear perviousness and attenuates autophagic capacity, creating an environment conducive to unrestrained accumulation of HPV capsids. Our identification of lower lamin B1 levels and nuclear BAF foci in the basal epithelial layer of several human cervix samples suggests that this pathway may contribute to an increased individual susceptibility to HPV infection.

Cells infected with human papilloma pseudovirus display nuclear reorganization and heterogenous infection kinetics.

Human papillomaviruses (HPV) are small, non-enveloped DNA viruses, which upon chronic infection can provoke cervical and head-and-neck cancers. Although the infectious life cycle of HPV has been studied and a vaccine is available for the most prevalent cancer-causing HPV types, there are no antiviral agents to treat infected patients. Hence, there is a need for novel therapeutic entry points and a means to identify them. In this work, we have used high-content microscopy to quantitatively investigate the early phase of HPV infection. Human cervical cancer cells and immortalized keratinocytes were exposed to pseudoviruses (PsV) of the widespread HPV type 16, in which the viral genome was replaced by a pseudogenome encoding a fluorescent reporter protein. Using the fluorescent signal as readout, we measured differences in infection between cell lines, which directly correlated with host cell proliferation rate. Parallel multiparametric analysis of nuclear organization revealed that HPV PsV infection alters nuclear organization and inflates promyelocytic leukemia protein body content, positioning these events at the early stage of HPV infection, upstream of viral replication. Time-resolved analysis revealed a marked heterogeneity in infection kinetics even between two daughter cells, which we attribute to differences in viral load. Consistent with the requirement for mitotic nuclear envelope breakdown, pharmacological inhibition of the cell cycle dramatically blunted infection efficiency. Thus, by systematic image-based single cell analysis, we revealed phenotypic alterations that accompany HPV PsV infection in individual cells, and which may be relevant for therapeutic drug screens.

Zebrafish (Danio rerio) larvae as a model for real-time studies of propagating VHS virus infection, tissue tropism and neutrophil activity.

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96 hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.

Ecopathogenic complexes of American trypanosomiasis in endemic areas of Venezuela: Diagnosis and variability of Trypanosoma cruzi.

BACKGROUND & OBJECTIVES: Trypanosoma cruzi, the causative agent of American trypanosomiasis, has been reported in 180 mammalian species and 154 triatomines species of Neotropic. This is a clonal parasite with variable biological behaviour, associated with the genetics of the parasite and its hosts. To know the eco-pathogenic complex of this zoonosis, it was proposed to characterize T. cruzi isolates obtained from triatomines and domestic, peridomestic and wild mammals of the eastern and central-western regions of Venezuela. METHODS: The positivity to T. cruzi was established and the isolates were genetically characterized by PCR amplification of the mini-exon gene, the DNA coding for 24Sa and 18S rRNA, and polymorphic sequences-RFLPs. The sampling sites were georeferenced using the MapSource Software and ArcGis 9.3 programs to generate distribution maps of the isolates. RESULTS: Of the 460 hosts (205 triatomines and 255 mammals), 49% were positive for the parasite. On the other hand, 38 isolates obtained from the triatomines and 23 isolates obtained from mammals were evaluated. The TcI genotype predominated in most of the isolates; however, in those obtained from triatomines the presence of the TcIII genotype in single infections and TcI + TcIII or TcI + TcIV in mixed infections was also evidenced. INTERPRETATION & CONCLUSION: There is a possibility that the triatomines act as biological syringes for these genotypes associated exclusively to them. The heterogeneity in T. cruzi isolates demonstrated the complexity of parasitosis in these regions, presenting its control and prevention as a challenge.

Slow Infection due to Lowering the Amount of Intact versus Empty Particles Is a Characteristic Feature of Coxsackievirus B5 Dictated by the Structural Proteins.

Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from fast-cytolysis-causing echovirus 1 (EV1) with nonstructural proteins from coxsackievirus B5 (CVB5), which shows persistent infection in RD cells. The results showed that the chimera behaved similarly to parental EV1, leading to efficient cytolysis in both permissive A549 and semipermissive RD cells. In contrast to EV1 and the chimera, CVB5 replicated slowly in permissive cells and showed persistent infection in semipermissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 batches constantly contained significant amounts of empty capsids, also in comparison to CVB5's close relative CVB3. During successive passaging of batches containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increase in the amount of empty particles and the lowering of the amount of infective particles are dictated by the CVB5 structural proteins, leading to slowing down of the infection between passages. Furthermore, the key factor for persistent infection is the small amount of infective particles produced, not the high number of empty particles that accumulate.IMPORTANCE Enteroviruses cause several severe diseases, with lytic infections that lead to rapid cell death but also persistent infections that are more silent and lead to chronic states of infection. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera with the structural proteins of echovirus 1 and the nonstructural proteins of coxsackievirus B5. Coxsackievirus B5 infection was found to lead to the production of a high number of empty viruses (empty capsids) that do not contain genetic material and are unable to continue the infection. Coinciding with the high number of empty capsids, the amount of infective virions decreased. This characteristic property was not observed in the constructed chimera virus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infections is essential in understanding virus-caused pathologies.

Aeromonas salmonicida infection kinetics and protective immune response to vaccination in sablefish (Anoplopoma fimbria).

Effective vaccine programs against Aeromonas salmonicida have been identified as a high priority area for the sablefish (Anoplopoma fimbria) aquaculture. In this study, we established an A. salmonicida infection model in sablefish to evaluate the efficacy of commercial vaccines and an autogenous vaccine preparation. Groups of 40 fish were intraperitoneally (ip) injected with different doses of A. salmonicida J410 isolated from infected sablefish to calculate the median lethal dose (LD50). Samples of blood, head kidney, spleen, brain, and liver were also collected at different time points to determine the infection kinetics. The LD50 was estimated as ~3 × 105 CFU/dose. To evaluate the immune protection provided by an autogenous vaccine and two commercial vaccines in a common garden experimental design, 140 fish were PIT-tagged, vaccinated and distributed equally into 4 tanks (35 fish for each group, including a control group). Blood samples were taken every 2 weeks to evaluate IgM titers. At 10 weeks post-immunization, all groups were ip challenged with 100 times the calculated LD50 for A. salmonicida J410. A. salmonicida was detected after 5 days post-infection (dpi) in all collected tissues. At 30 days post-challenge the relative percentage survival (RPS) with respect to the control group was calculated for each vaccine. The RPS for the bacterin mix was 65.22%, for Forte Micro 4® vaccine was 56.52% and for Alpha Ject Micro 4® was 30.43%, and these RPS values were reflected by A. salmonicida tissue colonization levels at 10 days post-challenge. Total IgM titers peaked at 6-8 weeks post-immunization, where the autogenous vaccine group showed the highest IgM titers and these values were consistent with the RPS data. Also, we determined that the A. salmonicida A-layer binds to immunoglobulins F(ab)' in a non-specific fashion, interfering with immune assays and potentially vaccine efficacy. Our results indicate that vaccine design influences sablefish immunity and provide a guide for future sablefish vaccine programs.

Baculovirus transit through insect cell membranes: A mechanistic approach.

Baculovirus systems are used for various purposes, but the kinetics of the infection process is not fully understood yet. We investigated the dynamics of virion movement from a medium toward the interior of insect cells and established a mechanistic model that shows an excellent fit to experimental results. It also makes possible a description of the viral dynamics on the cell surface. A novel measurement method was used to distinguish between infected cells that carry virions on their surfaces, cells that carry virions in their interior, and those carrying virions both inside and on their surface. The maximum number of virions carried by a cell: 55 viruses/cell, and the time required for viral internalization, 0.8 h , are reported. This information is particularly useful for assessing the infection efficacy and the required number of virions needed to infect a given cell population. Although our model specifically concerns the infection process of Sf9 insect cells by baculovirus, it describes general features of viral infection. Some of the model features may eventually be applicable in the studies towards palliation of the COVID-19 outbreak.

Efficiencies and kinetics of infection in different cell types/lines by African and Asian strains of Zika virus.

After recent outbreaks, Zika virus (ZIKV) was linked to severe neurological diseases including Guillain-Barré syndrome in adults and microcephaly in newborns. The severities of pathological manifestations have been associated with different ZIKV strains. To better understand the tropism of ZIKV, we infected 10 human and four nonhuman cell lines (types) with two African (IbH30656 and MR766) and two Asian (PRVABC59 and H/FP/2013) ZIKV strains. Cell susceptibility to ZIKV infection was determined by examining viral titers, synthesis of viral proteins, and replication of positive and negative strands of viral genome. Among nonhuman cell lines, only Vero cells were efficiently infected by ZIKV. Among human cell lines, all were permissive to ZIKV infection. However, 293T and HeLa cells showed differential susceptibility towards African strains. In 293T cells, the NS1 protein was expressed at the high level by African strains but was almost not expressed by Asian strains though there was no obvious difference in viral genome replication, suggesting that the differential susceptibility might be controlled at the stage of viral protein translation. This study provides comprehensive results of the permissiveness of different cell types to both African and Asian ZIKV strains, which might help clarify their different pathogenesis.

Fungal primary and opportunistic pathogens: an ecological perspective.

Fungal primary pathogenicity on vertebrates is here described as a deliberate strategy where the host plays a role in increasing the species' fitness. Opportunism is defined as the coincidental survival of an individual strain in host tissue using properties that are designed for life in an entirely different habitat. In that case, the host's infection control is largely based on innate immunity, and the etiologic agent is not transmitted after infection, and thus fungal evolution is not possible. Primary pathogens encompass two types, depending on their mode of transmission. Environmental pathogens have a double life cycle, and tend to become enzootic, adapted to a preferred host in a particular habitat. In contrast, pathogens that have a host-to-host transmission pattern are prone to shift to a neighboring, immunologically naive host, potentially leading to epidemics. Beyond these prototypical life cycles, some environmental fungi are able to make large leaps between dissimilar hosts/habitats, probably due to the similarity of key factors enabling survival in an entirely different niche, and thus allowing a change from opportunistic to primary pathogenicity. Mostly, such factors seem to be associated with extremotolerance.

A nanoluciferase-encoded bacteriophage illuminates viral infection dynamics of Pseudomonas aeruginosa cells.

Bacteriophages (phages) are increasingly considered for both treatment and early detection of bacterial pathogens given their specificity and rapid infection kinetics. Here, we exploit an engineered phage expressing nanoluciferase to detect signals associated with Pseudomonas aeruginosa lysis spanning single cells to populations. Using several P. aeruginosa strains we found that the latent period, burst size, fraction of infected cells, and efficiency of plating inferred from fluorescent light intensity signals were consistent with inferences from conventional population assays. Notably, imaging-based traits were obtained in minutes to hours in contrast to the use of overnight plaques, which opens the possibility to study infection dynamics in spatial and/or temporal contexts where plaque development is infeasible. These findings support the use of engineered phages to study infection kinetics of virus-cell interactions in complex environments and potentially accelerate the determination of viral host range in therapeutically relevant contexts.

Differential intra-host infection kinetics in Aedes aegypti underlie superior transmissibility of African relative to Asian Zika virus.

IMPORTANCE: The recent Zika virus (ZIKV) epidemic in the Americas highlights its potential public health threat. While the Asian ZIKV lineage has been identified as the main cause of the epidemic, the African lineage, which has been primarily confined to Africa, has shown evidence of higher transmissibility in Aedes mosquitoes. To gain a deeper understanding of this differential transmissibility, our study employed a combination of tissue-level infection kinetics and single-cell-level infection kinetics using in situ immunofluorescent staining. We discovered that the African ZIKV lineage propagates more rapidly and spreads more efficiently within mosquito cells and tissues than its Asian counterpart. This information lays the groundwork for future exploration of the viral and host determinants driving these variations in propagation efficiency.

Inhalation of virus-loaded droplets as a clinically plausible pathway to deep lung infection.

Respiratory viruses, such as SARS-CoV-2, preliminarily infect the nasopharyngeal mucosa. The mechanism of infection spread from the nasopharynx to the deep lung-which may cause a severe infection-is, however, still unclear. We propose a clinically plausible mechanism of infection spread to the deep lung through droplets, present in the nasopharynx, inhaled and transported into the lower respiratory tract. A coupled mathematical model of droplet, virus transport and virus infection kinetics is exercised to demonstrate clinically observed times to deep lung infection. The model predicts, in agreement with clinical observations, that severe infection can develop in the deep lung within 2.5-7 days of initial symptom onset. Results indicate that while fluid dynamics plays an important role in transporting the droplets, infection kinetics and immune responses determine infection growth and resolution. Immune responses, particularly antibodies and T-lymphocytes, are observed to be critically important for preventing infection severity. This reinforces the role of vaccination in preventing severe infection. Managing aerosolization of infected nasopharyngeal mucosa is additionally suggested as a strategy for minimizing infection spread and severity.

Cocktail, a Computer Program for Modelling Bacteriophage Infection Kinetics.

Cocktail is an easy-to-use computer program for mathematical modelling of bacteriophage (phage) infection kinetics in a chemostat. The infection of bacteria by phages results in complicated dynamic processes as both have the ability to multiply and change during the course of an infection. There is a need for a simple way to visualise these processes, not least due to the increased interest in phage therapy. Cocktail is completely self-contained and runs on a Windows 64-bit operating system. By changing the publicly available source code, the program can be developed in the directions that users see fit. Cocktail's models consist of coupled differential equations that describe the infection of a bacterium in a vessel by one or two (interfering) phages. In the models, the bacterial population can be controlled by sixteen parameters, for example, through different growth rates, phage resistance, metabolically inactive cells or biofilm formation. The phages can be controlled by eight parameters each, such as different adsorption rates or latency periods. As the models in Cocktail describe the infection kinetics of phages in vitro, the program is primarily intended to generate hypotheses, but the results can however be indicative in the application of phage therapy.

Kinetics of Placental Infection by Different Smooth Brucella Strains in Mice.

Abortion and reproductive failures induced by Brucella are the main symptoms of animal brucellosis. Laboratory animal models are essential tools of research to study the Brucella pathogenesis before experimentation in natural hosts. To extend the existing knowledge, we studied B. melitensis 16M (virulent) and Rev1 (attenuated) as well as B. suis bv2 infections in pregnant mice. Here, we report new information about kinetics of infection (in spleens, blood, placentas, vaginal shedding, and foetuses), serum cytokine profiles, and histopathological features in placentas and the litter throughout mice pregnancy. Both B. melitensis strains showed a marked placental tropism and reduced viability of pups (mainly in 16M infections), which was preceded by an intense Th1-immune response during placental development. In contrast, B. suis bv2 displayed lower placental tropism, mild proinflammatory immune response, and scarce bacterial transmission to the litter, thus allowing foetal viability. Overall, our studies revealed three different smooth Brucella patterns of placental and foetal pathogenesis in mice, providing a useful animal model for experimental brucellosis.

In Vivo Tracking of Bacterial Colonization in Different Murine Models Using Bioluminescence: The Example of Salmonella.

Applications of bioluminescence for the in vivo study of pathogenic microorganisms are numerous, ranging from the quantification of virulence gene expression to measuring the effect of antimicrobial molecules on the colonization of tissues and organs by the pathogen. Most studies are performed in mice, but recent works demonstrate that this technique is applicable to larger animals like fish, guinea pigs, ferrets, and chickens. Here, we describe the construction and the utilization of a constitutively luminescent strain of Salmonella Typhimurium to monitor in vivo and ex vivo the colonization of mice in the gastroenteritis, typhoid fever, and asymptomatic carriage models of Salmonella infection.

BTV-14 Infection in Sheep Elicits Viraemia with Mild Clinical Symptoms.

In 2011, Bluetongue virus serotype 14 (BTV-14) was detected in Russia during routine surveillance, and was subsequently found in a number of European countries. The strain had high sequence similarity to a BTV-14 vaccine strain. We aimed to determine the risk of this BTV-14 strain causing disease in a UK sheep breed. Four Poll Dorset sheep were infected with a Polish isolate of BTV-14 and infection kinetics were monitored over 28 days. BTV RNA was detected in EDTA blood by 4 days post-infection (dpi) and remained detectable at 28 days post-infection (dpi). Peak viraemia occurred at 6 and 7 dpi with Ct values ranging between 24.6 and 27.3 in all infected animals. BTV antibodies were detected by 10 dpi using a commercial ELISA and neutralising antibodies were detected from 10 dpi. BTV was isolated between 6 and 12 dpi. All infected sheep developed mild clinical signs such as reddening of conjunctiva and mucosal membranes, with one sheep demonstrating more overt clinical signs. Two uninoculated control animals remained clinically healthy and did not have detectable BTV RNA or antibodies. The overall mild clinical symptoms caused by this BTV-14 in this highly susceptible sheep breed were in accordance with the asymptomatic infections observed in the affected countries.

A SYBR green I-based quantitative RT-PCR assay for bovine ephemeral fever virus and its utility for evaluating viral kinetics in cattle.

We developed a SYBR green I-based reverse-transcription quantitative PCR (RT-qPCR) assay for bovine ephemeral fever virus (BEFV). Analytical sensitivity of the assay was ~ 100 times higher than conventional RT-PCR. The precision of the RT-qPCR established for RNA standards was high, with intra-assay and inter-assay coefficients of variation of 0.23-0.89% and 0.23-1.02%, respectively. The test was highly specific for BEFV strains, with no cross-reactivity with other viruses of veterinary significance. The assay detected BEFV RNA as early as 1 d post-infection (dpi) and up to 7-8 dpi in the blood samples of experimentally infected cattle. The most stable reference gene, peptidylprolyl isomerase A (PPIA), was selected for the quantification of BEFV. Viral RNA loads reached peak level at 3-5 dpi and then decreased rapidly through 7-8 dpi. Our assay provides a reliable approach for the detection of BEFV in the early infection stage and for use in the profiling of BEFV kinetics in vivo.

Infection Kinetics and Phylogenetic Analysis of vB_EcoD_SU57, a Virulent T1-Like Drexlerviridae Coliphage.

The morphology, infection kinetics, genome sequence and phylogenetic characterization of the previously isolated bacteriophage vB_EcoD_SU57 are presented. The phage vB_EcoD_SU57 was isolated on Escherichia coli strain ECOR57 from the E. coli reference collection and was shown to produce four mm clear plaques with halos. Infection kinetics, as assessed by one-step growth analyses, suggest that vB_EcoD_SU57 is a virulent phage with an adsorption rate of 8.5 × 10-10 mL × min-1, a latency period of 14 min, and a burst size of 13 PFU per bacterium. Transmission electron microscopy confirmed vB_EcoD_SU57 to be a phage that used to be classified as a Siphoviridae phage. Bioinformatics analyses showed that the genome was 46,150 base pairs long, contained 29 genes with predicted protein functions, and 51 open reading frames encoding proteins with unknown function, many of which were gathered in clusters. A putative tRNA gene was also identified. Phylogenetic analyses showed that vB_EcoD_SU57 is a Braunvirinae phage of the newly formed Drexlerviridae family and closely related to T1-like E. coli phages vB_EcoS_ACG-M12 (Guelphvirus) and Rtp (Rtpvirus) as well as the unclassified phages vB_EcoS_CEB_EC3a and ECH1.

Dynamics of Mammalian Cell Infection by Trypanosoma cruzi trypomastigotes.

In a recent work we demonstrated that Trypanosoma cruzi trypomastigotes change their motility patterns in the presence of mammalian cells, that the extent of the changes depends on the cell line, and that this extent is positively correlated with the efficiency with which parasites invade the different cell lines. These results open the question of what cellular characteristics are relevant for parasite identification and invasion. In the present work, we tackled such question. We performed infection-kinetics experiments on various cell lines, and developed a mathematical model to simulate the experimental outcomes. An analysis of the cell-parasite mechanisms included in the model, together with the parameter values that allowed it to replicate the experimental results, suggests that a process related to the cell replication rate may strongly influence the parasite invasion efficiency, and the infection dynamics in general.

Shotgun proteomics analysis of SARS-CoV-2-infected cells and how it can optimize whole viral particle antigen production for vaccines.

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.