Moderate Virulence Research Articles

Comparative Cross-Kingdom DDA- and DIA-PASEF Proteomic Profiling Reveals Novel Determinants of Fungal Virulence and a Putative Druggable Target.

Accurate and reliable detection of fungal pathogens presents an important hurdle to manage infections, especially considering that fungal pathogens, including the globally important human pathogen, Cryptococcus neoformans, have adapted diverse mechanisms to survive the hostile host environment and moderate virulence determinant production during coinfections. These pathogen adaptations present an opportunity for improvements (e.g., technological and computational) to better understand the interplay between a host and a pathogen during disease to uncover new strategies to overcome infection. In this study, we performed comparative proteomic profiling of an in vitro coinfection model across a range of fungal and bacterial burden loads in macrophages. Comparing data-dependent acquisition and data-independent acquisition enabled with parallel accumulation serial fragmentation technology, we quantified changes in dual-perspective proteome remodeling. We report enhanced and novel detection of pathogen proteins with data-independent acquisition-parallel accumulation serial fragmentation (DIA-PASEF), especially for fungal proteins during single and dual infection of macrophages. Further characterization of a fungal protein detected only with DIA-PASEF uncovered a novel determinant of fungal virulence, including altered capsule and melanin production, thermotolerance, and macrophage infectivity, supporting proteomics advances for the discovery of a novel putative druggable target to suppress C. neoformans pathogenicity.

Procymidone Application Contributes to Multidrug Resistance of Botrytis cinerea.

The necrotrophic pathogen Botrytis cinerea infects a broad range of plant hosts and causes substantial economic losses to many crops. Although resistance to procymidone has been observed in the field, it remains uncertain why procymidone is usually involved in multidrug resistance (MDR) together with other fungicides. Nine mutants derived from the B. cinerea strain B05.10 through procymidone domestication exhibited high resistance factors (RFs) against both procymidone and fludioxonil. However, the fitness of the mutants was reduced compared to their parental strain, showing non-sporulation and moderate virulence. Furthermore, the RFs of these mutants to other fungicides, such as azoxystrobin, fluazinam, difenoconazole, and pyrimethanil, ranged from 10 to 151, indicating the occurrence of MDR. Transcriptive expression analysis using the quantitative polymerase chain reaction (qPCR) revealed that the mutants overexpressed ABC transporter genes, ranging from 2 to 93.7-fold. These mutants carried single-point mutations W647X, R96X, and Q751X within BcBos1 by DNA sequencing. These alterations in BcBos1 conferred resistance to procymidone and other fungicides in the mutants. Molecular docking analysis suggested distinct interactions between procymidone and Bos1 in the B. cinerea standard strain B05.10 or the resistant mutants, suggesting a higher affinity of the former towards binding with the fungicide. This study provides a comprehensive understanding of the biological characteristics of the resistant mutants and conducts an initial investigation into its fungicide resistance traits, providing a reference for understanding the causes of multidrug resistance of B. cinerea in the field.

The serine-threonine protein kinase Snf1 orchestrates the expression of plant cell wall-degrading enzymes and is required for full virulence of the maize pathogen Colletotrichum graminicola

Colletotrichum graminicola, the causal agent of maize leaf anthracnose and stalk rot, differentiates a pressurized infection cell called an appressorium in order to invade the epidermal cell, and subsequently forms biotrophic and necrotrophic hyphae to colonize the host tissue. While the role of force in appressorial penetration is established (Bechinger et al., 1999), the involvement of cell wall-degrading enzymes (CWDEs) in this process and in tissue colonization is poorly understood, due to the enormous number and functional redundancy of these enzymes. The serine/threonine protein kinase gene SNF1 identified in Sucrose Non-Fermenting yeast mutants mediates de-repression of catabolite-repressed genes, including many genes encoding CWDEs. In this study, we identified and functionally characterized the SNF1 homolog of C. graminicola. Δsnf1 mutants showed reduced vegetative growth and asexual sporulation rates on media containing polymeric carbon sources. Microscopy revealed reduced efficacies in appressorial penetration of cuticle and epidermal cell wall, and formation of unusual medusa-like biotrophic hyphae by Δsnf1 mutants. Severe and moderate virulence reductions were observed on intact and wounded leaves, respectively. Employing RNA-sequencing we show for the first time that more than 2,500 genes are directly or indirectly controlled by Snf1 in necrotrophic hyphae of a plant pathogenic fungus, many of which encode xylan- and cellulose-degrading enzymes. The data presented show that Snf1 is a global regulator of gene expression and is required for full virulence.

Differences in the pathogenicity and molecular characteristics of fowl adenovirus serotype 4 epidemic strains in Guangxi Province, southern China.

Starting in 2015, the widespread prevalence of hydropericardium-hepatitis syndrome (HHS) has led to considerable financial losses within China's poultry farming industry. In this study, pathogenicity assessments, whole-genome sequencing, and analyses were conducted on 10 new isolates of the novel genotype FAdV-4 during a HHS outbreak in Guangxi Province, China, from 2019 to 2020. The results indicated that strains GX2019-010 to GX2019-013 and GX2019-015 to GX2019-018 were highly virulent, while strain GX2020-019 exhibited moderate virulence. Strain GX2019-014 was characterized as a wild-type strain with low virulence, displaying no pathogenic effects when 0.5 mL containing 106 TCID50 virus was inoculated into the muscle of specific pathogen-free (SPF) chickens at 4 weeks of age, while 107 TCID50 and 108 TCID50 resulted in mortality rates of 80 and 100%, respectively. The whole genomes of strains GX2019-010 to GX2019-013, GX2019-015 to GX2019-018, and GX2020-019 showed high homology with other Chinese newly emerging highly pathogenic FAdV-4 strains, whereas GX2019-014 was closer to nonmutant strains and shared the same residues with known nonpathogenic strains (B1-7, KR5, and ON1) at positions 219AA and 380AA of the Fiber-2 protein. Our work enriches the research on prevalent strains of FAdV-4 in China, expands the knowledge on the virulence diversity of the novel genotype FAdV-4, and provides valuable reference material for further investigations into the key virulence-associated genetic loci of FAdV-4.

Transcriptional Host Responses to Infection with Streptococcus suis in a Porcine Precision-Cut Lung Slice Model: Between-Strain Differences Suggest Association with Virulence Potential.

Streptococcus suis is a porcine and zoonotic pathogen in the upper respiratory tract, expressing different capsular serotypes and virulence-associated factors. Given its genomic and phenotypic diversity, the virulence potential of S. suis cannot be attributed to a single factor. Since strong inflammatory response is a hallmark of S. suis infection, the objective of this study was to investigate the differences in transcriptional host responses to two serotype 2 and one serotype 9 strains. Both serotypes are frequently found in clinical isolates. We infected porcine precision-cut lung slices (PCLSs) with two serotype 2 strains of high (strain S10) and low (strain T15) virulence, and a serotype 9 strain 8067 of moderate virulence. We observed higher expression of inflammation-related genes during early infection with strains T15 and 8067, in contrast to infection with strain 10, whose expression peaked late. In addition, bacterial gene expression from infected PCLSs revealed differences, mainly of metabolism-related and certain virulence-associated bacterial genes amongst these strains. We conclude that the strain- and time-dependent induction of genes involved in innate immune response might reflect clinical outcomes of infection in vivo, implying rapid control of infection with less virulent strains compared to the highly virulent strain S10.

Pathogenicity assessment of a bovine viral diarrhea virus type 1l (BVDV-1l) strain in experimentally infected calves

Bovine viral diarrhea is a widespread and economically important viral disease for livestock which can cause clinically diverse manifestations. The number of established BVDV subgenotypes has increased, not only the serological relationships of recently described subgenotypes but virulence and pathogenic characteristics have not yet been mostly elaborated. The dominant BVDV subgenotype in Turkiye was elaborated to be BVDV-1l, that involves more than half of field strains and there is no scientific data to identify the pathogenicity of this strain so far. This study investigated the pathogenicity of a selected field strain (TR-72) from subgenotype BVDV-1l. Experimental infection was implemented by intranasal inoculation of the strain TR-72 (10 ×105.5) to four young calves which were previously not vaccinated and were free both for BVDV antibodies and antigens. Clinical changes as well as blood parameters, body temperature, and viremia were monitored for 14 days. Only mild clinical signs associated with respiratory signs of BVDV infection were observed. Detected clinical signs included nasal discharge, conjunctivitis, cough, fatigue, high rectal temperature reaching 40.7 ℃, and white blood cell counts depression started from the 2nd day and 40.4% decreased between the 12th and 14th days post-infection (poi). The presence of viremia was investigated by virus isolation, RT-PCR, and real-time RT-PCR from blood samples. The efficiency of experimental infection was established not only by observed clinical signs but also by virus isolation from blood leukocytes between the 5th and 8th days poi., virus detection was obtained by real-time PCR between the 3rd − 13th days poi. Besides, the recorded mild clinical signs, high fever, long duration of viremia , and high decrease in blood parameters obtained in this study, it was shown that the noncytopathogenic BVDV-1l strain TR-72 has a moderate virulence in naïve cattle.

Isolation, Identification, and Pathogenicity of Vibrio gigantis Retrieved from European Seabass (Dicentrarchus labrax) Farmed in Türkiye.

In this study, V. gigantis strain C24 was isolated from cases of winter mortalities of hatchery-reared European seabass (Dicentrarchus labrax) broodstock in Türkiye. The first mortalities were reported in September 2016 and occurred annually in early autumn/late winter until the end of February 2019, when 15% of accumulated mortality was recorded. Diseased moribund fish exhibited general septicemic signs, including dermal ulcerations with hemorrhagic margins, distended abdomens, and hemorrhages below the pectorals, pelvic fins, and at the operculum. Postmortem findings showed congestion in several internal organs, hemorrhagic ascitic fluid, and congested prolapsed anal openings. The representative bacterial isolate V. gigantis strain C24 was characterized as Gram-negative, motile, nitrite-producing, and as vibrio static agent O/129-sensitive. The full-length 16S rRNA sequence (Accession No. ON778781) and gyrB gene sequence (Accession No. ON792326) of the C24 strain showed high similarity to V. gigantis strains. Moreover, the whole-genome average nucleotide identity (ANI) values (ANI > 97.7%) against four V. gigantis strains above the species demarcation limit unambiguously identified the C24 isolate as a member of this species. A preliminary virulence-gene analysis showed that the V. gigantis isolate C24 encoded at least three exotoxins, including two aerolysins and a thermolabile hemolysin. The experimental infection showed that the C24 isolate exhibited low to moderate virulence in experimentally infected European seabass juveniles. Interestingly, antimicrobial susceptibility testing revealed that the C24 isolate was susceptible to nalidixic acid, ciprofloxacin, and several other antibiotics but resistant to tilmicosin, kanamycin, streptomycin, and ampicillin. To our knowledge, this study is the first to report that V. gigantis could be considered an emerging bacterial pathogen in Türkiye, and it may threaten the international European seabass production.

Complete viral protein coding genes NS1 and VP1 sequences and phylogenetic analysis of a novel recombinant Mink Enteritis Virus isolated in China

The primary objective of this study was to detect the presence of MEV in mink samples and investigate the genetic characteristics of the virus. In 2017, a newly identified strain of MEV, named MEV-HLJ, was isolated. The virus’s complete NS1 and VP1 gene sequences were amplified using PCR and subjected to phylogenetic analysis. The results indicated that despite moderate virulence, MEV- HLJ belonged to the attenuated group. Recombination analysis using the GARD programme identified a putative recombination breakpoint at nucleotide position 2,075 between the NS1 and VP1 genes. Separate phylogenetic trees were constructed to confirm the recombinant event. This study provides insights into the genetic diversity and evolution of MEV isolates.

A multifaceted interplay between virulence, drug resistance, and the phylogeographic landscape of Mycobacterium tuberculosis.

Latin-American Mediterranean (LAM) family is one of the most significant and global genotypes of Mycobacterium tuberculosis. Here, we used the murine model to study the virulence and lethality of the genetically and epidemiologically distinct LAM strains. The pathobiological characteristics of the four LAM strains (three drug resistant and one drug susceptible) and the susceptible reference strain H37Rv were studied in the C57BL/6 mouse model. The whole-genome sequencing was performed using the HiSeq Illumina platform, followed by bioinformatics and phylogenetic analysis. The susceptible strain H37Rv showed the highest virulence. Drug-susceptible LAM strain (spoligotype SIT264) was more virulent than three multidrug-resistant (MDR) strains (SIT252, SIT254, and SIT266). All three MDR isolates were low lethal, while the susceptible isolate and H37Rv were moderately/highly lethal. Putting the genomic, phenotypic, and virulence features of the LAM strains/spoligotypes in the context of their dynamic phylogeography over 20 years reveals three types of relationships between virulence, resistance, and transmission. First, the most virulent and more lethal drug-susceptible SIT264 increased its circulation in parts of Russia. Second, moderately virulent and pre-XDR SIT266 was prevalent in Belarus and continues to be visible in North-West Russia. Third, the low virulent and MDR strain SIT252 previously considered as emerging has disappeared from the population. These findings suggest that strain virulence impacts the transmission, irrespective of drug resistance properties. The increasing circulation of susceptible but more virulent and lethal strains implies that personalized TB treatment should consider not only resistance but also the virulence of the infecting M. tuberculosis strains. IMPORTANCE The study is multidisciplinary and investigates the epidemically/clinically important and global lineage of Mycobacterium tuberculosis, named Latin-American-Mediterranean (LAM), yet insufficiently studied with regard to its pathobiology. We studied different LAM strains (epidemic vs endemic and resistant vs susceptible) in the murine model and using whole-genome analysis. We also collected long-term, 20-year data on their prevalence in Eurasia. The findings are both expected and unexpected. (i) We observe that a drug-susceptible but highly virulent strain increased its prevalence. (ii) By contrast, the multidrug-resistant (MDR) but low-virulent, low-lethal strain (that we considered as emerging 15 years ago) has almost disappeared. (iii) Finally, an intermediate case is the MDR strain with moderate virulence that continues to circulate. We conclude that (i) the former and latter strains are the most hazardous and require close epidemiological monitoring, and (ii) personalized TB treatment should consider not only drug resistance but also the virulence of the infecting strains and development of anti-virulence drugs is warranted.

The major role of Listeria monocytogenes folic acid metabolism during infection is the generation of N-formylmethionine.

Folic acid is an essential vitamin for bacteria, plants, and animals. The lack of folic acid leads to various consequences such as a shortage of amino acids and nucleotides that are fundamental building blocks for life. Though antifolate drugs are widely used for antimicrobial treatments, the underlying mechanism of bacterial folate deficiency during infection is unclear. This study compares the requirements of different folic acid end-products during the infection of Listeria monocytogenes, a facultative intracellular pathogen of animals and humans. The results reveal the critical importance of N-formylmethionine, the amino acid used by bacteria to initiate protein synthesis. This work extends the current understanding of folic acid metabolism in pathogens and potentially provides new insights into antifolate drug development in the future.

Assessing spatial distribution, genetic variants, and virulence of pathogen Mycoplasma agassizii in threatened Mojave desert tortoises.

Mojave desert tortoises (Gopherus agassizii), a threatened species under the US Endangered Species Act, are long-lived reptiles that experience a chronic respiratory disease. The virulence of primary etiologic agent, Mycoplasma agassizii, remains poorly understood, but it exhibits temporal and geographic variability in causing disease outbreaks in host tortoises. Multiple attempts to culture and characterize the diversity of M. agassizii have had minimal success, even though this opportunistic pathogen chronically persists in nearly every population of Mojave desert tortoises. The current geographic range and the molecular mechanisms of virulence of the type-strain, PS6T, are unknown, and the bacterium is thought to have low-to-moderate virulence. We designed a quantitative polymerase chain reaction (qPCR) targeting three putative virulence genes annotated on the PS6T genome as exo-α-sialidases, enzymes which facilitate growth in many bacterial pathogens. We tested 140 M. agassizii-positive DNA samples collected from 2010 to 2012 across the range of Mojave desert tortoises. We found evidence of multiple-strain infections within hosts. We also found the prevalence of these sialidase-encoding genes to be highest in tortoise populations surrounding southern Nevada, the area from which PS6T was originally isolated. We found a general pattern of loss or reduced presence of sialidase among strains, even within a single host. However, in samples that were positive for any of the putative sialidase genes, one particular gene (528), was positively associated with bacterial loads of M. agassizii and may act as a growth factor for the bacterium. Our results suggest three evolutionary patterns: (1) high levels of variation, possibly due to neutral changes and chronic persistence, (2) a trade-off between moderate virulence and transmission, and (3) selection against virulence in environmental conditions known to be physiologically stressful to the host. Our approach of quantifying genetic variation via qPCR represents a useful model of studying host-pathogen dynamics.

Pathogenicity and molecular characteristics of fowl adenovirus serotype 4 with moderate virulence in Guangxi Province, China.

The GX2020-019 strain of fowl adenovirus serotype 4 (FAdV-4) was isolated from the liver of chickens with hydropericardium hepatitis syndrome in Guangxi Province, China, and was purified by plaque assay three times. Pathogenicity studies showed that GX2020-019 can cause typical FAdV-4 pathology, such as hydropericardium syndrome and liver yellowing and swelling. Four-week-old specific pathogen-free (SPF) chickens inoculated with the virus at doses of 103 median tissue culture infectious dose (TCID50), 104 TCID50, 105 TCID50, 106 TCID50, and 107 TCID50 had mortality rates of 0, 20, 60, 100, and 100%, respectively, which were lower than those of chickens inoculated with other highly pathogenic Chinese isolates, indicating that GX2020-019 is a moderately virulent strain. Persistent shedding occurred through the oral and cloacal routes for up to 35 days postinfection. The viral infection caused severe pathological damage to the liver, kidney, lung, bursa of Fabricius, thymus, and spleen. The damage to the liver and immune organs could not be fully restored 21 days after infection, which continued to affect the immune function of chickens. Whole genome analysis indicated that the strain belonged to the FAdV-C group, serotype 4, and had 99.7-100% homology with recent FAdV-4 strains isolated from China. However, the amino acid sequences encoded by ORF30 and ORF49 are identical to the sequences found in nonpathogenic strains, and none of the 32 amino acid mutation sites that appeared in other Chinese isolates were found. Our research expands understanding of the pathogenicity of FAdV-4 and provides a reference for further studies.

Molecular identification of endophytic fungi in lawn grass (Axonopus compressus) and their pathogenic ability

Lawn grass (Axonopus compressus) is a widely distributed grass species from the family Poaceae that is ubiquitous in Malaysia. We isolated endophytic fungi from the leaves of A. compressus and molecularly identified them as Fusarium parceramosum, Colletotrichum siamense, C. gigasporum, C. endophyticum, Curvularia lunata, Stagonospora bicolor, Calonectria gracilis, and Albifimbria verrucari. These fungal endophytes are considered host generalists, as they have been isolated from other plants and have also been reported to be latent plant pathogens. We tested the pathogenicity of selected endophytic fungal isolates on A. compressus leaves, chili (Capsicum annum), and tomato (Solanum lycopersicum), and found that they were pathogenic to wounded A. compressus leaves with low to moderate virulence, and several were pathogenic to wounded and unwounded chili and tomato fruits. This indicated that the endophytes could infect both vegetable fruits with low to very high virulence. Pathogenicity tests demonstrated that endophytic fungi from the leaves of A. compressus can become pathogenic and infect the host and other plant species. The findings also indicated that leaves of A. compressus may harbor pathogens with latent ability that can become active due to changes in environmental conditions, thereby disrupting the balance between host-endophyte antagonism.

Trypanosoma cruzi Isolates Naturally Adapted to Congenital Transmission Display a Unique Strategy of Transplacental Passage.

Chagas disease is mainly transmitted by vertical transmission (VT) in nonendemic areas and in endemic areas where vector control programs have been successful. For the present study, we isolated natural Trypanosoma cruzi strains vertically transmitted through three generations and proceeded to study their molecular mechanism of VT using mice. No parasitemia was detected in immunocompetent mice, but the parasites were able to induce an immune response and colonize different organs. VT experiments revealed that infection with different strains did not affect mating, pregnancy, or resorption, but despite low parasitemia, VT strains reached the placenta and resulted in higher vertical transmission rates than strains of either moderate or high virulence. While the virulent strain modulated more than 2,500 placental genes, VT strains modulated 150, and only 29 genes are shared between them. VT strains downregulated genes associated with cell division and replication and upregulated immunomodulatory genes, leading to anti-inflammatory responses and tolerance. The virulent strain stimulated a strong proinflammatory immune response, and this molecular footprint correlated with histopathological analyses. We describe a unique placental response regarding the passage of T. cruzi VT isolates across the maternal-fetal interphase, challenging the current knowledge derived mainly from studies of laboratory-adapted or highly virulent strains. IMPORTANCE The main findings of this study are that we determined that there are Trypanosoma cruzi strains adapted to transplacental transmission and completely different from the commonly used laboratory reference strains. This implies a specific strategy for the vertical transmission of Chagas disease. It is impressive that the strains specialized for vertical transmission modify the gene expression of the placenta in a totally different way than the reference strains. In addition, we describe isolates of T. cruzi that cannot be transmitted transplacentally. Taken together, these results open up new insights into the molecular mechanisms of this insect vector-independent transmission form.

Virulence Variability in Xylella fastidiosa and Disease Susceptibility of Cultivated and Wild Grape Species

Five isolates of Xylella fastidiosa subsp. fastidiosa were evaluated for virulence differences using the grape cultivar Cabernet Sauvignon. Significant variation in virulence was observed among isolates. Two isolates representing high and moderate virulence in grape were used for evaluating susceptibility on 21 grape lines (cultivars and breeding lines). Lines showed significant differences in symptom development and bacterial populations when inoculated with one of two isolates. Table grape cultivars Scarlet Royal, Solbrio, and Red Globe were among the cultivars with the highest disease symptoms and bacterial populations, whereas Flame Seedless showed moderate or low symptoms and had lower bacterial populations in this study than most other commercial table grape cultivars. Sources of Pierce's disease resistance or tolerance commonly used in breeding programs showed few symptoms when inoculated with either isolate, but levels of bacteria within the plant varied. A new source of Pierce's disease resistance/tolerance, IAC 572, was identified.

Cytokine profile in <i>in vitro</i> mouse macrophage culture infected with <i>Bacillus anthracis</i> spores with varying plasmid composition

Bacillus anthracis, the causative agent of anthrax, is able to exist both in environmental conditions (soil) and in the macroorganism. The manifestation of pathogenic properties of B. anthracis strains is determined by relevant plasmid composition, because the main toxin and the capsule-related virulence factors are located in bacterial plasmid. Modeling anthrax infection in vitro in macrophage culture might reveal an influence of individual B. anthracis strain characteristics on infection and development of infectious process. The aim of this study was to analyze cytokine level during infection of in vitro macrophage cell cultures with spores of anthrax microbe strains bearing varying plasmid composition. The dependence of the macrophage cell cytokine profile on the plasmid composition of B. anthracis strains was revealed while modeling anthrax infection in vitro. The presence of the toxin-producing plasmid pXO1 in anthrax microbe strains has a powerful stimulating effect on the production of macrophages J774A cell line cytokines. B. anthracis strains lacking the pXO1 plasmid virtually stimulated no production of IL-1, caused very low secretion of IL-1, IL-6, MCP-1, MIP-1, MIP-1, IL-12 (p70) and active G-CSF products. The low cytokine response of macrophage cells infected with monoplasmid strains bearing only the capsule-forming plasmid was due not only to the absence of a binary toxin, but also to disturbed regulation of capsule production associated with the absence of the atxA gene. The capsule, along with lethal and edematous toxins, belongs to the main virulence factors of B. anthracis, but strains lacking the pXO1 virulence plasmid, had its production impaired, because the main regulator of capsule synthesis is the atxA gene localized on the pXO1 plasmid being positively regulated by the acpA and acpB genes, so that strains lacking the toxin-forming plasmid, even in the presence of the encapsulation plasmid, elicit a weak cytokine response in infected cells. Diplasmid strains of B. anthracis, due to produced main virulence factors a two-component toxin and a capsule, enforce macrophages (in the experiment) to actively produce IL-1, IL-6, MCP-1, G-CSF, MIP-1; MIP-1, IL-12 (p70). Strains with moderate virulence and capable of capsulation in air virtually did not differ from highly virulent strains in terms of their effect on in vitro macrophage culture.

Deciphering the virulence potential of Listeria monocytogenes in the Norwegian meat and salmon processing industry by combining whole genome sequencing and in vitro data

Whole genome sequencing (WGS) of foodborne pathogens such as Listeria monocytogenes is globally on the rise in the food industry. It provides an improvement for proactive surveillance and source-tracking and allows in-depth genetic characterization of the pathogen. In the present study, the virulence gene profile including 99 virulence genes of 767 L. monocytogenes isolates from the Norwegian meat and salmon processing industry was characterized. The isolate collection comprised 28 clonal complexes (CCs) that occur globally. We additionally determined the in vitro virulence potential for 13 major CCs in human intestinal epithelial Caco2 cells using cocktails of three to six representative isolates. Our aim was to test whether the virulence potential could be predicted from the virulence gene profiles to estimate the application potential of WGS in risk assessment in the food industry.The virulence gene profiles were highly conserved within the individual CCs and similar among phylogenetically closely related CCs. We observed a CC-associated distribution of accessory virulence genes in addition to different length polymorphisms. Furthermore, we detected different premature stop codons (PMSC) in the inlA gene, which were mainly present in CC9, CC121 and CC5 isolates. Accordingly, CC9 and CC5 were unable to invade Caco2 cells, whereas CC121 showed moderate virulence potential due to the presence of an isolate harboring full-length inlA. The highest invasion was observed for CC403 and CC415, potentially due to the presence of accessory virulence genes. We demonstrated that CC14, which harbored full-length inlA, was unable to invade Caco2 cells due to a low inlA gene expression. Reconstruction of inlA in CC9 and CC121 isolates showed that without the presence of InlA on the cell wall (as detected in the CC9 isolates), invasion into host cells failed.Our study showed that predicting the virulence potential based on genetic virulence profiles provides valuable information for risk assessment in the food industry but also has its limitations. The mere presence of a full-length inlA gene is not sufficient for virulence, but gene expression and the presence of the protein on the cell wall is required for the successful invasion of L. monocytogenes into host cells. Moreover, hypovirulent CCs like CC121 were among the most abundant human clinical isolates in Norway despite harboring a PMSC mutation in the inlA gene. In conclusion, our study highlights that combining genotypic and phenotypic data is of great importance to improve the informative value of applying WGS in the food industry.

Proteome Analysis of Swine Macrophages after Infection with Two Genotype II African Swine Fever Isolates of Different Pathogenicity

Since the introduction of a highly pathogenic genotype II isolate of the African swine fever virus (ASFV) into Georgia in 2007, African swine fever (ASF) has gone panzootic. Outbreaks have been reported in Europe, Asia and, more recently, Latin America. Thus, ASFV has become a major threat to the pig industry worldwide, as broadly applicable vaccines are not available. While the majority of ASFV strains show high virulence in domestic pigs and wild boar, variations within the ASFV genome have resulted in the emergence of attenuated strains with low or moderate virulence. However, the molecular basis of the differences in virulence has not yet been discovered. To reveal virulence-associated protein expression patterns, we analysed the proteomes of the natural target cells of ASFV, primary porcine macrophages, after infection with two genotype II ASFV strains displaying high (Armenia 2008) and moderate (Estonia 2014) virulence using quantitative mass spectrometry. Very similar expression patterns were observed for the viral genes, and any differences were limited to the deletions within the Estonia 2014 genome. In addition to the canonical ASFV proteins, twelve novel protein products from recently described transcripts were confirmed in both isolates. Pathway analyses showed that both isolates evoked a similar host proteome response, despite their difference in virulence. However, subtle differences in the manipulation of the proteins involved in the proinflammatory response mediated by the MAPK14/p38 signalling cascade were observed.

Genotypic and phenotypic quantitative microbial risk assessment model of human salmonellosis related to the consumption of chicken meat in the central region of Mexico

Chicken meat is often associated withSalmonella entericacontamination worldwide. This study proposes a risk assessment model for human salmonellosis linked to the domestic consumption of chicken meat in the central region of Mexico, incorporating genotypic and phenotypic data. SixS. entericagroups were used, considering the presence of specific virulence genes and multidrug resistance (MDR). Sixteen exposure scenarios were established considering retail point (RP1 = fresh market/butcher shop; RP2 = mini-super/supermarket), transportation, home storage, cooking, and cross-contamination. The model predicted a mean annual salmonellosis cases of 66,754 due to chicken consumption (CI95% 10775–231606). The mean probability of illness (Pill) among the exposure scenarios ranged from 2.5 × 10-9 to 3.7 × 10-6, 7.7 × 10-8 to 1.1 × 10-4, and 6.7 × 10-4 to 7.8 × 10-2 for low, moderate, and high virulence groups. Exposure scenarios with the highest Pill were not responsible for most cases due to their low frequency of occurrence. The high virulence/ MDR group was responsible for most cases (66.5 %), despite the low S. enterica prevalence (RP1 0.5 % and RP2 5.0 %). The years lost due to disability (YLD) value for MDR was 2.6 × higher than for non-MDR. Spearman rank showed that the inputs with higher influence on the variability of salmonellosis depended on the type of exposure scenario. For example, the cooking temperature and time had the most significant influence in the scenarios where S. enterica can survive after cooking.Including the microbial genotypic and phenotypic characteristics in risk assessment modeling highlights the importance of focusing on high-virulent and MDR strains, which are not the most frequent but represent the highest public health risk.

Gene expression in primary hemocyte culture of the Pacific white shrimp Penaeus vannamei infected with different white spot syndrome virus (WSSV) strains

Five previously analyzed white spot syndrome virus (WSSV) strains from northwest Mexico, differing in their genome architecture as well as in virulence, were selected (high virulence JP and LG strains; moderate virulence GVE and DIV strains; and low virulence LC10 strain) to evaluate pathogenesis response in vitro. Expression of phagocytosis-activating protein PAP, manganese superoxide dismutase MnSOD and peroxiredoxin PRX, and two genes of immediate-early expression (IE1 and WSSV304) were measured by qPCR in a primary hemocyte cell culture from Penaeus vannamei at 1, 3, 6, 12, and 24 h post-infection (hpi). PAP expression was significantly higher at 1 and 3 hpi, and JP and LC10 strains induced the highest expression. The response of MnSOD was high at 1 hpi, and a significant increase in PRX expression was detected at 3 hpi, probably due to the occurrence of an oxidative burst; expression levels of MnSOD and PRX were significantly higher at 1 and 3 hpi, respectively, induced by the LG strain (high virulence), suggesting an acute response. In general, expression of most immune-related - genes decreased after the initial hours of infection. Expression levels of IE1 and WSSV304 were exceptionally high at 1 hpi in almost all five WSSV analyzed strains, confirming their efficient mechanism for replication and viral fitness. The results of this study do not show an accurate link between the genome size and WSSV virulence of the strains, albeit the strain with the smallest genome showed the highest virulence. All strains induced an early immune response in heterogeneous ways.