Editor’s Choice Articles

Fusarium species are common pathogens on maize and reduce the product quality through contamination with mycotoxins thus jeopardizing safety of both animal feed and human food products. Monitoring of Fusarium infected maize ears and stalks was conducted in Germany to determine the range of Fusarium species present in the field and to assess the impact of tillage, crop rotation, and weather conditions on the frequency of Fusarium species. From 2016 till 2018, a total of 387 infected ears and 190 stalk segments from 58 locations in Germany were collected. For each sample location, site-specific agronomic data on tillage and previous crops as well as meteorological data on precipitation, air temperature, and relative humidity during the vegetation period were recorded. The most frequent Fusarium species detected in maize ears were Fusarium graminearum, F. verticillioides and F. temperatum, whereas, F. graminearum, F. equiseti, F. culmorum, and F. temperatum were the species prevailing on maize stalks. Differences in the local species composition were found to be primarily associated with weather variations between the years and the microclimate at the different locations. The results indicate that mean temperature and precipitation in July, during flowering, has the strongest impact on the local range of Fusarium spp. on ears, whereas the incidence of Fusarium species on stalks is mostly affected by weather conditions during September. Ploughing significantly reduced the infection with F. graminearum and F. temperatum, while crop rotation exerted only minor effects. Full article

Coronavirus infections are a continuous threat raised time and again. With the recent emergence of novel virulent strains, these viruses can have a large impact on human and animal health. Porcine epidemic diarrhea (PED) is considered to be a reemerging pig disease caused by the enteropathogenic alphacoronavirus PED virus (PEDV). In the absence of effective vaccines, infection prevention and control through diagnostic testing and quarantine are critical. Early detection and differential diagnosis of PEDV infections increase the chance of successful control of the disease. Therefore, there is a continuous need for development of reduced assay-step protocols, no-wash, high-throughput immunoassays. This study described the characterization of the humoral immune response against PEDV under experimental and field conditions using a rapid, sensitive, luminescent proximity homogenous assay (AlphaLISA). PEDV IgG and IgA antibodies were developed toward the beginning of the second week of infection. PEDV IgG antibodies were detected for at least 16 weeks post-exposure. Remarkably, the serum IgA levels remained high and relatively stable throughout the study, lasting longer than the serum IgG response. Overall, AlphaLISA allows the detection and characterization of pathogen-specific antibodies with new speed, sensitivity, and simplicity of use. Particularly, the bridge assay constitutes a rapid diagnostic that substantially improves upon the “time to result” metric of currently available immunoassays. Full article

Toxoplasma gondii is a ubiquitous parasitic protist found in a wide variety of hosts, including a large proportion of the human population. Beyond an acute phase which is generally self-limited in immunocompetent individuals, the ability of the parasite to persist as a dormant stage, called bradyzoite, is an important aspect of toxoplasmosis. Not only is this stage not eliminated by current treatments, but it can also reactivate in immunocompromised hosts, leading to a potentially fatal outcome. Yet, despite its critical role in the pathology, the bradyzoite stage is relatively understudied. One main explanation is that it is a considerably challenging model, which essentially has to be derived from in vivo sources. However, recent progress on genetic manipulation and in vitro differentiation models now offers interesting perspectives for tackling key biological questions related to this particularly important developmental stage. Full article

In December 2019, a cluster of fatal pneumonia cases presented in Wuhan, China. They were caused by a previously unknown coronavirus. All patients had been associated with the Wuhan Wholefood market, where seafood and live animals are sold. The virus spread rapidly and public health authorities in China initiated a containment effort. However, by that time, travelers had carried the virus to many countries, sparking memories of the previous coronavirus epidemics, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), and causing widespread media attention and panic. Based on clinical criteria and available serological and molecular information, the new disease was called coronavirus disease of 2019 (COVID-19), and the novel coronavirus was called SARS Coronavirus-2 (SARS-CoV-2), emphasizing its close relationship to the 2002 SARS virus (SARS-CoV). The scientific community raced to uncover the origin of the virus, understand the pathogenesis of the disease, develop treatment options, define the risk factors, and work on vaccine development. Here we present a summary of current knowledge regarding the novel coronavirus and the disease it causes. Full article

Tomato bacterial wilt caused by Ralstonia solanacearum (RS) is one of the most devastating soil-borne diseases, and compost is to be considered as a resource-saving and environment-friendly measure to control the disease. Herein, a pot experiment was implemented to explore the effects of vinegar residue matrix amendments on the growth performances of tomato seedlings and to examine the suppression ability against bacterial wilt under vinegar residue substrate (VRS), and peat substrate (Peat) with RS inoculation. The results revealed that VRS effectively suppressed the disease incidence of bacterial wilt, increased the number of bacteria and actinomycetes, decreased fungi populations, promoted soil microbial populations and microbial activities, enhanced the growths of tomato seedlings, and modulated defense mechanism. In addition, VRS efficiently inhibited the oxidative damage in RS inoculated leaves via the regulation of excess reactive oxide species (O₂^•− and H₂O₂) production, lessening of malondialdehyde (MDA) content, and causing less membrane injury; resulting in enhancements of antioxidants enzymes activities accompanying with modulating their encoding gene expression. The transcription levels of NPR1, PIN2, PR1b, ACO1, EDS1, PR1B, MAPK3, PIN2, and RRS1 were also modulated with the pathogens inoculated in tomato leaves both in VRS and Peat treatments, which indicated that systemic-acquired resistance possesses cross-talk between salicylic acid, jasmonic acid, and the ethylene-dependent signaling pathway. Besides, the RS inoculation significantly inhibited the growth of tomato seedlings, and all growth indices of plants grown in VRS were considerably higher than those produced in Peat. Taken together, VRS represents a new strategy to control tomato bacterial wilt through boosting the soil microbial populations and microbial activities. Furthermore, VRS promotes the plant immune response to provide a better growth environment for plants surviving in disease conditions. Full article

Porcine circovirus 2 (PCV-2) is one of the most impactful and widespread pathogens of the modern swine industry. Unlike other DNA viruses, PCV-2 is featured by a remarkable genetic variability, which has led to the emergence and recognition of different genotypes, some of which (PCV-2a, 2b, and 2d) have alternated over time. Currently, PCV-2d is considered the most prevalent genotype, and some evidence of differential virulence and vaccine efficacy have been reported. Despite the potential practical relevance, the data on PCV-2 epidemiology in Italy are quite outdated and do not quantify the actual circulation of this genotype in Italy. In the present study, 82 complete ORF2 sequences were obtained from domestic pigs and wild boars sampled in Northern Italy in the period 2013–2018 and merged with those previously obtained from Italy and other countries. A combination of phylogenetic, haplotype network, and phylodynamic analyses were used to genotype the collected strains and evaluate the temporal trend and the spatial and host spread dynamics. A rising number of PCV-2d detections was observed in domestic pigs, particularly since 2013, reaching a detection frequency comparable to PCV-2b. A similar picture was observed in wild boars, although a lower sequence number was available. Overall, the present study demonstrates the extreme complexity of PCV-2 molecular epidemiology in Italy, the significant spread across different regions, the recurrent introduction from foreign countries, and the frequent occurrence of recombination events. Although a higher viral flux occurred from domestic to wild populations than vice versa, wild boars seem to maintain PCV-2 infection and spread it over relatively long distances. Full article

The term “prion disease” encompasses a group of neurodegenerative diseases affecting both humans and animals. Currently, there is no effective therapy and all forms of prion disease are invariably fatal. Because of (a) the outbreak of bovine spongiform encephalopathy in cattle and variant Creutzfeldt–Jakob disease in humans; (b) the heated debate about the prion hypothesis; and (c) the availability of a natural prion disease in rodents, the understanding of the pathogenic process in prion disease is much more advanced compared to that of other neurodegenerative disorders, which inspired many attempts to develop therapeutic strategies against these fatal diseases. In this review, we focus on immunotherapy against prion disease. We explain our rationale for immunotherapy as a plausible therapeutic choice, review previous trials using either active or passive immunization, and discuss potential strategies for overcoming the hurdles in developing a successful immunotherapy. We propose that immunotherapy is a plausible and practical therapeutic strategy and advocate more studies in this area to develop effective measures to control and treat these devastating disorders. Full article

Alfalfa plants in the field can display a range of virus-like symptoms, especially when grown over many years for seed production. Most known alfalfa viruses have RNA genomes, some of which can be detected using diagnostic assays, but many viruses of alfalfa are not well characterized. This study aims to identify the RNA and DNA virus complexes associated with alfalfa plants in Australia. To maximize the detection of RNA viruses, we purified double-stranded RNA (dsRNA) for high throughput sequencing and characterized the viromes of ten alfalfa samples that showed diverse virus-like symptoms. Using Illumina sequencing of tagged cDNA libraries from immune-captured dsRNA, we identified sequences of the single-stranded RNA viruses, alfalfa mosaic virus (AMV), bean leafroll virus, a new emaravirus tentatively named alfalfa ringspot-associated virus, and persistent dsRNA viruses belonging to the families Amalgaviridae and Partitiviridae. Furthermore, rolling circle amplification and restriction enzyme digestion revealed the complete genome of chickpea chlorosis Australia virus, a mastrevirus (family Geminiviridae) previously reported only from chickpea and French bean that was 97% identical to the chickpea isolate. The sequence data also enabled the assembly of the first complete genome (RNAs 1–3) of an Australian AMV isolate from alfalfa. Full article

Rodents are known to be reservoir hosts for at least 60 zoonotic diseases and are known to play an important role in their transmission and spread in different ways. We sampled different rodent communities within and around human settlements in Northern Senegal, an area subjected to major environmental transformations associated with global changes. Herein, we conducted an epidemiological study on their bacterial communities. One hundred and seventy-one (171) invasive and native rodents were captured, 50 from outdoor trapping sites and 121 rodents from indoor habitats, consisting of five species. The DNA of thirteen pathogens was successfully screened on the rodents’ spleens. We found: 2.3% of spleens positive to Piroplasmida and amplified one which gave a potentially new species Candidatus “Theileria senegalensis”; 9.35% of Bartonella spp. and amplified 10, giving three genotypes; 3.5% of filariasis species; 18.12% of Anaplasmataceae species and amplified only 5, giving a new potential species Candidatus “Ehrlichia senegalensis”; 2.33% of Hepatozoon spp.; 3.5% of Kinetoplastidae spp.; and 15.2% of Borrelia spp. and amplified 8 belonging all to Borrelia crocidurae. Some of the species of pathogens carried by the rodents of our studied area may be unknown because most of those we have identified are new species. In one bacterial taxon, Anaplasma, a positive correlation between host body mass and infection was found. Overall, male and invasive rodents appeared less infected than female and native ones, respectively. Full article

Lassa virus (LASV), the causative agent of Lassa fever, is estimated to be responsible for up to 300,000 new infections and 5000 deaths each year across Western Africa. The most recent 2018 and 2019 Nigerian outbreaks featured alarmingly high fatality rates of up to 25.4%. In addition to the severity and high fatality of the disease, a significant population of survivors suffer from long-term sequelae, such as sensorineural hearing loss, resulting in a huge socioeconomic burden in endemic regions. There are no Food and Drug Administration (FDA)-approved vaccines, and therapeutics remain extremely limited for Lassa fever. Development of countermeasures depends on relevant animal models that can develop a disease strongly mimicking the pathogenic features of Lassa fever in humans. The objective of this review is to evaluate the currently available animal models for LASV infection with an emphasis on their pathogenic and histologic characteristics as well as recent advances in the development of a suitable rodent model. This information may facilitate the development of an improved animal model for understanding disease pathogenesis of Lassa fever and for vaccine or antiviral testing. Full article

Coronaviruses (CoVs) are RNA viruses that have become a major public health concern since the Severe Acute Respiratory Syndrome-CoV (SARS-CoV) outbreak in 2002. The continuous evolution of coronaviruses was further highlighted with the emergence of the Middle East Respiratory Syndrome-CoV (MERS-CoV) outbreak in 2012. Currently, the world is concerned about the 2019 novel CoV (SARS-CoV-2) that was initially identified in the city of Wuhan, China in December 2019. Patients presented with severe viral pneumonia and respiratory illness. The number of cases has been mounting since then. As of late February 2020, tens of thousands of cases and several thousand deaths have been reported in China alone, in addition to thousands of cases in other countries. Although the fatality rate of SARS-CoV-2 is currently lower than SARS-CoV, the virus seems to be highly contagious based on the number of infected cases to date. In this review, we discuss structure, genome organization, entry of CoVs into target cells, and provide insights into past and present outbreaks. The future of human CoV outbreaks will not only depend on how the viruses will evolve, but will also depend on how we develop efficient prevention and treatment strategies to deal with this continuous threat. Full article

Bartonella spp. are increasingly implicated in association with a spectrum of zoonotic infectious diseases. One hundred sanitary workers in La Rioja, Spain, completed a questionnaire and provided blood specimens for Bartonella spp. serology and Bartonella Alpha-Proteobacteria growth medium (BAPGM) enrichment blood culture/PCR. Six immunofluorescence assays (IFA) were performed and aseptically obtained blood specimens were inoculated into liquid BAPGM and subcultured onto blood agar plates. Bartonella DNA was amplified using conventional and real-time PCR assays. The Bartonella spp., strain, or genotype was determined by DNA sequencing. Bartonella seroreactivity was documented in 83.1% and bloodstream infection in 21.6% of participants. Bartonella henselae, B. vinsonii subsp. berkhoffii genotypes I and III, and B. quintana were identified. IFA seroreactivity and PCR positivity were not statistically associated with self-reported symptoms. Our results suggest that exposure to and non-clinical infection with Bartonella spp. may occur more often than previously suspected in the La Rioja region. Full article

A serum neutralization test (SNT) is an essential method for the serological diagnosis of pestivirus infections, including classical swine fever, because of the cross reactivity of antibodies against pestiviruses and the non-quantitative properties of antibodies in an enzyme-linked immunosorbent assay. In conventional SNTs, an immunoperoxidase assay or observation of cytopathic effect after incubation for 3 to 7 days is needed to determine the SNT titer, which requires labor-intensive or time-consuming procedures. Therefore, a new SNT, based on the luciferase system and using classical swine fever virus, bovine viral diarrhea virus, and border disease virus possessing the 11-amino-acid subunit derived from NanoLuc luciferase was developed and evaluated; this approach enabled the rapid and easy determination of the SNT titer using a luminometer. In the new method, SNT titers can be determined tentatively at 2 days post-infection (dpi) and are comparable to those obtained by conventional SNTs at 3 or 4 dpi. In conclusion, the luciferase-based SNT can replace conventional SNTs as a high-throughput antibody test for pestivirus infections. Full article

Small ruminant lentiviruses (SRLV) are a group of highly divergent viruses responsible for global and fatal infections in sheep and goats. Since the current phylogenetic classification of these viruses was proposed in 2004, it nowadays consists out of 5 genotypes and 28 subtypes. In support of our national SRLV control program, we performed the genetic characterization of SRLV strains circulating in the Belgian sheep and goat population. Fourteen sheep and 9 goat strains were sequenced in the gag-pol and pol regions using the method described by Shah. Most SRLV strains from sheep and goats belonged to prototype A1 and B1 subtypes, respectively. We, however, also found indications for cross-species transmission of SRLV strains between sheep and goats and vice versa, and identified a new subtype designated as B5. An in-depth analysis of the current SRLV phylogeny revealed that many subtypes have been defined over the years based on limited sequence information. To keep phylogeny as a useful tool, we advocate to apply more rigorous sequencing standards to ensure the correct classification of current and new emerging strains. The genetic characterization of Belgian SRLV strains will help in the development of appropriate diagnostic tools to assist the national control program. Full article

Aggregatibacter actinomycetemcomitans, the focus of this review, was initially proposed as a microbe directly related to a phenotypically distinct form of periodontitis called localized juvenile periodontitis. At the time, it seemed as if specific microbes were implicated as the cause of distinct forms of disease. Over the years, much has changed. The sense that specific microbes relate to distinct forms of disease has been challenged, as has the sense that distinct forms of periodontitis exist. This review consists of two components. The first part is presented as a detective story where we attempt to determine what role, if any, Aggregatibacter plays as a participant in disease. The second part describes landscape ecology in the context of how the host environment shapes the framework of local microbial dysbiosis. We then conjecture as to how the local host response may limit the damage caused by pathobionts. We propose that the host may overcome the constant barrage of a dysbiotic microbiota by confining it to a local tooth site. We conclude speculating that the host response can confine local damage by restricting bacteremic translocation of members of the oral microbiota to distant organs thus constraining morbidity and mortality of the host. Full article

Despite the high burden of vector-borne disease in (sub)tropical areas, few information are available regarding the diversity of tick and tick-borne pathogens circulating in the Caribbean. Management and control of vector-borne disease require actual epidemiological data to better assess and anticipate the risk of (re)emergence of tick-borne diseases in the region. To simplify and reduce the costs of such large-scale surveys, we implemented a high-throughput microfluidic real-time PCR system suitable for the screening of the main bacterial and parasitic genera involved in tick-borne disease and potentially circulating in the area. We used the new screening tool to perform an exploratory epidemiological study on 132 adult specimens of Amblyomma variegatum and 446 of Rhipicephalus microplus collected in Guadeloupe and Martinique. Not only the system was able to detect the main pathogens of the area—Ehrlichia ruminantium, Rickettsia africae, Anaplasma marginale, Babesia bigemina and Babesia bovis—but the system also provided evidence of unsuspected microorganisms in Caribbean ticks, belonging to the Anaplasma, Ehrlichia, Borrelia and Leishmania genera. Our study demonstrated how high-throughput microfluidic real-time PCR technology can assist large-scale epidemiological studies, providing a rapid overview of tick-borne pathogen and microorganism diversity, and opening up new research perspectives for the epidemiology of tick-borne pathogens. Full article

The emergence and spread of drug-resistant Mycobacterium tuberculosis strains (including MDR, XDR, and TDR) force scientists worldwide to search for new anti-tuberculosis drugs. We have previously reported a number of imidazo[1,2-b][1,2,4,5]tetrazines – putative inhibitors of mycobacterial eukaryotic-type serine-threonine protein-kinases, active against M. tuberculosis. Whole genomic sequences of spontaneous drug-resistant M. smegmatis mutants revealed four genes possibly involved in imidazo[1,2-b][1,2,4,5]tetrazines resistance; however, the exact mechanism of resistance remain unknown. We used different approaches (construction of targeted mutants, overexpression of the wild-type (w.t.) and mutant genes, and gene-expression studies) to assess the role of the previously identified mutations. We show that mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis, thus providing resistance to imidazo[1,2-b][1,2,4,5]tetrazines by increased efflux through the MmpS5-MmpL5 system, similarly to the mechanisms of resistance described for M. tuberculosis and M. abscessus. Mycobacterial MmpS5-MmpL5 transporters should be considered as an MDR-efflux system and they should be taken into account at early stages of anti-tuberculosis drug development. Full article

Here, we examined the pathogenicity and genetic differences between classical swine fever viruses (CSFV) isolated on pig farms in North Vietnam from 2014–2018. Twenty CSFV strains from 16 pig farms were classified as genotype 2 (sub-genotypes 2.1b, 2.1c, and 2.2). The main sub-genotype, 2.1c, was classified phylogenetically as belonging to the same cluster as viruses isolated from the Guangdong region in South China. Strain HY58 (sub-genotype 2.1c), isolated from pigs in Vietnam, caused higher mortality (60%) than the Vietnamese ND20 strain (sub-genotype 2.2). The Vietnamese strain of sub-genotype 2.1b was estimated to have moderate virulence; indeed, genetic analysis revealed that it belongs to the same cluster as Korean CSFV sub-genotype 2.1b. Most CSFVs circulating in North Vietnam belong to sub-genotype 2.1c. Geographical proximity means that this genotype might continue to circulate in both North Vietnam and Southern China (Guangdong, Guangxi, and Hunan). Full article

African swine fever (ASF) is a viral disease of domestic and wild suids for which there is currently no vaccine or treatment available. The recent spread of ASF virus (ASFV) through Europe and Asia is causing enormous economic and animal losses. Unfortunately, the measures taken so far are insufficient and an effective vaccine against ASFV needs to be urgently developed. We hypothesized that immunization with a cocktail of thirty-five rationally selected antigens would improve the protective efficacy of subunit vaccine prototypes given that the combination of fewer immunogenic antigens (between 2 and 22) has failed to elicit protective efficacy. To this end, immunogenicity and efficacy of thirty-five adenovirus-vectored ASFV antigens were evaluated in wild boar. The treated animals were divided into different groups to test the use of BioMize adjuvant and different inoculation strategies. Forty-eight days after priming, the nine treated and two control wild boar were challenged with the virulent ASFV Arm07 isolate. All animals showed clinical signs and pathological findings consistent with ASF. This lack of protection is in line with other studies with subunit vaccine prototypes, demonstrating that there is still much room for improvement to obtain an effective subunit ASFV vaccine. Full article

Zika virus (ZIKV) is a mosquito-borne flavivirus that has emerged and caused global outbreaks since 2007. Although ZIKV proteins have been shown to suppress early anti-viral innate immune responses, little is known about the exact mechanisms. This study demonstrates that infection with either the African or Asian lineage of ZIKV leads to a modulated expression of suppressor of cytokine signaling (SOCS) genes encoding SOCS1 and SOCS3 in the following cell models: A549 human lung adenocarcinoma cells; JAr human choriocarcinoma cells; human neural progenitor cells. Studies of viral gene expression in response to SOCS1 or SOCS3 demonstrated that the knockdown of these SOCS proteins inhibited viral NS5 or ZIKV RNA expression, whereas overexpression resulted in an increased expression. Moreover, the overexpression of SOCS1 or SOCS3 inhibited the retinoic acid-inducible gene-I-like receptor-mediated activation of both type I and III interferon pathways. These results imply that SOCS upregulation following ZIKV infection modulates viral replication, possibly via the regulation of anti-viral innate immune responses. Full article

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that has received an increasing amount of attention from virologists, clinicians, veterinarians, and epidemiologists over the past decade. The host range and animal reservoirs of HEV are rapidly expanding and a plethora of emerging HEV variants have been recently identified, some of which have the potential for interspecies infection. In this review, the detection of genetically diverse HEV variants, classified into and presumably associated with the species Orthohepevirus C, currently comprising HEV genotypes C1 and C2, by either serological or molecular approach is summarized. The distribution, genomic variability, and evolution of Orthohepevirus C are analyzed. Moreover, the potential risk of cross-species infection and zoonotic transmission of Orthohepevirus C are discussed. Full article

Streptococcus suis serotype 2 is an important porcine bacterial pathogen and zoonotic agent causing sudden death, septic shock and meningitis. These pathologies are the consequence of an exacerbated inflammatory response composed of various mediators including interleukin (IL)-1β. Elevated levels of the toxin suilysin (SLY) were demonstrated to play a key role in S. suis-induced IL-1β production. However, 95% of serotype 2 strains isolated from diseased pigs in North America, many of which are virulent, do not produce SLY. In this study, we demonstrated that SLY-negative S. suis induces elevated levels of IL-1β in systemic organs, with dendritic cells contributing to this production. SLY-negative S. suis-induced IL-1β production requires MyD88 and TLR2 following recognition of lipoproteins. However, the higher internalization rate of the SLY-negative strain results in intracellularly located DNA being recognized by the AIM2 inflammasome, which promotes IL-1β production. Finally, the role of IL-1 in host survival during the S. suis systemic infection is beneficial and conserved, regardless of SLY production, via modulation of the inflammation required to control bacterial burden. In conclusion, this study demonstrates that SLY is not required for S. suis-induced IL-1β production. Full article

Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments. Full article

Introduction: Reliable and cost-effective diagnostics for hepatitis E virus (HEV) infection are necessary. The aim of our study was to investigate which diagnostic test is most accurate to detect HEV infection in immunocompetent and immunosuppressed patients in a real world setting. Patients and Methods: We performed a retrospective analysis of 1165 patients tested for HEV antibodies and HEV PCR at the same time point. Clinical, laboratory and virological data were taken from patient charts. HEV IgA was measured in a subgroup of 185 patients. Results: HEV RNA was detectable in 61 patients (5.2%); most of them (n = 49, 80.3%/n = 43, 70.5%) were HEV IgM+ and IgG+; however, 12 patients (19.6%) were HEV RNA positive/HEV IgM negative and 17 patients (27.8%) were HEV RNA positive/HEV IgG negative. Ten HEV RNA positive patients (16.4%) had neither HEV IgG nor IgM antibodies. Importantly, all of them were immunosuppressed. HEV IgA testing was less sensitive than HEV IgM for HEV diagnosis. Conclusions: HEV infection can be overlooked in patients without HEV specific antibodies. Performing PCR is necessary to diagnose or exclude HEV infection in immunocompromised hosts. In immunocompetent patients, a screening based on HEV antibodies (IgG/IgM) is sufficient. Full article

Porcine epidemic diarrhea virus (PEDV) is a highly virulent re-emerging enteric coronavirus that causes acute diarrhea, dehydration, and up to 100% mortality in neonatal suckling piglets. Despite this, a safe and effective PEDV vaccine against highly virulent strains is unavailable, making PEDV prevention and control challenging. Lactogenic immunity induced via the gut-mammary gland-secretory IgA (sIgA) axis, remains the most promising and effective way to protect suckling piglets from PEDV. Therefore, a successful PEDV vaccine must induce protective maternal IgA antibodies that passively transfer into colostrum and milk. Identifying variables that influence lymphocyte migration and IgA secretion during gestation and lactation is imperative for designing maternal immunization strategies that generate the highest amount of lactogenic immune protection against PEDV in suckling piglets. Because pregnancy-associated immune alterations influence viral pathogenesis and adaptive immune responses in many different species, a better understanding of host immune responses to PEDV in pregnant swine may translate into improved maternal immunization strategies against enteric pathogens for multiple species. In this review, we discuss the role of host factors during pregnancy on antiviral immunity and their implications for generating protective lactogenic immunity in suckling neonates. Full article

Tuberculosis, caused by Mycobacterium tuberculosis complex bacteria, remains one of the most pressing health problems. Despite the general trend towards reduction of the disease incidence rate, the situation remains extremely tense due to the distribution of the resistant forms. Most often, these strains emerge through the intra-host microevolution of the pathogen during treatment failure. In the present study, the focus was on three serial clinical isolates of Mycobacterium tuberculosis Beijing B0/W148 cluster from one patient with pulmonary tuberculosis, to evaluate their changes in metabolism during anti-tuberculosis therapy. Using whole genome sequencing (WGS), 9 polymorphisms were determined, which occurred in a stepwise or transient manner during treatment and were linked to the resistance (GyrA D94A; inhA t-8a) or virulence. The effect of the inhA t-8a mutation was confirmed on both proteomic and transcriptomic levels. Additionally, the amount of RpsL protein, which is a target of anti-tuberculosis drugs, was reduced. At the systemic level, profound changes in metabolism, linked to the evolution of the pathogen in the host and the effects of therapy, were documented. An overabundance of the FAS-II system proteins (HtdX, HtdY) and expression changes in the virulence factors have been observed at the RNA and protein levels. Full article

Epigenetic reprogramming in macrophages is termed trained innate immunity, which regulates immune tolerance and limits tissue damage during infection. Neisseria gonorrhoeae is a strict human pathogen that causes the sexually transmitted infection termed gonorrhea. Here, we report that this pathogen harbors a gene that encodes a histone deacetylase-like enzyme (Gc-HDAC) that shares high 3D-homology to human HDAC1, HDAC2 and HDAC8. A Gc-HDAC null mutant was constructed to determine the biologic significance of this gene. The results showed that WT gonococci reduced the expression of host defense peptides LL-37, HBD-1 and SLPI in macrophages when compared to its Gc-HDAC-deficient isogenic strain. The enrichment of epigenetic marks in histone tails control gene expression and are known to change during bacterial infections. To investigate whether gonococci exert epigenetic modifications on host chromatin, the enrichment of acetylated lysine 9 in histone 3 (H3K9ac) was investigated using the TLR-focused ChIP array system. The data showed that infection with WT gonococci led to higher H3K9ac enrichment at the promoters of pro-inflammatory mediators’ genes, many TLRs, adaptor proteins and transcription factors, suggesting gene activation when compared to infection with the Gc-HDAC-deficient mutant. Taken together, the data suggest that gonococci can exert epigenetic modifications on host cells to modulate certain macrophage defense genes, leading to a maladaptive state of trained immunity. Full article

Human papillomavirus (HPV) E6 and E7 oncoproteins are critical for development and maintenance of the malignant phenotype in HPV-induced cancers. These two viral oncoproteins interfere with a plethora of cellular pathways, including the regulation of cell cycle and the control of apoptosis, which are critical in maintaining normal cellular functions. E6 and E7 bind directly with certain components of the Ubiquitin Proteasome System (UPS), enabling them to manipulate a number of important cellular pathways. These activities are the means by which HPV establishes an environment supporting the normal viral life cycle, however in some instances they can also lead to the development of malignancy. In this review, we have discussed how E6 and E7 oncoproteins from alpha and beta HPV types interact with the components of the UPS, and how this interplay contributes to the development of cancer. Full article

Amdoparvovirus and Protoparvovirus are monophyletic viral genera that infect carnivores. We performed surveillance for and sequence analyses of parvoviruses in mustelids in insular British Columbia to investigate parvoviral maintenance and cross-species transmission among wildlife. Overall, 19.1% (49/256) of the tested animals were parvovirus-positive. Aleutian mink disease virus (AMDV) was more prevalent in mink (41.6%, 32/77) than martens (3.1%, 4/130), feline panleukopenia virus (FPV) was more prevalent in otters (27.3%, 6/22) than mink (5.2%, 4/77) or martens (2.3%, 3/130), and canine parvovirus 2 (CPV-2) was found in one mink, one otter, and zero ermines (N = 27). Viruses were endemic and bottleneck events, founder effects, and genetic drift generated regional lineages. We identified two local closely related AMDV lineages, one CPV-2 lineage, and five FPV lineages. Highly similar viruses were identified in different hosts, demonstrating cross-species transmission. The likelihood for cross-species transmission differed among viruses and some species likely represented dead-end spillover hosts. We suggest that there are principal maintenance hosts (otters for FPV, raccoons for CPV-2/FPV, mink for AMDV) that enable viral persistence and serve as sources for other susceptible species. In this multi-host system, viral and host factors affect viral persistence and distribution, shaping parvoviral ecology and evolution, with implications for insular carnivore conservation. Full article

Polymerase chain reaction (PCR) is a unique technique in molecular biology and biotechnology for amplifying target DNA strands, and is also considered as a gold standard for the diagnosis of many canine diseases as well as many other infectious diseases. However, PCR still faces many challenges and issues related to its sensitivity, specificity, efficiency, and turnaround time. To address these issues, we described the use of unique ZnO nanoflowers in PCR reaction and an efficient ZnO nanoflower-based PCR (nanoPCR) for the molecular diagnosis of canine vector-borne diseases (CVBDs). A total of 1 mM of an aqueous solution of ZnO nanoflowers incorporated in PCR showed a significant enhancement of the PCR assay with respect to its sensitivity and specificity for the diagnosis of two important CVBDs, Babesia canis vogeli and Hepatozoon canis. Interestingly, it drastically reduced the turnaround time of the PCR assay without compromising the yield of the amplified DNA, which can be of benefit for veterinary practitioners for the improved management of diseases. This can be attributed to the favorable adsorption of ZnO nanoflowers to the DNA and thermal conductivity of ZnO nanoflowers. The unique ZnO nanoflower-assisted nanoPCR greatly improved the yield, purity, and quality of the amplified products, but the mechanism behind these properties and the effects and changes due to the different concentrations of ZnO nanoflowers in the PCR system needs to be further studied. Full article

Enterovirus 71 (EV71) has become an important public health problem in the Asia-Pacific region in the past decades. EV71 infection might cause neurological and psychiatric complications and even death. Although an EV71 vaccine has been currently approved, there is no effective therapy for treating EV71-infected patients. Virus infections have been reported to shape host T cell receptor (TCR) repertoire. Therefore, understanding of host TCR repertoire in EV71 infection could better the knowledge in viral pathogenesis and further benefit the anti-viral therapy development. In this study, we used a mouse-adapted EV71 (mEV71) model to observe changes of host TCR repertoire in an EV71-infected central nervous system. Neonate mice were infected with mEV71 and mouse brainstem TCRβ repertoires were explored. Here, we reported that mEV71 infection impacted host brainstem TCRβ repertoire, where mEV71 infection skewed TCRβ diversity, changed VJ combination usages, and further expanded specific TCRβ CDR3 clones. Using bioinformatics analysis and ligand-binding prediction, we speculated the expanded TCRβ CDR3 clone harboring CASSLGANSDYTF sequence was capable of binding cleaved EV71 VP1 peptides in concert with major histocompatibility complex (MHC) molecules. We observed that mEV71 infection shaped host TCRβ repertoire and presumably expanded VP1-specific TCRβ CDR3 in mEV71-infected mouse brainstem that integrated EV71 pathogenesis in central nervous system. Full article

The correct management of diseases that are transmitted between wildlife and livestock requires a reliable estimate of the pathogen transmission rate. The calculation of this parameter is a challenge for epidemiologists, since transmission can occur through multiple pathways. The social network analysis is a widely used tool in epidemiology due to its capacity to identify individuals and communities with relevant roles for pathogen transmission. In the present work, we studied the dynamic network of interactions in a complex epidemiological scenario using information from different methodologies. In 2015, nine red deer, seven fallow deer, six wild boar and nine cattle were simultaneously monitored using GPS-GSM-Proximity collars in Doñana National Park. In addition, 16 proximity loggers were set in aggregation points. Using the social network analysis, we studied the dynamic network of interactions, including direct and indirect interactions, between individuals of different species and the potential transmission of pathogens within this network. The results show a high connection between species through indirect interactions, with a marked seasonality in the conformation of new interactions. Within the network, we differentiated four communities that included individuals of all the species. Regarding the transmission of pathogens, we observed the important role that fallow deer could be playing in the maintenance and transmission of pathogens to livestock. The present work shows the need to consider different types of methodologies in order to understand the complete functioning of the network of interactions at the wildlife/livestock interface. It also provides a methodological approach applicable to the management of shared diseases. Full article

Introduction: Strongyloidiasis is a prevailing helminth infection ubiquitous in tropical and subtropical areas, however, seroprevalence data are scarce in migrant populations, particularly for those coming for Asia. Methods: This study aims at evaluating the prevalence of S. stercoralis at the hospital level in migrant populations or long term travellers being attended in out-patient and in-patient units as part of a systematic screening implemented in six Spanish hospitals. A cross-sectional study was conducted and systematic screening for S. stercoralis infection using serological tests was offered to all eligible participants. Results: The overall seroprevalence of S. stercoralis was 9.04% (95%CI 7.76–10.31). The seroprevalence of people with a risk of infection acquired in Africa and Latin America was 9.35% (95%CI 7.01–11.69), 9.22% (7.5–10.93), respectively. The number of individuals coming from Asian countries was significantly smaller and the overall prevalence in these countries was 2.9% (95%CI −0.3–6.2). The seroprevalence in units attending potentially immunosuppressed patients was significantly lower (5.64%) compared with other units of the hospital (10.20%) or Tropical diseases units (13.33%) (p < 0.001). Conclusions: We report a hospital-based strongyloidiasis seroprevalence of almost 10% in a mobile population coming from endemic areas suggesting the need of implementing strongyloidiasis screening in hospitalized patients coming from endemic areas, particularly if they are at risk of immunosuppression. Full article

The outbreaks of viral encephalopathy and retinopathy (VER), caused by nervous necrosis virus (NNV), represent one of the main infectious threats for marine aquaculture worldwide. Since the first description of the disease at the end of the 1980s, a considerable amount of research has gone into understanding the mechanisms involved in fish infection, developing reliable diagnostic methods, and control measures, and several comprehensive reviews have been published to date. This review focuses on host–virus interaction and epidemiological aspects, comprising viral distribution and transmission as well as the continuously increasing host range (177 susceptible marine species and epizootic outbreaks reported in 62 of them), with special emphasis on genotypes and the effect of global warming on NNV infection, but also including the latest findings in the NNV life cycle and virulence as well as diagnostic methods and VER disease control. Full article

Epstein–Barr virus (EBV) and Helicobacter pylori (H. pylori) are two pathogens associated with the development of various human cancers. The coexistence of both microorganisms in gastric cancer specimens has been increasingly reported, suggesting that crosstalk of both pathogens may be implicated in the carcinogenesis process. Considering that chronic inflammation is an initial step in the development of several cancers, including gastric cancer, we conducted a systematic review to comprehensively evaluate publications in which EBV and H. pylori co-infection has been documented in patients with non-malignant gastroduodenal disorders (NMGDs), including gastritis, peptic ulcer disease (PUD), and dyspepsia. We searched the PubMed database up to August 2019, as well as publication references and, among the nine studies that met the inclusion criteria, we identified six studies assessing EBV infection directly in gastric tissues (total 949 patients) and three studies in which EBV infection status was determined by serological methods (total 662 patients). Due to the substantial methodological and clinical heterogeneity among studies identified, we could not conduct a meta-analysis. The overall prevalence of EBV + H. pylori co-infection in NMGDs was 34% (range 1.8% to 60%). A higher co-infection rate (EBV + H. pylori) was reported in studies in which EBV was documented by serological methods in comparison with studies in which EBV infection was directly assessed in gastric specimens. The majority of these studies were conducted in Latin-America and India, with most of them comparing NMGDs with gastric cancer, but there were no studies comparing the co-infection rate in NMGDs with that in asymptomatic individuals. In comparison with gastritis caused by only one of these pathogens, EBV + H. pylori co-infection was associated with increased severity of gastric inflammation. In conclusion, only relatively small studies testing EBV and H. pylori co-infection in NMGDs have been published to date and the variable report results are likely influenced by geographic factors and detection methods. Full article

To monitor drug resistance in Plasmodium vivax, a multidrug resistance 1 (Pvmdr1) gene and a putative transporter protein (Pvcrt-o) gene were used as molecular markers for chloroquine resistance. The biomarkers, the dihydrofolate reductase (Pvdhfr) gene and the dihydropteroate synthetase (Pvdhps) gene, were also used for the detection of resistance to sulphadoxine-pyrimethamine (SP); this drug is often accidentally used to treat P. vivax infections. Clinical blood samples (n = 120) were collected from patients who had been to one of eight malaria-endemic countries and diagnosed with P. vivax infection. The chloroquine resistance marker, the Pvmdr1 gene, showed F976:L1076 mutations and L1076 mutation. A K10 insertion in the Pvcrt-o gene was also found among the samples successfully sequenced. A combination of L/I57:R58:M61:T117 mutations in the Pvdhfr gene and G383:G553 mutations in the Pvdhps gene were also observed. Mutations found in these genes indicate that drug resistance is present in these eight countries. Whether or not countries are using chloroquine to treat P. vivax, there appears to be an increase in mutation numbers in resistance gene markers. The detected changes in mutation rates of these genes do suggest that there is still a trend towards increasing P. vivax resistance to chloroquine. The presence of the mutations associated with SP resistance indicates that P. vivax has had exposure to SP and this may be a consequence of either misdiagnosis or coinfections with P. falciparum in the past. Full article

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory pathogen of swine that has become extremely costly to the swine industry worldwide, often causing losses in production and animal life due to their ease of spread. However, the intracellular changes that occur in pigs following viral respiratory infections are still scantily understood for PRRSV, as well as other viral respiratory infections. The aim of this study was to acquire a better understanding of the PRRS disease by comparing gene expression changes that occur in tracheobronchial lymph nodes (TBLN) of pigs infected with either porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV-2), or swine influenza A virus (IAV-S) infections. The study identified and compared gene expression changes in the TBLN of 80 pigs following infection by PRRSV, PCV-2, IAV-S, or sham inoculation. Total RNA was pooled for each group and time-point (1, 3, 6, and 14 dpi) to make 16 libraries—analyses are by Digital Gene Expression Tag Profiling (DGETP). The data underwent standard filtering to generate a list of sequence tag raw counts that were then analyzed using multidimensional and differential expression statistical tests. The results showed that PRRSV, IAV-S and PCV-2 viral infections followed a clinical course in the pigs typical of experimental infection of young pigs with these viruses. Gene expression results echoed this course, as well as uncovered genes related to intersecting and unique host immune responses to the three viruses. By testing and observing the host response to other respiratory viruses, our study has elucidated similarities and differences that can assist in the development of vaccines and therapeutics that shorten or prevent a chronic PRRSV infection. Full article

The replication of virus in secondary lymphoid organs is crucial for the activation of antigen-presenting cells. Balanced viral replication ensures the sufficient availability of antigens and production of cytokines, and both of which are needed for virus-specific immune activation and viral elimination. Host factors that regulate coordinated viral replication are not fully understood. In the study reported here, we identified Map3k14 as an important regulator of enforced viral replication in the spleen while performing genome-wide association studies of various inbred mouse lines in a model of lymphocytic choriomeningitis virus (LCMV) infection. When alymphoplasia mice (aly/aly, Map3k14^aly/aly, or Nik^aly/aly), which carry a mutation in Map3k14, were infected with LCMV or vesicular stomatitis virus (VSV), they display early reductions in early viral replication in the spleen, reduced innate and adaptive immune activation, and lack of viral control. Histologically, scant B cells and the lack of CD169⁺ macrophages correlated with reduced immune activation in Map3k14^aly/aly mice. The transfer of wildtype B cells into Map3k14^aly/aly mice repopulated CD169⁺ macrophages, restored enforced viral replication, and resulted in enhanced immune activation and faster viral control. Full article

Infectious pancreatic necrosis (IPN) is a disease of great concern in aquaculture, mainly among salmonid farmers, since losses in salmonid fish—mostly very young rainbow trout (Salmo gairdnery) fry and Atlantic salmon (Salmo salar) post-smolt—frequently reach 80–90% of stocks. The virus causing the typical signs of the IPN disease in salmonids, named infectious pancreatic necrosis virus (IPNV), has also been isolated from other fish species either suffering related diseases (then named IPNV-like virus) or asymptomatic; the general term aquabirnavirus is used to encompass all these viruses. Aquabirnaviruses are non-enveloped, icosahedral bisegmented dsRNA viruses, whose genome codifies five viral proteins, three of which are structural, and one of them is an RNA-dependent RNA polymerase. Due to the great importance of the disease, there have been great efforts to find a way to predict the level of virulence of IPNV isolates. The viral genome and proteins have been the main focus of research. However, to date such a reliable magic marker has not been discovered. This review describes the processes followed for decades in the attempts to discover the viral determinants of virulence, and to help the reader understand how viral components can be involved in virulence modulation in vitro and in vivo. There is also a brief description of the disease, of host defenses, and of the molecular structure and function of the virus and its viral components. Full article

The genus Naegleria, of the free-living amoeba (FLA) group, has been investigated mainly due to its human health impact, resulting in deadly infections and their worldwide distribution on freshwater systems. Naegleria fowleri, colloquially known as the “brain-eating amoeba,” is the most studied Naegleria species because it causes primary amoebic meningoencephalitis (PAM) of high lethality. The assessment of FLA biodiversity is fundamental to evaluate the presence of pathogenic species and the possibility of human contamination. However, the knowledge of FLA distribution in Brazil is unknown, and to rectify this situation, we present research on identifying Naegleria spp. in the Monjolinho River as a model study. The river is a public Brazilian freshwater source that crosses the city of São Carlos, in São Paulo state, Brazil. Five distinct sampling sites were examined through limnological features, trophozoites culturing, and PCR against internal transcribed spacer (ITS) regions and 5.8S rRNA sequences. The results identified N. philippinensis, N. canariensisi, N. australiensis, N. gruberi, N. dobsoni sequences, as well as a Hartmannella sequence. The methodology delineated here represents the first Brazilian Naegleria spp. study on a freshwater system. Our results stress the urgency of a large scale evaluation of the presence of free-living amoebas in Brazil. Full article

The ideal laboratory test to detect Neisseria gonorrhoeae (Ng) should be sensitive, specific, easy to use, rapid, and affordable and should provide information about susceptibility to antimicrobial drugs. Currently, such a test is not available and presumably will not be in the near future. Thus, diagnosis of gonococcal infections presently includes application of different techniques to address these requirements. Microscopy may produce rapid results but lacks sensitivity in many cases (except symptomatic urogenital infections in males). Highest sensitivity to detect Ng was shown for nucleic acid amplification technologies (NAATs), which, however, are less specific than culture. In addition, comprehensive analysis of antibiotic resistance is accomplished only by in vitro antimicrobial susceptibility testing of cultured isolates. As a light at the end of the tunnel, new developments of molecular techniques and microfluidic systems represent promising opportunities to design point-of-care tests for rapid detection of Ng with high sensitivity and specificity, and there is reason to hope that such tests may also provide antimicrobial resistance data in the future. Full article

Seasonal influenza epidemics remain one of the largest public health burdens nowadays. The best and most effective strategy to date in preventing influenza infection is a worldwide vaccination campaign. Currently, two vaccines are available to the public for the treatment of influenza infection, the chemically Inactivated Influenza Vaccine (IIV) and the Live Attenuated Influenza Vaccine (LAIV). However, the LAIV is not recommended for parts of the population, such as children under the age of two, immunocompromised individuals, the elderly, and pregnant adults. In order to improve the safety of the LAIV and make it available to more of the population, we sought to further attenuate the LAIV. In this study, we demonstrate that the influenza A virus (IAV) master donor virus (MDV) A/Ann Arbor/6/60 H2N2 LAIV can inhibit host gene expression using both the PA-X and NS1 proteins. Furthermore, we show that by removing PA-X, we can limit the replication of the MDV LAIV in a mouse model, while maintaining full protective efficacy. This work demonstrates a broadly applicable strategy of tuning the amount of host antiviral responses induced by the IAV MDV for the development of newer and safer LAIVs. Moreover, our results also demonstrate, for the first time, the feasibility of genetically manipulating the backbone of the IAV MDV to improve the efficacy of the current IAV LAIV. Full article

Living beings spend their lives and carry out their daily activities interacting with environmental situations that present space-time variations and that involve contact with other life forms, which may behave as commensals or as invaders and/or parasites. The characteristics of the environment, as well as the processes that support the maintenance of life and that characterize the execution of activities of daily life generally present periodic variations, which are mostly synchronized with the light–dark cycle determined by Earth’s rotation on its axis. These rhythms with 24-h periodicity, defined as circadian, influence events linked to the interaction between hosts and hosted microorganisms and can dramatically determine the outcome of this interplay. As for the various pathological conditions resulting from host–microorganism interactions, a particularly interesting scenario concerns infections by viruses. When a viral agent enters the body, it alters the biological processes of the infected cells in order to favour its replication and to spread to various tissues. Though our knowledge concerning the mutual influence between the biological clock and viruses is still limited, recent studies start to unravel interesting aspects of the clock–virus molecular interplay. Three different aspects of this interplay are addressed in this mini-review and include the circadian regulation of both innate and adaptive immune systems, the impact of the biological clock on viral infection itself, and finally the putative perturbations that the virus may confer to the clock leading to its deregulation. Full article

Leishmania are obligate intracellular parasites known to have developed successful ways of efficient immunity evasion. Because of this, leishmaniasis, a disease caused by these flagellated protists, is ranked as one of the most serious tropical infections worldwide. Neither prophylactic medication, nor vaccination has been developed thus far, even though the infection has usually led to strong and long-lasting immunity. In this paper, we describe a “suicidal” system established in Leishmania mexicana, a human pathogen causing cutaneous leishmaniasis. This system is based on the expression and (de)stabilization of a basic phospholipase A2 toxin from the Bothrops pauloensis snake venom, which leads to the inducible cell death of the parasites in vitro. Furthermore, the suicidal strain was highly attenuated during macrophage infection, regardless of the toxin stabilization. Such a deliberately weakened parasite could be used to vaccinate the host, as its viability is regulated by the toxin stabilization, causing a profoundly reduced pathogenesis. Full article

Achromobacter xylosoxidans can cause chronic infections in the lungs of patients with cystic fibrosis (CF) by adapting to the specific environment. The study of longitudinal isolates allows to investigate its within-host evolution to unravel the adaptive mechanisms contributing to successful colonization. In this study, four clinical isolates longitudinally collected from two chronically infected patients underwent whole genome sequencing, de novo assembly and sequence analysis. Phenotypic assays were also performed. The isolates coming from one of the patients (patient A) presented a greater number of genetic variants, diverse integrative and conjugative elements, and different protease secretion. In the first of these isolates (strain A1), we also found a large deletion in the mutS gene, involved in DNA mismatch repair (MMR). In contrast, isolates from patient B showed a lower number of variants, only one integrative and mobilizable element, no phenotypic changes, and no mutations in the MMR system. These results suggest that in the two patients the establishment of a chronic infection was mediated by different adaptive mechanisms. While the strains isolated from patient B showed a longitudinal microevolution, strain A1 can be clearly classified as a hypermutator, confirming the occurrence and importance of this adaptive mechanism in A. xylosoxidans infection. Full article

Leukotoxin (LtxA), from oral pathogen Aggregatibacter actinomycetemcomitans, is a secreted membrane-damaging protein. LtxA is internalized by β2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes. Full article

Background: The cationic amphiphilic drug U18666A inhibits the proliferation of type I FIPV in vitro. In this study, we evaluated the in vivo antiviral effects of U18666A by administering it to SPF cats challenged with type I FIPV. Methods: Ten SPF cats were randomly assigned to two experimental groups. FIPV KU-2 were inoculated intraperitoneally to cats. The control group was administered PBS, and the U18666A-treated group was administered U18666A subcutaneously at 2.5 mg/kg on day 0, and 1.25 mg/kg on days 2 and 4 after viral inoculation. Results: Two of the five control cats administered PBS alone developed FIP. Four of the five cats administered U18666A developed no signs of FIP. One cat that temporarily developed fever, had no other clinical symptoms, and no gross lesion was noted on an autopsy after the end of the experiment. The FIPV gene was detected intermittently in feces and saliva regardless of the development of FIP or administration of U18666A. Conclusions: When U18666A was administered to cats experimentally infected with type I FIPV, the development of FIP might be suppressed compared with the control group. However, the number of animals with FIP is too low to establish anti-viral effect of U18666A in cats. Full article

Appropriate vaccine selection is crucial in the control of foot-and-mouth disease (FMD). Vaccination can prevent clinical disease and reduces viral shedding, but there is a lack of cross-protection between the seven serotypes and their sublineages, making the selection of an adequately protective vaccine difficult. Since the exact composition of their vaccines is not consistently disclosed by all manufacturers, incompatibility of the strains used for vaccination with regionally circulating strains can cause vaccination campaigns to fail. Here, we present a deep sequencing approach for polyvalent inactivated FMD vaccines that can identify all component strains by their genome sequences. The genomes of all strains of a commercial pentavalent FMD vaccine were de novo assembled and the vaccine composition determined semi-quantitatively. The genome assembly required high stringency parameters to prevent misassemblies caused by conserved regions of the genome shared by related strains. In contrast, reference-guided assembly is only recommended in cases where the number of strains is previously known and appropriate reference sequences are available. The presented approach can be applied not only to any inactivated whole-virus FMD vaccine but also to vaccine quality testing in general and allows for better decision-making for vaccines with an unknown composition. Full article

Background: equine sarcoids are the most frequent skin tumors in equidae worldwide. It is well known that delta bovine papillomaviruses are their causative agents. We have recently shown the presence in equine sarcoids of abnormal vessel structures, which could cause a hypoxic condition. The aim of this study was to analyze the expression of hypoxia-inducible factor-1 alpha (HIF-1α) in a subset of BPV positive equine sarcoids and explore the relationship with vascular endothelial growth factor (VEGF) expression. Results: 80% of equine sarcoids showed strong cytoplasmic staining in >60% of neoplastic fibroblasts, while 20% of samples showed a moderate cytoplasmic staining in 40–60% of neoplastic fibroblasts for HIF-1α. Results of Western blotting (WB) were consistent with immunohistochemistry (IHC). Moreover, a positive correlation between HIF-1α and VEGF expression (r = 0.60, p < 0.01) was observed. Conclusion: we have shown that HIF-1α was strongly expressed in equine sarcoid. The upregulation of HIF-1α has been described in numerous tumors and can be modulated by many proteins encoded by transforming viruses. Thus, it is also possible that BPV could have a relevant role in HIF-1α pathway regulation, contributing to the development of equine sarcoids by promoting HIF-1α/VEGF mediated tumor angiogenesis. Full article

Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and Salmonella pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the cas3 deletion mutant (Δcas3). The bacterial invasive and intracellular capacity of Δcas3 to host cells was also reduced, thereby increasing the survival of infected host cells and live chickens. By the transcriptome-wide screen (RNA-Seq), we found that the cas3 gene impacts a series of genes related to QS, the flagellum, and SPI-1-T3SS system, thereby altering the virulence phenotypes. As QS SPI-1-T3SS and CRISPR-Cas systems are widely distributed in the bacteria kingdom, our findings extend our understanding of virulence regulation and pathogenicity in mammalian hosts for Salmonella and potentially other bacteria. Full article