Infected Cultures Research Articles

Reduced treatment frequencies with bumped kinase inhibitor 1369 are effective against porcine cystoisosporosis

Bumped kinase inhibitors (BKIs) are a new class of antiprotozoal drugs that target calcium-dependent protein kinase 1 (CDPK1) in various apicomplexan parasites. A multiple dose regimen of BKI 1369 has been shown to be highly effective against Cystoisospora suis (syn. Isospora suis), the causative agent of neonatal porcine coccidiosis. However, multiple dosing may not be widely applicable in the field. The present study aimed to determine the efficacy of reduced treatment frequencies with BKI 1369 against porcine cystoisosporosis in vitro and in vivo. Pre-incubation of sporozoites with BKI 1369 completely failed to inhibit the infection in vitro unless treatment was prolonged post-infection. Notably, a single treatment of infected cell cultures 2 days post-infection (dpi) resulted in a significant reduction of merozoite replication. In an experimental infection model, treatment of suckling piglets experimentally infected with C. suis 2 and 4 dpi with 20 mg BKI 1369/kg body weight completely suppressed oocyst excretion. A single treatment on the day of infection or 2 dpi suppressed oocyst excretion in 50% and 82% of the piglets and reduced the quantitative excretion in those that shed oocysts by 95.2% and 98.4%, respectively. Moreover, a significant increase in body weight gain and reduced number of diarrhea days were observed in BKI 1369 treated piglets compared to the control piglets, irrespective of time points and frequencies of treatment. Given that reduced treatment frequencies with BKI 1369 are comparable in efficacy to repeated applications without any adverse effects, this could be considered as a practical therapeutic alternative against porcine cystoisosporosis.

A Comparative Study of High-Dose Colistin Administration for the Management of Multidrug-Resistant Gram-Negative Infections in the ICU

The aim of this study was to assess the clinical and microbiological efficacy and toxicity of different high-dose regimens of colistin in the ICU patients with colistin-sensitive MDR Gram-negative infections. In this prospective, open label, randomized clinical trial, patients with clinical features of infection and positive culture for MDR colistin-sensitive Gram-negative bacteria were randomly allocated to receive colistin, 3 or 9 million units every 8 and 24 hours, (groups A and B), respectively. For each dose regimen, clinical and microbiological response, the rates of nephrotoxicity, and its risk factors were analyzed. Forty-three patients were enrolled, and 35 completed the study protocol, of whom 30 (88.2%) had ventilator-associated pneumonia (VAP) and 5 (14%) had bacteremia. Although there were no statistically significant differences in the clinical or microbiological response between the study groups, the microbiological response favored the divided dose group numerically ( p = 0.40 ). Clinical response was achieved in 27 of 35 (77.1%) patients ( p = 0.999 ). Twelve (34.28%) patients developed AKI during colistin treatment, 4 and 8 in groups A and B, respectively ( p value =0.193). Significant risk factors for nephrotoxicity related to colistin were age, hyperbilirubinemia, and coadministration of other nephrotoxic agents. In multivariate regression analysis, the only independent risk factor for CMS-associated AKI was hyperbilirubinemia ( p value =0.008). Although the clinical and microbiological responses to colistin administration were not statistically different in two groups, the microbiological response favored the divided dose regimen group numerically. We did not observe any significant safety concerns with high-dose colistin administration in this population.

A Modular Microscale Granuloma Model for Immune-Microenvironment Signaling Studies in vitro.

Tuberculosis (TB) is one of the most potent infectious diseases in the world, causing more deaths than any other single infectious agent. TB infection is caused by inhalation of Mycobacterium tuberculosis (Mtb) and subsequent phagocytosis and migration into the lung tissue by innate immune cells (e.g., alveolar macrophages, neutrophils, and dendritic cells), resulting in the formation of a fused mass of immune cells known as the granuloma. Considered the pathological hallmark of TB, the granuloma is a complex microenvironment that is crucial for pathogen containment as well as pathogen survival. Disruption of the delicate granuloma microenvironment via numerous stimuli, such as variations in cytokine secretions, nutrient availability, and the makeup of immune cell population, can lead to an active infection. Herein, we present a novel in vitro model to examine the soluble factor signaling between a mycobacterial infection and its surrounding environment. Adapting a newly developed suspended microfluidic platform, known as Stacks, we established a modular microscale infection model containing human immune cells and a model mycobacterial strain that can easily integrate with different microenvironmental cues through simple spatial and temporal “stacking” of each module of the platform. We validate the establishment of suspended microscale (4 μL) infection cultures that secrete increased levels of proinflammatory factors IL-6, VEGF, and TNFα upon infection and form 3D aggregates (granuloma model) encapsulating the mycobacteria. As a proof of concept to demonstrate the capability of our platform to examine soluble factor signaling, we cocultured an in vitro angiogenesis model with the granuloma model and quantified morphology changes in endothelial structures as a result of culture conditions (P < 0.05 when comparing infected vs. uninfected coculture systems). We envision our modular in vitro granuloma model can be further expanded and adapted for studies focusing on the complex interplay between granulomatous structures and their surrounding microenvironment, as well as a complementary tool to augment in vivo signaling and mechanistic studies.

Time to positivity of acute and chronic periprosthetic joint infection cultures

IntroductionA prolonged incubation time is generally recommended for diagnosing periprosthetic joint infections (PJI). However, in literature, no distinction is made between acute and chronic infections. MethodsAll patients with a PJI that underwent surgical debridement between November 2015 and February 2019 with or without revision of the prosthesis were retrospectively evaluated. Synovial fluid, 5 intraoperative periprosthetic tissue samples, and the sonicated prosthesis were cultured. ResultsFifty-nine patients were analyzed, including 21 acute PJIs (33 isolates) and 38 chronic PJIs (46 isolates). In acute PJIs, all isolates grew within 5 days, while this took 11 days for chronic PJIs. Sonication fluid showed the shortest time to positivity (78% at day 2) for chronic PJIs, but no difference was observed for acute PJIs compared to tissue cultures. ConclusionIn contrast to cultures from chronic PJIs, acute PJIs do not need a prolonged incubation time and no clear benefit is observed for sonication.

Isolation and characterisation of an ISKNV-genotype megalocytivirus from imported angelfish Pterophyllum scalare.

Using cultures of the SKF-9 cell line, megalocytivirus AFIV-16 was isolated from imported angelfish Pterophyllum scalare held in quarantine at the Australian border. The cytopathic effect caused by isolate AFIV-16 presented as cell rounding and enlargement, but complete destruction of the infected cell cultures did not occur. The infected cells demonstrated immunocytochemical reactivity with monoclonal antibody M10, which is used for diagnosis of OIE-listed red sea bream iridoviral disease. Using electron microscopy, the virus particles, consisting of hexagonal nucleocapsids, were observed in the cytoplasm of SKF-9 cells. The replication of AFIV-16 in cultured SKF-9 cells was significantly greater at 28°C incubation than at 22 and 25°C incubation, whereas no difference in growth characteristics was observed for red sea bream iridovirus (RSIV) isolate KagYT-96 across this temperature range. Whole genome sequencing demonstrated that AFIV-16 has a 99.96% similarity to infectious spleen and kidney necrosis virus (ISKNV), the type species in the genus Megalocytivirus. AFIV-16 was classified into ISKNV genotype Clade 1 by phylogenetic analysis of the major capsid protein gene nucleotide sequence. This is the first report of whole genome sequencing of an ISKNV genotype megalocytivirus isolated from ornamental fish.

Diagnostic performance of suction drainage fluid culture for acute surgical site infection after aseptic instrumented spine surgery: a retrospective analysis of 363 cases.

Analyze the diagnostic performance of suction drainage fluid culture for acute surgical site infection, which has not been specifically reported in spine surgery patients. This was a retrospective single-center observational study including data from 363 patients who underwent aseptic instrumented spine surgery between 2015 and 2017. A suction drain was inserted in all cases. Data analyzed were patient age, gender, ASA score, indication for surgery (degenerative disease, tumor, trauma), spine level (cervical, thoracic, lumbar), procedure performed and spine level, operative time, body temperature, postoperative C-reactive protein time-curve, clinical aspect of surgical scar, bacteriology results of suction drainage fluid, and in case of revision surgery, lavage fluid. Major criteria for periprosthetic infection proposed by the Musculoskeletal Infection Society (MSIS) were accepted as the gold standard for the diagnosis of acute surgical site infection. The overall rate of surgical site infection was 6.9% (5.76% for 1- or 2-level fusion, 5.81% for 3- or 4-level fusion, and 15.6% for 5-level fusion and above). The suction drain was withdrawn on the second postoperative day in 44.1% of cases and the third day in 39.1%. The sensitivity of suction drainage fluid culture for the diagnosis of surgical site infection was 20% [95%CI 6.8-40.7%] with a 96.2% [95%CI 93.2-97.9] specificity. The diagnostic performance of suction drainage fluid culture after aseptic instrumented spine surgery for acute surgical site infection is insufficient to warrant its use in routine practice.

Microbiology of Implant-Based Breast Reconstruction Infections: A Systematic Review.

Postsurgical complications have been an increasing concern for hospitals, particularly in light of payment reform. The costs to the health care system are increasing in light of Medicare penalties for readmissions for certain conditions. Surgical site infection following implant-based breast reconstruction (IBR) remains challenging. The rates of positive cultures and isolated microorganisms in IBR are unknown. This systematic review summarizes the reported microorganisms and positive culture rates in the existing literature. A systematic review was performed using the guidelines outlined in Preferred Reporting Items for Systematic Reviews and Meta-analyses. Articles were included if breast implant infection rates and culture results were reported. Further subgroup analysis of culture positive infections was performed. A total of 25 studies were included, encompassing 25,177 IBR. Implant infections were reported in 1356 reconstructions (5.4%). Cultures were positive in 74.5% of infections. Gram-positive bacteria were the most common (68.6%), of which Staphylococcus species (51%) was the most isolated pathogen, followed by Pseudomonas. A subgroup analysis showed that early infections (63% vs 88%), radiotherapy (82% vs 93%), and acellular dermal matrix use (90% vs 100%) were associated with lower positive culture rates than their respective counterparts. Patients who received chemotherapy had higher positive culture results (94% vs 83%). Isolated microorganisms also varied among the subgroups. This systematic review outlines reported microorganisms in IBR. Staphylococcus species and Pseudomonas were the most frequently reported microorganism. Negative cultures were reported in up to 25.5% of infections. Patients with early infections, radiotherapy, and acellular dermal matrix demonstrated higher negative culture rates. This review can help guide the use of empirical antimicrobial therapy in IBR.

Dual antibiotic prevention bundle is associated with decreased surgical site infections

BackgroundGynecologic oncology surgery is associated with a wide variation in surgical site infection risk. The optimal method for infection prevention in this heterogeneous population remains uncertain.Study DesignA retrospective cohort study...

Ivacaftor or lumacaftor/ivacaftor treatment does not alter the core CF airway epithelial gene response to rhinovirus

BackgroundAberrant responses by the cystic fibrosis airway epithelium during viral infection may underly the clinical observations. Whether CFTR modulators affect antiviral responses by CF epithelia is presently unknown. We tested the hypothesis that treatment of CF epithelial cells with ivacaftor (Iva) or ivacaftor/lumacaftor (Iva/Lum) would improve control of rhinovirus infection. MethodsNineteen CF epithelial cultures (10 homozygous for p.Phe508del as CFTR Class 2, 9 p.Phe508del/p.Gly551Asp as Class 3) were infected with rhinovirus 1B at multiplicity of infection 12 for 24 h. Culture RNA and supernatants were harvested to assess gene and protein expression respectively. ResultsRNA-seq analysis comparing rhinovirus infected cultures to control identified 796 and 629 differentially expressed genes for Class 2 and Class 3, respectively. This gene response was highly conserved when cells were treated with CFTR modulators and were predicted to be driven by the same interferon-pathway transcriptional regulators (IFNA, IFNL1, IFNG, IRF7, STAT1). Direct comparisons between treated and untreated infected cultures did not yield any differentially expressed genes for Class 3 and only 68 genes for Class 2. Changes were predominantly related to regulators of lipid metabolism and inflammation, aspects of epithelial biology known to be dysregulated in CF. In addition, CFTR modulators did not affect viral copy number, or levels of pro-inflammatory cytokines produced post-infection. ConclusionsThough long-term clinical data is not yet available, results presented here suggest that first generation CFTR modulators do not interfere with core airway epithelial responses to rhinovirus infection. Future work should investigate the latest triple modulation therapies.

Visualization of Viral Infection Dynamics in a Unicellular Eukaryote and Quantification of Viral Production Using Virus Fluorescence in situ Hybridization.

One of the major challenges in viral ecology is to assess the impact of viruses in controlling the abundance of specific hosts in the environment. To this end, techniques that enable the detection and quantification of virus-host interactions at the single-cell level are essential. With this goal in mind, we implemented virus fluorescence in situ hybridization (VirusFISH) using as a model the marine picoeukaryote Ostreococcus tauri and its virus Ostreococcus tauri virus 5 (OtV5). VirusFISH allowed the visualization and quantification of the proportion of infected cells during an infection cycle in experimental conditions. We were also able to quantify the abundance of free viruses released during cell lysis, discriminating OtV5 from other mid-level fluorescence phages in our non-axenic infected culture that were not easily distinguishable with flow cytometry. Our results showed that although the major lysis of the culture occurred between 24 and 48 h after OtV5 inoculation, some new viruses were already produced between 8 and 24 h. With this work, we demonstrate that VirusFISH is a promising technique to study specific virus-host interactions in non-axenic cultures and establish a framework for its application in complex natural communities.

Evaluation of Sociodemographic Factors among Diabetic Patients with Urinary Tract Infections in Kisii Referral Hospital, Kenya.

People with noninsulin-dependent diabetes mellitus are prone to urinary tract infections. There is a wide gap of information in developing countries regarding the sociodemographic factors linked to UTI among diabetics and the gender disparity among the same. Sociodemographic factors differ with geographical location and many other factors, and this makes them an important aspect that can influence the social burden of UTI among diabetics. The objective of this study was to determine the association between sociodemographic factors and UTI among diabetics. The study was carried out in the Kisii Teaching and Referral Hospital in Kenya. One hundred and eighty diabetic patients were enrolled in cross-sectional study design. Clean-catch midstream urine was collected from all participants and cultured in cysteine lactose electrolyte deficient agar for bacterial isolation. Classification of a positive culture for urinary tract infection was based on more than 100,000 (≥105) colony-forming units of a single bacterial species. The data were analyzed using frequencies, chi-square (p < 0.05), and logic regression with the help of the Statistical Package for the Social Sciences (SPSS) version 20 to find the odds ratio. One hundred and seven participants were male (59.4%), and 73 (40.6%) were female. The majority of the participants were between the age of 55 and 59 years old (77.2%), and 125 participants (69.4%) had attained tertiary education as the highest level of education. The overall prevalence of urinary tract infections was 20.6% with 37 participants testing positive for urinary tract infection. Age was found to have a significant association with urinary tract infection (p=0.002) while gender (p=0.45) and level of education (p=0.11) showed no significant association with urinary tract infections among diabetic patients. These findings suggest that age was the biggest association factor that influenced urinary tract infections among diabetic patients.

Heat Inactivation of Different Types of SARS-CoV-2 Samples: What Protocols for Biosafety, Molecular Detection and Serological Diagnostics?

Standard precautions to minimize the risk of SARS-CoV-2 transmission implies that infected cell cultures and clinical specimens may undergo some sort of inactivation to reduce or abolish infectivity. We evaluated three heat inactivation protocols (56 °C-30 min, 60 °C-60 min and 92 °C-15 min) on SARS-CoV-2 using (i) infected cell culture supernatant, (ii) virus-spiked human sera (iii) and nasopharyngeal samples according to the recommendations of the European norm NF EN 14476-A2. Regardless of the protocol and the type of samples, a 4 Log10 TCID50 reduction was observed. However, samples containing viral loads > 6 Log10 TCID50 were still infectious after 56 °C-30 min and 60 °C-60 min, although infectivity was < 10 TCID50. The protocols 56 °C-30 min and 60 °C-60 min had little influence on the RNA copies detection, whereas 92 °C-15 min drastically reduced the limit of detection, which suggests that this protocol should be avoided for inactivation ahead of molecular diagnostics. Lastly, 56 °C-30 min treatment of serum specimens had a negligible influence on the results of IgG detection using a commercial ELISA test, whereas a drastic decrease in neutralizing titers was observed.

Clinical Utilization of Blood and Urine Cultures and Incidences of Bacteremia and Bacteriuria in a Hospital in Thailand.

BackgroundTo effectively treat sepsis and urinary tract infection (UTI), blood and urine cultures should be used appropriately and relative to incidences of bacteremia and bacteriuria. This study aimed to investigate the use of blood and urine cultures and incidences of bacteremia and bacteriuria in a hospital in Thailand.Material/MethodsMedical records of patients admitted from 2016 to 2018 were randomly selected and data in the records were anonymously extracted for investigation.ResultsFrom 12 000 records, data on blood and urine cultures were extracted from 9% and 4% of them, respectively. The negative rate of blood culture was 87.48%. Bacteremia was detected in 10.22%. The positive rate of urine culture was 27.38% and the contamination rate was 31.26%. Escherichia coli was the most common cause of community-onset bacteremia and bacteriuria. Methicillin-resistant coagulase-negative staphylococci and Acinetobacter baumannii were the most common cause of hospital-acquired bacteremia while yeasts were the most common cause of hospital-acquired UTI.ConclusionsA high negative rate of blood culture may result not only from its low sensitivity but also from liberal test use to identify sepsis in some conditions. Improper urine collection is the main problem with use of urine culture.

FREQUENCY AND ANTIMICROBIAL RESISTANCE PATTERNS OF BACTERIA ISOLATED FROM ASPIRATION SPECIMENS OF PATIENTS WITH HIP AND KNEE INFECTIONS AT A HOSPITAL IN BAGHDAD

The present study was carried out to determine the frequency of bacteria responsible for hip and knee infections at a hospital in Baghdad/Iraq and to get an updated knowledge about their antibiotic resistance pattern during a period of study which extended from 1 October, 2016 till 1 October, 2017. Identification of bacteria at species level was done by using Analytic Profile Index (API) system (Bio-Merieux, France). The antimicrobial susceptibility test was performed according to Kirby-Bauer (disk diffusion) technique. The frequency of positive aspiration of hip and knee joint infections cultures was 25(65.79%) cases out of 38 hip and knee joint aspiration. Out of 25 positive aspirations of hip and knee joint infections cultures, 3(12%) cultures gave mix growth of bacteria. Frequency of positive hip and knee joint infections cultures was higher in male than female patients, 19(76%) vs. 6(24%) (P-value ≤ 0.05).&#x0D; Out of 28 isolated bacteria, Gram positive was the most frequently isolated bacteria in hip and knee joint infection cultures, 22(78.6%) vs. 6(21.4%) (P-value &lt; 0.05). Staphylococcus epidermidis was the most frequently isolated 13(46.5%) isolates followed by Staphylococcus aureus isolates 7(25%). On other hand Pseudomonas aeruginosa was the most frequently isolated Gram negative bacteria 3(10.7 %%) in hip and knee joint infection cultures.&#x0D; The most effective antibiotic on Gram-positive isolates was Vancomycin which showed 95.5%. In Gram negative bacteria the most effective antibiotics were Imipenem (100%) followed by Amikacin which showed sensitivity rate 83.33.

Imaging mass spectrometry of interspecies metabolic exchange revealed the allelopathic interaction between Microcystis aeruginosa and its antagonist

The frequent outbreaks of cyanobacterial blooms which caused serious societal and economic loss have become a worldwide problem. Interactions between toxic cyanobacteria and heterotrophic bacteria competitors play a pivotal role in the formation of toxic cyanobacterial bloom, but the underlying mechanisms of interactions between them await further research. The antagonist activity of Pseudomonas grimontii (P.grimontii) was confirmed by reduction in chlorophyll a concentration of Microcystis aeruginosa (M. aeruginosa) in an infected culture for a 7d period. The initial concentration of P.grimontii affected the M. aeruginosa activity significantly. When the 10% (V/V) concentration of P.grimontii A01 and P.grimontii A14 cultures were infected, the reduction of M. aeruginosa reached to 91.81% and 78.25% after 7 days, respectively. While a 0.1% (v/v) concentration of P.grimontii A01 and P.grimontii A14 cultures were infected, the M. aeruginosa increased 31.13% and 16.67% occurred, respectively. The content of reactive oxygen species (ROS) and malondialdehyde (MDA) increased with increasing of P.grimontii fermentation liquid, indicating the M. aeruginosa underwent oxidative stress. Using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry (IMS) profiling of co-cultures of M. aeruginosa and its antagonist P.grimontii, we revealed novel interspecies allelopathic interactions and compete molecule. We showed the spatial secondary metabolites may mediate the interactions in which P.grimontii inhibits growth of M. aeruginosa. Additionally, we revealed how M. aeruginosa feedback to the P.grimontii, which stimulates secondary metabolites such as [D-Asp3]-microcystin-LR released by M. aeruginosa. IMS method highlights the significance of allelopathic interactions between a widely distributed toxic cyanobacteria and its bacteria competitors.

Development of BacMam Induced Hepatitis E Virus Replication Model in Hepatoma Cells to Study the Polyprotein Processing.

The processing of polyprotein(s) to form structural and non-structural components remains an enigma due to the non-existence of an efficient and robust Hepatitis E Virus (HEV) culture system. We used the BacMam approach to construct an HEV replication model in which the HEV genome was cloned in the BacMam vector under the CMV promoter. The recombinant BacMam was used to infect Huh7 cells to transfer the HEV genome. HEV replication was authenticated by the presence of RNAs of both the polarity (+) and (−) and formation of hybrid RNA, a replication intermediate. The presence of genes for Papain-like Cysteine Protease (PCP), methyltransferase (MeT), RNA dependent RNA polymerase (RdRp), and ORF2 was confirmed by PCR amplification. Further, the infectious nature of the culture system was established as evidenced by the cross-infection of uninfected cells using the cell lysate from the infected cells. The HEV replication model was validated by detection of the ORF1 (Open Reading Frame1) encoded proteins, identified by Western blotting and Immunofluorescence by using epitope-specific antibodies against each protein. Consequently, discrete bands of 18, 35, 37, and 56 kDa corresponding to PCP, MeT, RdRp, and ORF2, respectively, were seen. Besides demonstrating the presence of non-structural enzymes of HEV along with ORF2, activity of a key enzyme, HEV-methyltransferase has also been observed. A 20% decrease in the replicative forms of RNA could be seen in presence of 100 μM Ribavirin after 48 h of treatment. The inhibition gradually increased from 0 to 24 to 48 h post-treatment. Summarily, infectious HEV culture system has been established, which could demonstrate the presence of HEV replicative RNA forms, the structural and non-structural proteins and the methyltransferase in its active form. The system may also be used to study the mechanism of action of Ribavirin in inhibiting HEV replication and develop a therapy.

The hyperpolarization-activated cyclic nucleotide-gated 4 channel as a potential anti-seizure drug target.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by four genes (HCN1-4) with distinct biophysical properties and functions within the brain. HCN4 channels activate slowly at robust hyperpolarizing potentials, making them more likely to be engaged during hyperexcitable neuronal network activity seen during seizures. HCN4 channels are also highly expressed in thalamic nuclei, a brain region implicated in seizure generalization. Here, we assessed the utility of targeting the HCN4 channel as an anti-seizure strategy using pharmacological and genetic approaches. The impact of reducing HCN4 channel function on seizure susceptibility and neuronal network excitability was studied using an HCN4 channel preferring blocker (EC18) and a conditional brain specific HCN4 knockout mouse model. EC18 (10 mg·kg-1 ) and brain-specific HCN4 channel knockout reduced seizure susceptibility and proconvulsant-mediated cortical spiking recorded using electrocorticography, with minimal effects on other mouse behaviours. EC18 (10 μM) decreased neuronal network bursting in mouse cortical cultures. Importantly, EC18 was not protective against proconvulsant-mediated seizures in the conditional HCN4 channel knockout mouse and did not reduce bursting behaviour in AAV-HCN4 shRNA infected mouse cortical cultures. These data suggest the HCN4 channel as a potential pharmacologically relevant target for anti-seizure drugs that is likely to have a low side-effect liability in the CNS.

Development and use of indirect liquid-phase ELISA test system for detection of PRRS virus antigen during in-process control of raw materials intended for vaccine production

Porcine reproductive and respiratory syndrome (PRRS) being endemic and reported in the most countries in the world remains one of the most challenging diseases in pig industry. The main disease control measures include preventive vaccination and animal movement control within and outside the country as well as diagnostic testing of pigs in the population. Live and inactivated vaccines are used for specific prevention of porcine reproductive and respiratory syndrome. Complete and irreversible infectious agent inactivation with maximum epitope preservation and protective immunity in immunized animals are the main requirements for inactivated vaccines. Therefore, continuous improvement of methods for vaccine quality control at various vaccine production stages is of current importance. Results of development of the test system based on indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) for PRRS virus antigen detection and activity testing in infectious and inactivated virus-containing cell cultures at intermediate stages of the vaccine production process are described in the paper. The test-system development process included purified and concentrated virus antigen as well as hyperimmune rabbit sera preparation. Specificity of purified and concentrated virus antigen was confirmed with real-time polymerase chain reaction. The developed test-system was shown to detect the virus antigen at initial infectivity titre of 4.87–7.21 lg TCID50/cm3 corresponding to ELISA titre (dilution) of 1:4 up to 1:64. Methodical Guidelines for detection of porcine reproductive and respiratory syndrome virus antigen with indirect liquid-phase enzyme-linked immunosorbent assay (ELISA) (2019) were developed based on the work results, commissioned and approved by the FGBI “ARRIAH” Scientific Board.

The Andes Orthohantavirus NSs Protein Antagonizes the Type I Interferon Response by Inhibiting MAVS Signaling.

The small messenger RNA (SmRNA) of the Andes orthohantavirus (ANDV), a rodent-borne member of the Hantaviridae family of viruses of the Bunyavirales order, encodes a multifunctional nucleocapsid (N) protein and for a nonstructural (NSs) protein of unknown function. We have previously shown the expression of the ANDV-NSs, but only in infected cell cultures. In this study, we extend our early findings by confirming the expression of the ANDV-NSs protein in the lungs of experimentally infected golden Syrian hamsters. Next, we show, using a virus-free system, that the ANDV-NSs protein antagonizes the type I interferon (IFN) induction pathway by suppressing signals downstream of the melanoma differentiation-associated protein 5 (MDA5) and the retinoic acid-inducible gene 1 (RIG-I) and upstream of TBK1. Consistent with this observation, the ANDV-NSs protein antagonized mitochondrial antiviral-signaling protein (MAVS)-induced IFN-β, NF-κB, IFN-regulatory factor 3 (IRF3), and IFN-sensitive response element (ISRE) promoter activity. Results demonstrate that ANDV-NSs binds to MAVS in cells without disrupting the MAVS-TBK-1 interaction. However, in the presence of the ANDV-NSs ubiquitination of MAVS is reduced. In summary, this study provides evidence showing that the ANDV-NSs protein acts as an antagonist of the cellular innate immune system by suppressing MAVS downstream signaling by a yet not fully understand mechanism. Our findings reveal new insights into the molecular regulation of the hosts' innate immune response by the Andes orthohantavirus.IMPORTANCEAndes orthohantavirus (ANDV) is endemic in Argentina and Chile and is the primary etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in South America. ANDV is distinguished from other hantaviruses by its unique ability to spread from person to person. In a previous report, we identified a novel ANDV protein, ANDV-NSs. Until now, ANDV-NSs had no known function. In this new study, we established that ANDV-NSs acts as an antagonist of cellular innate immunity, the first line of defense against invading pathogens, hindering the cellular antiviral response during infection. This study provides novel insights into the mechanisms used by ANDV to establish its infection.

Infection of filamentous phytoplankton by fungal parasites enhances herbivory in pelagic food webs

AbstractChytrid fungal parasites are ubiquitous in aquatic ecosystems and infect a wide array of aquatic organisms, including all phytoplankton groups. In addition to their role as parasites, chytrids serve as food to zooplankton, thereby establishing an alternative trophic link between primary and secondary production in pelagic food webs, the so‐called mycoloop. We hypothesized that, in addition to the mycoloop, chytrid infection facilitates grazing of filamentous phytoplankton by rendering it more edible to zooplankton consumers through infection‐induced fragmentation. We undertook grazing assays to compare the ability of the key zooplankter Daphnia to graze on a filamentous cyanobacterium in the presence or absence of chytrid infection. A near doubling in mean clearance rates was consistently recorded when Daphnia were fed with infected cultures of the cyanobacterium as compared to uninfected ones. Infected filaments were shorter than noninfected ones, indicating that infection‐induced fragmentation undermines resistance of filamentous phytoplankton to grazing. We propose an extended conceptualization of the mycoloop that includes both direct effects (i.e., transfer via grazing of chytrid zoospores) and indirect effects (i.e., trophic upgrading and facilitated grazing on phytoplankton via fragmentation) of chytrid infection on trophic transfer at the base of pelagic food webs.