Experimental challenge of flatfishes (Pleuronectidae) with salmonid alphavirus (SAV): Observations on tissue tropism and pathology in common dab Limanda limanda L.Abstract
Salmonid alphavirus (SAV) is the aetiological agent of pancreas disease (PD), a serious viral disease in salmonids. For several decades, SAV was known to infect salmonid species only, until SAV was detected using real-time PCR in several species of wild-caught flatfishes in Scotland in 2010. The presence of SAV in wild flatfishes has been confirmed by further surveys from Ireland and Scotland. The role of flatfishes in SAV-spread and epizootiology has not been elucidated, and no experimental challenges have been conducted to examine virus tissue tropism, virulence and pathology in flatfishes. Wild-caught flatfishes (common dab; Limanda limanda, European plaice; Pleuronectes platessa, European flounder; Platichthys flesus and lemon sole; Microstomus kitt) were either intramuscularly (i.m.) or intraperitoneally (i.p.) challenged with SAV3 or exposed to SAV3 through cohabitation with i.p. injected salmon. SAV-infections were seen in i.m. and i.p. injected dab and i.p. injected salmon but did not result in a transmissible infection in dab although several routes of entry were assessed (oral route not tested). SAV was detected in several tissues of eight common dab (not from cohabitants), with high SAV-levels in pancreas. No viraemia was detected in the SAV-positive common dab and no virus shedding were detected in the tanks. However, pathology in exocrine pancreas and hearts consistent with SAV-replication were seen. This is the first study reporting SAV-induced pathology in a non-salmonid species. The results from the present challenge study supports evidence for common dab being susceptible hosts for SAV. The study also demonstrates that flatfishes are less susceptible to SAV3-infection than salmon.
Characterization of Winter Dysentery Bovine Coronavirus Isolated from Cattle in IsraelAbstract
Bovine coronavirus (BCoV) is the causative agent of winter dysentery (WD). In adult dairy cattle, WD is characterized by hemorrhagic diarrhea and a reduction in milk production. Therefore, WD leads to significant economic losses in dairy farms. In this study, we aimed to isolate and characterize local BCoV strains. BCoV positive samples, collected during 2017–2021, were used to amplify and sequence the S1 domain of S glycoprotein and the full hemagglutinin esterase gene. Based on our molecular analysis, local strains belong to different genetic variants circulating in dairy farms in Israel. Phylogenetic analysis revealed that all local strains clustered together and in proximity to other BCoV circulating in the area. Additionally, we found that local strains are genetically distant from the reference enteric strain Mebus. To our knowledge, this is the first report providing molecular data on BCoV circulating in Israel.
Crystal digital RT-PCR for the detection and quantification of norovirus and hepatitis A virus RNA in frozen raspberriesAbstract
Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15–145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.
CHIKV infection reprograms codon optimality to favor viral RNA translation by altering the tRNA epitranscriptomeAbstract
Ample evidence indicates that codon usage bias regulates gene expression. How viruses, such as the emerging mosquito-borne Chikungunya virus (CHIKV), express their genomes at high levels despite an enrichment in rare codons remains a puzzling question. Using ribosome footprinting, we analyze translational changes that occur upon CHIKV infection. We show that CHIKV infection induces codon-specific reprogramming of the host translation machinery to favor the translation of viral RNA genomes over host mRNAs with an otherwise optimal codon usage. This reprogramming was mostly apparent at the endoplasmic reticulum, where CHIKV RNAs show high ribosome occupancy. Mechanistically, it involves CHIKV-induced overexpression of KIAA1456, an enzyme that modifies the wobble U34 position in the anticodon of tRNAs, which is required for proper decoding of codons that are highly enriched in CHIKV RNAs. Our findings demonstrate an unprecedented interplay of viruses with the host tRNA epitranscriptome to adapt the host translation machinery to viral production.
Monitoring SARS-CoV-2 RNA in Wastewater with RT-qPCR and Chip-Based RT-dPCR: Sewershed-Level Trends and Relationships to COVID-19Abstract
We evaluated the performance of reverse transcription quantitative PCR (uniplex and duplex RT-qPCR) and chip-based digital PCR (duplex RT-dPCR) using CDC N1 and CDC N2 assays for longitudinal monitoring of SARS-CoV-2 RNA in influent wastewater samples (n = 281) from three wastewater plants in Ohio from January 2021 to January 2022. Human fecal virus (PMMoV) and wastewater flow rate were used to normalize SARS-CoV-2 concentrations. SARS-CoV-2 measurements and COVID-19 cases were strongly correlated, but normalization effects on correlations varied between sewersheds. SARS-CoV-2 measurements by RT-qPCR were strongly correlated with 7-day moving average COVID-19 cases (average Spearman’s ρ = 0.58, p < 0.05). SARS-CoV-2 was detected more frequently in samples with duplex RT-dPCR than with duplex RT-qPCR during periods of low COVID-19 cases. Duplex and uniplex RT-qPCR N1 concentrations were more strongly correlated with cases (ρ = 0.62) than N2 (ρ = 0.52). RT-dPCR correlations (average ρ = 0.21) were weaker than those of RT-qPCR (average ρ = 0.58). We also share practical experience from establishing wastewater surveillance. Per sample, RT-qPCR had a lower cost ($6 vs $18) and sample turnaround time (3–4 h vs 7–9 h) than RT-dPCR. These findings reinforce selection and use of PCR-based wastewater surveillance tools.
Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse Transcription-qPCRAbstract
RNA interference (RNAi) is a powerful therapeutic approach for messenger RNA (mRNA) level regulation in human cells. RNAi can be triggered by small interfering RNAs (siRNAs) which are delivered by non-viral carriers, e.g., dendriplexes. siRNA quantification inside carriers is essential in drug delivery system development. However, current siRNA measuring methods either are not very sensitive, only semi-quantitative or not specific towards intact target siRNA sequences. We present a novel reverse transcription real-time PCR (RT-qPCR)-based application for siRNA quantification in drug formulations. It enables specific and highly sensitive quantification of released, uncomplexed target siRNA and thus also indirect assessment of siRNA stability and concentration inside dendriplexes. We show that comparison with a dilution series allows for siRNA quantification, exclusively measuring intact target sequences. The limit of detection (LOD) was 4.2 pM (±0.2 pM) and the limit of quantification (LOQ) 77.8 pM (±13.4 pM) for uncomplexed siRNA. LOD and LOQ of dendriplex samples were 31.6 pM (±0 pM) and 44.4 pM (±9.0 pM), respectively. Unspecific non-target siRNA sequences did not decrease quantification accuracy when present in samples. As an example of use, we assessed siRNA complexation inside dendriplexes with varying nitrogen-to-phosphate ratios. Further, protection of siRNA inside dendriplexes from RNase A degradation was quantitatively compared to degradation of uncomplexed siRNA. This novel application for quantification of siRNA in drug delivery systems is an important tool for the development of new siRNA-based drugs and quality checks including drug stability measurements.
Echovirus 9 genetic diversity detected in whole-capsid genome sequences obtained directly from clinical specimens using next generation sequencingAbstract
Echovirus 9 (E9) has been detected in an increased number of symptomatic patient samples received by the National Enterovirus Reference Laboratory in Hungary during 2018 compared to previously reported years.
Formerly identified E9 viruses from different specimen types detected from patients of various ages and showing differing clinical signs were chosen for the detailed analysis of genetic relationships and potential variations within the viral populations. We used next generation sequencing (NGS) analysis of 3,900 nucleotide long amplicons covering the entire capsid coding region of the viral genome without isolation, directly from clinical samples.
Compared to the E9 reference strain, the viruses showed about 79% nucleotide and around 93% amino acid sequence similarity. The four new viral genome sequences had 1-20 nucleotide differences between them also resulting in 6 amino acid variances in the coding region, including 3 in the structural VP1 capsid protein. One virus from a patient with hand, foot, and mouth disease had two amino acid changes in the VP1 capsid protein. An amino acid difference was also detected in the non-structural 2C gene of one virus sequenced from a throat swab sample from a patient with meningitis, compared to the faecal specimen taken two days later. Two amino acid changes, one in the capsid protein, were found between faecal samples of meningitis patients of different ages.
Sequencing the whole capsid genome revealed several nucleotide and amino acid differences between E9 virus strains detected in Hungary in 2018.
A fast extraction-free isothermal LAMP assay for detection of SARS-CoV-2 with potential use in resource-limited settingsAbstract
To retain the spread of SARS-CoV-2, fast, sensitive and cost-effective testing is essential, particularly in resource limited settings (RLS). Current standard nucleic acid-based RT-PCR assays, although highly sensitive and specific, require transportation of samples to specialised laboratories, trained staff and expensive reagents. The latter are often not readily available in low- and middle-income countries and this may significantly impact on the successful disease management in these settings. Various studies have suggested a SARS-CoV-2 loop mediated isothermal amplification (LAMP) assay as an alternative method to RT-PCR.
Four previously published primer pairs were used for detection of SARS-CoV-2 in the LAMP assay. To determine optimal conditions, different temperatures, sample input and incubation times were tested. Ninety-three extracted RNA samples from St. George's Hospital, London, 10 non-extracted nasopharyngeal swab samples from Great Ormond Street Hospital for Children, London, and 92 non-extracted samples from Queen Elisabeth Central Hospital (QECH), Malawi, which have previously been tested for SARS-Cov-2 by quantitative reverse-transcription RealTime PCR (qRT-PCR), were analysed in the LAMP assay.
In this study we report the optimisation of an extraction-free colourimetric SARS-CoV-2 LAMP assay and demonstrated that a lower limit of detection (LOD) between 10 and 100 copies/µL of SARS-CoV-2 could be readily detected by a colour change of the reaction within as little as 30 min. We further show that this assay could be quickly established in Malawi, as no expensive equipment is necessary. We tested 92 clinical samples from QECH and showed the sensitivity and specificity of the assay to be 86.7% and 98.4%, respectively. Some viral transport media, used routinely to stabilise RNA in clinical samples during transportation, caused a non-specific colour-change in the LAMP reaction and therefore we suggest collecting samples in phosphate buffered saline (which did not affect the colour) as the assay allows immediate sample analysis on-site.
SARS-CoV-2 LAMP is a cheap and reliable assay that can be readily employed in RLS to improve disease monitoring and management.
SARS-CoV-2 circulation in Croatian wastewaters and the absence of SARS-CoV-2 in bivalve molluscan shellfishAbstract
The circulation of SARS-CoV-2 in the environment has been confirmed numerous times, whilst research on the bioaccumulation in bivalve molluscan shellfish (BMS) has been rather scarce. The present study aimed to fulfil the knowledge gap on SARS-CoV-2 circulation in wastewaters and surface waters in this region and to extend the current knowledge on potential presence of SARS-CoV-2 contamination in BMS. The study included 13 archive wastewater and surface water samples from the start of epidemic and 17 influents and effluents from nine wastewater treatment plants (WWTP) of different capacity and treatment stage, sampled during the second epidemic wave. From that period are the most of 77 collected BMS samples, represented by mussels, oysters and warty venus clams harvested along the Dalmatian coast. All samples were processed according to EN ISO 15216-1 2017 using Mengovirus as a whole process control. SARS-CoV-2 detection was performed by real-time and conventional RT-PCR assays targeting E, N and nsp14 protein genes complemented with nsp14 partial sequencing. Rotavirus A (RVA) real-time RT-PCR assay was implemented as an additional evaluation criterion of virus concentration techniques. The results revealed the circulation of SARS-CoV-2 in nine influents and two secondary treatment effluents from eight WWTPs, while all samples from the start of epidemic (wastewaters, surface waters) were negative which was influenced by sampling strategy. All tertiary effluents and BMS were SARS-CoV-2 negative. The results of RVA amplification were beneficial in evaluating virus concentration techniques and provided insights into RVA dynamics within the environment and community. In conclusion, the results of the present study confirm SARS-CoV-2 circulation in Croatian wastewaters during the second epidemic wave while extending the knowledge on wastewater treatment potential in SARS-CoV-2 removal. Our findings represent a significant contribution to the current state of knowledge that considers BMS of a very low food safety risk regarding SARS-CoV-2.
Point-of-Care Platform for Rapid Multiplexed Detection of SARS-CoV-2 Variants and Respiratory PathogensAbstract
The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses to end the pandemic. Widespread detection of variants is critical to inform policy decisions to mitigate further spread, and postpandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, a portable, magnetofluidic cartridge platform for automated polymerase chain reaction testing in <30 min is developed. Cartridges are designed for multiplexed detection of SARS-CoV-2 with either identification of variant mutations or screening for Influenza A and B. Moreover, the platform can perform identification of B.1.1.7 and B.1.351 variants and the multiplexed SARS-CoV-2/Influenza assay using archived clinical nasopharyngeal swab eluates and saliva samples. This work illustrates a path toward affordable and immediate testing with potential to aid surveillance of viral variants and inform patient treatment.
A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian BornavirusesAbstract
Avian bornaviruses were first described in 2008 as the causative agents of proventricular
dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly
diverse avian bornaviruses covering at least five viral species have been discovered in different
bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional
or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays,
allowing the detection of one particular virus, or of assays with a broad detection spectrum, which,
however, do not allow for the simultaneous specification of the detected virus. To facilitate the
simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay
was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were
designed to recognize conserved regions within the overlapping X/P gene and probes were selected
to detect virus species-specific regions within the target region. The optimization of the assay resulted
in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic
birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field
samples from various avian species. This assay w
Innate and Adaptive Immune Genes Associated with MERS-CoV Infection in DromedariesAbstract
The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses, only eight years after the emergence of another zoonotic betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I (HLA-A-24-like) and II (HLA-DPB1-like), and innate immune response (PTPN4, MAGOHB), and in cilia coating the respiratory tract (DNAH7). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses, although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.
Correlation of SARS-CoV-2 RNA in wastewater with COVID-19 disease burden in sewershedsAbstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the coronavirus disease
(COVID-19), is shed in feces and the viral ribonucleic acid (RNA) is detectable inwastewater. A nine-weekwastewater
epidemiology study of tenwastewater facilities, serving 39% of the state of Utah or 1.26Mindividuals was
conducted in April andMay of 2020. COVID-19 cases were tabulated fromwithin each sewershed boundary. RNA
from SARS-CoV-2 was detectable in 61% of 126 wastewater samples. Urban sewersheds serving >100,000 individuals
and tourist communities had higher detection frequencies. An outbreak of COVID-19 across two communities
positively correlated with an increase in wastewater SARS-CoV-2 RNA, while a decline in COVID-19 cases
preceded a decline in RNA. SARS-CoV-2 RNA followed a first order decay rate in wastewater, while 90% of the
RNAwas present in the liquid phase of the influent. Infiltration and inflow, virus decay and sewershed characteristics
should be considered during correlation analysis of SAR-CoV-2 with COVID-19 cases. These results provide
evidence of the utility of wastewater epidemiology to assist in public health responses to COVID-19.
COVID-19 infection among emergency department healthcare providers in a large tertiary academic medical center following the peak of the pandemicAbstract
The COVID-19 pandemic has spread through the US during the last few months exposing healthcare providers to possible infection. Here we report testing of emergency department (ED) healthcare providers (HCP) for exposure to COVID-19 through lateral flow point of care (POC) and lab-based enzyme-linked immunosorbent assay (ELISA), and RTq-PCR for evidence of acute infection.
138 ED HCP were tested between May 26th (approximately one month after the peak of COVID-19 first wave of cases) and June 14th. Enrolled ED HCP represented about 70% of the total ED HCP workforce during the study period. Subjects were tested with a POC COVID-19 antibody test, and standard ELISA performed by a university-based research lab. Subjects also provided a mid-turbinate swab and a saliva specimen for RTq-PCR. All subjects provided demographic information, past medical history, information about personal protective equipment (PPE) use, COVID-19 symptoms, as well as potential COVID-19 exposures during the previous 4 weeks, both in the ED, and outside the clinical setting.
None of the HCP had positive RT-PCR results; 7 HCP (5%) had positive IgG for COVID-19; there was strong agreement between the lab-based ELISA (reference test) and the POC Ab test (P ≤ 0.0001). For the POC Ab test there were no false negatives and only one false positive among the 138 participants. There was no significant difference in demographic/ethnic variables, past medical history, hours worked in the ED, PPE use, or concerning exposures between seropositive and seronegative individuals. Moreover, there was no significant difference in reported symptoms between the two groups during the previous four weeks.
The rate of COVID-19 seroconversion in our ED was 5% during the month following the pandemic's first wave. Based on questionnaire responses, differences in demographics/ethnicity, medical history, COVID-19 exposures, and PPE use were not associated with ED HCP having been infected with SARS-CoV-2. In the setting of our limited cohort of subjects the COVID-19 POC Ab test performed comparably to the ELISA lab-based standard.
Investigation of pooling strategies using clinical COVID-19 samples for more efficient diagnostic testingAbstract
When testing large numbers of clinical COVID-19 samples for diagnostic purposes, pooling samples together for processing can offer significant reductions in the materials, reagents, time, and labor needed. We have evaluated two different strategies for pooling independent nasopharyngeal swab samples prior to testing with an EUA-approved SARS-CoV-2 RT-qPCR diagnostic assay. First, in the Dilution Study, we assessed the assay's ability to detect a single positive clinical sample diluted in multiple negative samples before the viral RNA extraction stage. We observed that positive samples with Ct values at ~30 can be reliably detected in pools of up to 30 independent samples, and positive samples with Ct values at ~35 can be detected in pools of 5 samples. Second, in the Reloading Study, we assessed the efficacy of reloading QIAamp viral RNA extraction columns numerous times using a single positive sample and multiple negative samples. We determined that one RNA extraction column can be reloaded with up to 20 clinical samples (1 positive and 19 negatives) sequentially without any loss of signal in the diagnostic assay. Furthermore, we found there was no significant difference in assay readout whether the positive sample was loaded first or last in a series of 20 samples. These results demonstrate that different pooling strategies can lead to increased process efficiencies for COVID-19 clinical diagnostic testing.
Antibody Screening Results for Anti-Nucleocapsid Antibodies Towards the Development of a SARS-CoV-2 Nucleocapsid Protein Antigen Detecting Lateral Flow AssayAbstract
The global COVID-19 pandemic has created an urgent demand for accurate rapid point of care diagnostic tests. Antigen-based assays are suitably inexpensive and can be rapidly mass-produced, but sufficiently accurate performance requires highly optimized antibodies and assay conditions. An automated liquid handling system,
customized to handle lateral flow immunoassay (LFA) arrays, was used for high-throughput antibody screening of anti-nucleocapsid antibodies that will perform optimally on an LFA. Six hundred seventy-three anti-nucleocapsid
antibody pairs were tested as both capture and detection reagents with the goal of finding those pairs that have the greatest affinity for unique epitopes of the nucleocapsid protein of SARS-CoV-2 while also performing
optimally in an LFA format. In contrast to traditional antibody screening methods (e.g. ELISA, bio-layer interferometry), the methods described here integrate real-time LFA reaction kinetics and binding directly on
nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2.
Multiplexed and extraction-free amplification for simplified SARS-CoV-2 RT-PCR testsAbstract
The rapid onset of the global COVID-19 pandemic has led to multiple challenges for
accurately diagnosing the infection. One of the main bottlenecks for COVID-19 detection is reagent and material shortages for sample collection, preservation, and purification prior to testing. Currently, most authorized diagnostic tests require RNA extraction from patient samples and detection by reverse transcription polymerase chain reaction (RT-PCR). However, RNA purification is expensive, time consuming, and requires technical expertise to perform. Additionally, there have been reported shortages of the RNA purification kits needed for most tests. With these challenges in mind, we report on extraction-free amplification of SARS-CoV-2 RNA directly from patient samples. In addition, we have developed a multiplex RT-PCR using the CDC singleplex targets. This multiplex has a limit of detection of 2 copies/µL. We have demonstrated these improvements to the current diagnostic workflow, which reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.
An efficient, reproducible and accurate RT-qPCR based method to determine mumps specific neutralizing antibodyAbstract
A resurgence of mumps among fully vaccinated adolescents and young adults globally has led to questions about the longevity of vaccine derived specific immunity. Unfortunately, the ideal serological correlate of immunity to mumps has yet to be identified. However, neutralising antibody titres in serum are used extensively as a surrogate marker of immunity to mumps. Conventional neutralisation tests are technically challenging, thus we developed and validated a high throughput, RT-qPCR microneutralisation (RT-qPCR-MN) method to determine serum neutralising antibody levels to mumps virus strains which avoids a number of the technical limitations of existing methods.
The qPCR-MN assays were thoroughly validated using human serum samples from patients with prior exposure to mumps infection or vaccination.
Each sample of pooled sera neutralised virus at a constant rate and without significant changes when tested against genotype A (MuV-A) and G (MuV-G) mumps virus concentrations from 200 to 3200 TCID50. The within run and between run variation of the RT-qPCR-MN assays for both genotypes were less than 3 % and 9 % for low and high titre samples, respectively. The correlation between the focus reduction neutralisation test and RT-qPCR-MN was excellent for both MuV-G (r2 = 0.80, 95CI: 0.67–1.00, p < 0.0001) and MuV-A genotypes (r2 = 0.88, 95 %CI 0.69–1.00, p < 0.0001) endpoint determinations.
We have developed a RT-qPCR MN assay for mumps virus that is simple, fast, scientifically objective and has high throughput. The assay can be used to provide key insights into the efficacy of mumps vaccination, to help explain the causes for the resurgence of mumps infection in vaccinated populations.
Characterization of Experimental Oro-Nasal Inoculation of Seba’s Short-Tailed Bats (Carollia perspicillata) with Bat Influenza A Virus H18N11Abstract
In 2012 and 2013, the genomic sequences of two novel influenza A virus (IAV) subtypes, designated H17N10 and H18N11, were identified via next-generation sequencing in the feces of the little yellow-shouldered fruit bat (Sturnira lilium) and the flat-faced fruit-eating bat (Artibeus planirostris), respectively. The pathogenesis caused by these viruses in their respective host species is currently insufficiently understood, which is primarily due to the inability to obtain and keep these bat species under appropriate environmental and biosafety conditions. Seba’s short-tailed bats (Carollia perspicillata), in contrast, are close relatives and a natural H18N11 reservoir species, with the advantage of established animal husbandry conditions in academic research. To study viral pathogenesis in more detail, we here oro-nasally inoculated Seba’s short-tailed bats with the bat IAV H18N11 subtype. Following inoculation, bats appeared clinically healthy, but the histologic examination of tissues revealed a mild necrotizing rhinitis. Consistently, IAV-matrix protein and H18-RNA positive cells were seen in lesioned respiratory and olfactory nasal epithelia, as well as in intestinal tissues. A RT-qPCR analysis confirmed viral replication in the conchae and intestines as well as the presence of viral RNA in the excreted feces, without horizontal transmission to naïve contact animals. Moreover, all inoculated animals seroconverted with low titers of neutralizing antibodies.
Different dynamics of Usutu virus infections in Austria and Hungary, 2017–2018Abstract
Usutu virus (USUV), a mosquito‐borne flavivirus closely related to West Nile virus, emerged in Austria in 2001, when it caused a considerable mass‐mortality of Eurasian blackbirds. Cases in birds increased until 2003 and quickly declined thereafter, presumably due to developing herd immunity. Since 2006, no further cases were recorded, until two blackbirds were tested positive in 2016. In Hungary, USUV first appeared in 2005 and has caused only sporadic infections since then. Initially, the only genetic USUV lineage found across both countries was Europe 1. This changed in 2015/2016, when Europe 2 emerged, which has since then become the prevalent lineage. Due to dispersal of these strains and introduction of new genetic lineages, USUV infections are now widespread across Europe. In 2009, the first cases of USUV‐related encephalitis were described in humans, and the virus has been frequently detected in blood donations since 2016. To monitor USUV infections among the Austrian wild bird population in 2017/2018, 86 samples were investigated by RT‐PCR. In 67 of them, USUV nucleic acid was detected (17 in 2017, 50 in 2018). The majority of succumbed birds were blackbirds, found in Vienna and Lower Austria. However, the virus also spread westwards to Upper Austria and southwards to Styria and Carinthia. In Hungary, 253 wild birds were examined, but only six of them were infected with USUV (five in 2017, one in 2018). Thus, in contrast to the considerable increase in USUV‐associated bird mortality in Austria, the number of infections in Hungary declined after a peak in 2016. Except for one case of USUV lineage Africa 3 in Austria in 2017, Europe 2 remains the most prevalent genetic lineage in both countries. Since USUV transmission largely depends on temperature, which affects vector populations, climate change may cause more frequent USUV outbreaks in the future.
Respiratory viral infections in Western Australians with cystic fibrosisAbstract
Viral respiratory infections (VRI) in people living with Cystic fibrosis (CF) is less well understood than respiratory bacterial infections, particularly adults with CF and few studies have compared children with adults. This study evaluated the frequency of respiratory viruses in patients with cystic fibrosis (CF) in Western Australia (WA). We determined the VRI in CF and compared them with non-CF patients. Further, we compared CF patients that were hospitalised with those that were not.
Nucleic acid from sputum of 157 CF and 348 non-CF patients was analysed for influenzavirus A (Flu A) and B, (Flu B), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), human rhinovirus (RV), and parainfluenza viruses (PIV 1-3) by RT-PCR, during the 2016 winter respiratory season.
No significant difference in the frequency of respiratory virus detection between CF and non-CF patients was found. RV was the most frequently detected virus in CF patients, and in hospitalised CF. RSV and hMPV were found less frequently in CF patients and RSV was not found in any hospitalised CF patient. A trend for fewer influenzavirus detections in adult CF patients was observed, however the trend was opposite for paediatric patients. RV and Flu A were the most common viruses detected in hospitalised CF patients.
There was no significant difference in VRI between CF and non-CF patients. RV and influenza A were most commonly found in hospitalised CF patients, suggesting that infection with these viruses may contribute to hospitalisation for CF respiratory exacerbations.
Human Norovirus Neutralized by a Monoclonal Antibody Targeting the Histo-Blood Group Antigen PocketAbstract
Temporal changes in the GII.4 human norovirus capsid sequences occasionally result in the emergence of genetic variants capable of causing new epidemics. The persistence of GII.4 is believed to be associated with the recognition of numerous histo-blood group antigen (HBGA) types and antigenic drift. We found that
one of the earliest known GII.4 isolates (in 1974) and a more recent epidemic GII.4
variant (in 2012) had varied norovirus-specific monoclonal antibody (MAb) reactivities but similar HBGA binding profiles. To better understand the binding interaction
of one MAb (10E9) that had varied reactivity with these GII.4 variants, we determined the X-ray crystal structure of the NSW-2012 GII.4 P domain 10E9 Fab complex.
We showed that the 10E9 Fab interacted with conserved and variable residues,
which could be associated with antigenic drift. Interestingly, the 10E9 Fab binding
pocket partially overlapped the HBGA pocket and had direct competition for conserved HBGA binding residues (i.e., Arg345 and Tyr444). Indeed, the 10E9 MAb
blocked norovirus virus-like particles (VLPs) from binding to several sources of HBGAs.
Moreover, the 10E9 antibody completely abolished virus replication in the human
norovirus intestinal enteroid cell culture system. Our new findings provide the first
direct evidence that competition for GII.4 HBGA binding residues and steric obstruction could lead to norovirus neutralization. On the other hand, the 10E9 MAb recognized residues flanking the HBGA pocket, which are often substituted as the virus
evolves. This mechanism of antigenic drift likely influences herd immunity and impedes the possibility of acquiring broadly reactive HBGA-blocking antibodies.
IMPORTANCE The emergence of new epidemic GII.4 norovirus variants is thought
to be associated with changes in antigenicity and HBGA binding capacity. Here, we
show that HBGA binding profiles remain unchanged between the 1974 and 2012
GII.4 variants, whereas these variants showed various levels of reactivity against a
panel of GII.4 MAbs. We identified a MAb that bound at the HBGA pocket, blocked
norovirus VLPs from binding to HBGAs, and neutralized norovirus virions in the cell
culture system. Raised against a GII.4 2006 strain, this MAb was unreactive to a GII.4
1974 isolate but was able to neutralize the newer 2012 strain, which has important
implications for vaccine design. Altogether, these new findings suggest that the
amino acid variations surrounding the HBGA pocket lead to temporal changes in antigenicity without affecting the ability of GII.4 variants to bind HBGAs, which are
known cofactors for infection.
Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of -Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexAbstract
Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-
9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a 150-fold upregulation of MexMN pump gene expression and a 50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several -lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.
Matrix composition in 3-D collagenous bioscaffolds modulates the survival and angiogenic phenotype of human chronic wound dermal fibroblastsAbstract
There is a substantial need for new strategies to stimulate cutaneous tissue repair in the treatment of chronic wounds. To address this challenge, our team is developing modular biomaterials termed “bead foams”, comprised of porous beads synthesized exclusively of extracellular matrix (ECM) and assembled into a cohesive three-dimensional (3-D) network. In the current study, bead foams were fabricated from human decellularized adipose tissue (DAT) or commercially-sourced bovine tendon collagen (COL) to explore the effects of ECM composition on human wound edge dermal fibroblasts (weDF) sourced from chronic wound tissues. The DAT and COL bead foams were shown to be structurally similar, but compositionally distinct, containing different levels of glycosaminoglycan content and collagen types IV, V, and VI. In vitro testing under conditions simulating stresses within the chronic wound microenvironment indicated that weDF survival and angiogenic marker expression were significantly enhanced in the DAT bead foams as compared to the COL bead foams. These findings were corroborated through in vivo assessment in a subcutaneous athymic mouse model. Taken together, the results demonstrate that weDF survival and paracrine function can be modulated by the matrix source applied in the design of ECM-derived scaffolds and that the DAT bead foams hold promise as cell-instructive biological wound dressings.
Statement of Significance
Biological wound dressings derived from the extracellular matrix (ECM) can be designed to promote the establishment of a more permissive microenvironment for healing in the treatment of chronic wounds. In the current work, we developed modular biomaterials comprised of fused networks of porous ECM-derived beads fabricated from human decellularized adipose tissue (DAT) or commercially-available bovine collagen. The bioscaffolds were designed to be structurally similar to provide a platform for investigating the effects of ECM composition on human dermal fibroblasts isolated from chronic wounds. Testing in in vitro and in vivo models demonstrated that cell survival and pro-angiogenic function were enhanced in the adipose-derived bioscaffolds, which contained higher levels of glycosaminoglycans and collagen types IV, V, and VI. Our findings support that the complex matrix composition within DAT can induce a more pro-regenerative cellular response for applications in wound healing.
The exonuclease Xrn1 activates transcription and translation of mRNAs encoding membrane proteinsAbstract
The highly conserved 5’–3’ exonuclease Xrn1 regulates gene expression in eukaryotes by coupling nuclear DNA transcription to cytosolic mRNA decay. By integrating transcriptome-wide analyses of translation with biochemical and functional studies, we demonstrate an unanticipated regulatory role of Xrn1 in protein synthesis. Xrn1 promotes translation of a specific group of transcripts encoding membrane proteins. Xrn1-dependence for translation is linked to poor structural RNA contexts for translation initiation, is mediated by interactions with components of the translation initiation machinery and correlates with an Xrn1-dependence for mRNA localization at the endoplasmic reticulum, the translation compartment of membrane proteins. Importantly, for this group of mRNAs, Xrn1 stimulates transcription, mRNA translation and decay. Our results uncover a crosstalk between the three major stages of gene expression coordinated by Xrn1 to maintain appropriate levels of membrane proteins.
Urine RNA Processing in a Clinical Setting: Comparison of 3 ProtocolsAbstract
Objective: The objective of this study was to compare quantitative and qualitative RNA extraction results from clinical voided urine samples between 3 commercially available extraction protocols.
Methods: For phase 1, fresh voided urine samples from 10 female subjects were collected and processed in clinic and transported to the laboratory with cold packs. RNA was purified with 1 of 3 RNA extraction protocols: (1) TRI Reagent Protocol; (2) Absolutely RNA Nanoprep Kit; and (3) ZR Urine RNA Isolation Kit. Real-time polymerase chain reactions (RT-PCR) were performed. As the ZR Urine RNA Isolation Kit provided the highest quality RNA in phase 1, for phase 2, RNA was extracted from 9 additional voided urine specimens using this kit to perform additional qualitative analyses.
Results: Median RNA yield was significantly higher with the TRI Reagent Protocol as compared with the other protocols (P = 0.007). However, there was a significantly lower median threshold cycle value from polymerase chain reaction (indicating improved downstream application performance) with the ZR Urine RNA Isolation Kit as compared with the other methods (P = 0.005). In phase 2, the median RNA integrity number of urine RNA was 2.5 (range, 1.6-5.9).
Conclusions: Although other methods may provide a higher quantity of RNA, when using clinical urine samples, the ZR Urine RNA Isolation Kit provided the highest quality of extracted RNA. This kit is especially attractive for the clinical setting because it does not require an initial centrifugation step. The urine RNA obtained with this kit may be useful for polymerase chain reaction but is not likely to be of high enough integrity for RNA sequencing.
Storage-Dependent Generation of Potent Anti-ZIKV Activity in Human Breast MilkAbstract
Zika virus (ZIKV) causes congenital neurologic birth defects, notably microcephaly, and has been associated with other serious complications in adults. The virus has been detected in human breast milk and possible transmissions via breastfeeding have been reported. Breast milk is rich in nutrients and bio-active substances that might directly affect viral infectivity. Thus, here, we analyzed the effect of human breast milk on ZIKV infection. We observed that fresh human breast milk had no effect on ZIKV, but found that upon storage, milk effectively suppressed infection. The antiviral activity is present in the fat-containing cream fraction of milk and results in the destruction of the structural integrity of viral particles, thereby abrogating infectivity. The release of the factor is time dependent but varies with donors and incubation temperatures. The viral titer of milk that was spiked with ZIKV decreased considerably upon storage at 37 °C for 8 h, was lost entirely after 2 days of 4 °C storage, but was not affected at −20 °C. This suggests that cold storage of milk inactivates ZIKV and that the antiviral factor in milk may also be generated upon breastfeeding and limit this transmission route of ZIKV.
Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virusAbstract
Human hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. Approximately 15–25 million people are chronically infected with HDV. As a satellite virus of the human hepatitis B virus (HBV), HDV uses the HBV-encoded envelope proteins for egress from and de novo entry into hepatocytes. So far, in vitro production of HDV particles is restricted to co-transfection of cells with HDV/HBV encoding cDNAs. This approach has several limitations. In this study, we established HuH7-END cells, which continuously secrete infectious HDV virions. The cell line was generated through stepwise stable integration of the cDNA of the HDV antigenome, the genes for the HBV envelope proteins and the HBV/HDV receptor NTCP. We found that HuH7-END cells release infectious HDV particles up to 400 million copies/milliliter and support virus spread to co-cultured cells. Due to the expression of NTCP, HuH7-END cells are also susceptible to de novo HDV entry. Virus production is stable for >16 passages and can be scaled up for preparation of large HDV virus stocks. Finally, HuH7-END cells are suitable for screening of antiviral drugs targeting HDV replication. In summary, the HuH7-END cell line provides a novel tool to study HDV replication in vitro.
Targeting the HIV-infected brain to improve ischemic stroke outcomeAbstract
HIV-associated cerebrovascular events remain highly prevalent even in the current era of antiretroviral therapy (ART). We hypothesize that low-level HIV replication and associated inflammation endure despite antiretroviral treatment and affect ischemic stroke severity and outcomes. Using the EcoHIV infection model and the middle cerebral artery occlusion as the ischemic stroke model in mice, we present in vivo analysis of the relationship between HIV and stroke outcome. EcoHIV infection increases infarct size and negatively impacts tissue and functional recovery. Ischemic stroke also results in an increase in EcoHIV presence in the affected regions, suggesting post-stroke reactivation that magnifies pro-inflammatory status. Importantly, ART with a high CNS penetration effectiveness (CPE) is more beneficial than low CPE treatment in limiting tissue injury and accelerating post-stroke recovery. These results provide potential insight for treatment of HIV-infected patients that are at risk of developing cerebrovascular disease, such as ischemic stroke.
Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settingsAbstract
Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.
Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settingsAbstract
Nucleic acid amplification technologies (NAATs) are high-performance tools for rapidly and accurately detecting infectious agents. They are widely used in high-income countries to diagnose disease and improve patient care. The complexities associated with test methods, reagents, equipment, quality control and assurance require dedicated laboratories with trained staff, which can exclude their use in low-resource and decentralized healthcare settings. For certain diseases, fully integrated NAAT devices and assays are available for use in environmentally-controlled clinics or emergency rooms where relatively untrained staff can perform testing. However, decentralized settings in many low- and middle-income countries with large burdens of infectious disease are challenged by extreme environments, poor infrastructure, few trained staff and limited financial resources. Therefore, there is an urgent need for low-cost, integrated NAAT tools specifically designed for use in low-resource settings (LRS). Two essential components of integrated NAAT tools are: 1) efficient nucleic acid extraction technologies for diverse and complex sample types; and 2) robust and sensitive nucleic acid amplification and detection technologies. In prior work we reported the performance and workflow capacity for the nucleic acid extraction component. In the current study we evaluated performance of eight novel nucleic acid amplification and detection technologies from seven developers using blinded panels of RNA and/or DNA from three pathogens to assess both diagnostic accuracy and suitability as an essential component for low-cost NAAT in LRS. In this exercise, we noted significant differences in performance among these technologies and identified those most promising for potential further development.
Arbovirus surveillance using FTATM cards in modified CO2‐baited encephalitis virus surveillance traps in the Northern Territory, AustraliaAbstract
In 2016, modified CO2‐baited encephalitis virus surveillance (EVS) traps were evaluated for flavivirus surveillance in the Northern Territory, Australia. The traps were fitted with honey‐soaked nucleic acid preservation cards (FTATM) for mosquitoes to expectorate virus while feeding on the cards. Cards were tested for the presence of selected arboviruses, with two cards testing positive for Kunjin virus and Alfuy, while sentinel chickens tested in parallel also showed Kunjin virus activity at the same time. The results from the cards and vector mosquito feeding rates indicate that CO2‐baited EVS traps coupled with honey‐baited FTATM cards are an effective tool for broad‐scale arbovirus surveillance.
Experimental Infection and Transmission Competence of Sindbis Virus in Culex torrentium and Culex pipiens Mosquitoes from Northern SwedenAbstract
Introduction: Sindbis virus (SINV) is a mosquito-borne Alphavirus known to infect birds and cause intermittent
outbreaks among humans in Fenno-Scandia. In Sweden, the endemic area has mainly been in central Sweden.
Recently, SINV infections have emerged to northern Sweden, but the vectorial efficiency for SINV of mosquito
species in this northern region has not yet been ascertained.
Objective: Mosquito larvae were sampled from the Umea˚ region in northern Sweden and propagated in a laboratory
to adult stage to investigate the infection, dissemination, and transmission efficiency of SINV in mosquitoes.
Materials and Methods: The mosquito species were identified by DNA barcoding of the cytochrome oxidase I
gene. Culex torrentium was the most abundant (82.2%) followed by Culex pipiens (14.4%), Aedes annulipes
(1.1%), Anopheles claviger (1.1%), Culiseta bergrothi (1.1%), or other unidentified species (1.1%). Mosquitoes
were fed with SINV-infected blood and monitored for 29 days to determine the viral extrinsic incubation period.
Infection and dissemination were determined by RT-qPCR screening of dissected body parts of individual
mosquitoes. Viral transmission was determined from saliva collected from individual mosquitoes at 7, 14, and
29 days. SINV was detected by cell culture using BHK-21 cells, RT-qPCR, and sequencing.
Results: Cx. torrentium was the only mosquito species in our study that was able to transmit SINV. The overall
transmission efficiency of SINV in Cx. torrentium was 6.8%. The rates of SINV infection, dissemination, and
transmission in Cx. torrentium were 11%, 75%, and 83%, respectively.
Conclusions: Cx. torrentium may be the key vector involved in SINV transmission in northern Sweden.
Indications for a vertical transmission pathway of piscine myocarditis virus in Atlantic salmon (Salmo salar L.)Abstract
Losses due to cardiomyopathy syndrome (CMS) keep increasing in salmon‐producing countries in the North‐Atlantic. Recently, Piscine myocarditis virus (PMCV) has been detected in post‐smolts shortly after sea‐transfer, indicating a possible carry‐over from the hatcheries. In addition, there are reports of prevalences of PMCV as high as 70%–90% in certain groups of broodfish, and a recent outbreak of CMS in the Faroe Islands has been linked to the importation of eggs from a CMS‐endemic area. Thus, there is a need to investigate whether PMCV can be transmitted vertically from infected broodstock to their progeny. In the present study, samples from eggs, larvae, fingerlings and presmolt originating from PMCV‐positive broodstock from two commercial Atlantic salmon producers were tested for PMCV. The prevalence of PMCV in the broodstock was 98% in the hearts, 69% in the roe and 59% in the milt. Piscine myocarditis virus was detected in all stages of the progeny until and including the 40 g stage. Piscine myocarditis virus was also detected in presmolt sampled for tissue tropism. This provides farmers with several options for minimizing the risk of transfer of PMCV from broodstock to progeny, including screening of broodstock and aiming to use only those that are negative for PMCV or have low levels of virus.
Monitoring infection with Piscine myocarditis virus and development of cardiomyopathy syndrome in farmed Atlantic salmon (Salmo salar L.) in NorwayAbstract
An epidemiological study was carried out in Norway in 2015–2018, investigating the development of infection with Piscine myocarditis virus (PMCV) and development of cardiomyopathy syndrome (CMS) in farmed Atlantic salmon. Cohorts from 12 sites were followed and sampled every month or every other month from sea transfer to slaughter. PMCV was detected at all sites and in all sampled cages, and fish in six sites developed clinical CMS. The initial infection happened between 1 and 7 months post‐sea transfer, and the median time from infection with PMCV until outbreak of CMS was 6.5 months. Generally, fish from sites with CMS had higher viral titre and a higher prevalence of PMCV, compared to sites that did not develop clinical CMS. The virus persisted until the point of slaughter at most (11 out of 12) of the sites. The detection of PMCV in all sites suggests that PMCV is more widespread than previously known. Screening for PMCV as a tool to monitor impending outbreaks of CMS must be supported by observations of the health status of the fish and risk factors for development of disease.
Rifamycin SV exhibits strong anti-inflammatory in vitro activity through pregnane X receptor stimulation and NFκB inhibitionAbstract
Rifamycin SV (rifamycin), is a member of the ansamycin family of antimicrobial compounds which kills bacteria commonly associated with infectious diarrhea and other enteric infections. Rifamycin has been found to be effective in experimental animal models of gut inflammation and its efficacy in these settings has been attributed partially to immunomodulatory non-bactericidal activities. This study aimed to further evaluate the anti-inflammatory activities of rifamycin by analyzing its effect on two key regulators of inflammation: PXR and NFκB. Rifamycin stimulated PXR transcriptional activity in two PXR reporter cell lines and induced expression of two genes known to be regulated by PXR and are directly involved in cellular detoxification: CYP3A4 and PgP. Moreover, CYP3A4 metabolic activity was induced by rifamycin in HepG2 cells. Rifamycin also antagonized TNFα and LPS-induced NFκB activities and inhibited IL1β-induced synthesis of inflammatory chemokine, IL8. Although reciprocal regulation of PXR and NFkB by rifamycin was not directly addressed, the data suggest that in the absence of PXR, inhibition of NFκB by rifamycin is not dependent on PXR stimulation. Thus, rifamycin exhibits potent anti-inflammatory activities, characterized by in vitro PXR activation and concomitant CYP3A4 and PgP induction, in parallel with potent NFκB inhibition and concomitant IL8 inhibition.
Development of a PPRV challenge model in goats and its use to assess the efficacy of a PPR vaccineAbstract
Peste des Petits Ruminants (PPR) is a severe disease of small ruminants and has high economic impacts in developing countries. Endemic in Africa, the Middle East and Asia, the disease is currently progressing with occurrences reported in North Africa, Turkey and in Georgia, and now threatens Europe. Much remains unknown about the infection dynamics, the virulence of the different strains and species/breed susceptibility. Robust experimental challenge models are needed to explore these fields and to confirm the efficacy of currently sold vaccines. We first assessed virulence of two PPR virus strains (CI89 and MA08) in Saanen goats. Whereas the MA08 strain led to classical severe clinical signs of PPR, the CI89 strain appeared to cause a mild disease in Saanen goats, highlighting the difference in virulence between strains in this animal model. We further demonstrated the importance of the inoculation route in the appearance of clinical signs and that ocular excretion is a better choice than blood for viral detection. After developing a robust challenge model, we assessed the efficacy of a vaccine (PPR-VAC®, BVI Botswana) against the MA08 strain and demonstrated that this vaccine blocked viral excretion and significantly reduced clinical signs. These results reinforce the paradigm that a strain from one lineage could protect against strains from other lineages.
The ubiquitin-protein ligase E6AP/UBE3A supports early encephalomyocarditis virus replicationAbstract
Many viruses make use of, and even direct, the ubiquitin-proteasome system to facilitate the generation of a cellular environment favorable for virus replication, while host cells use selected protein ubiquitylation pathways for antiviral defense. Relatively little information has been acquired, however, regarding the extent to which protein ubiquitylation determines the replication success of picornaviruses. Here we report that the ubiquitin-protein ligase E6AP/UBE3A, recently shown to be a participant in encephalomyocarditis virus (EMCV) 3C protease concentration regulation, also facilitates the early stages of EMCV replication, probably by a mechanism that does not involve 3C protease ubiquitylation. Using stably transfected E6 AP knockdown cells, we found that reduced E6AP concentration extends the time required for infected cells to undergo the morphological changes caused by virally induced pathogenesis and to begin the production of infectious virions. This lag in virion production is accompanied by a corresponding delay in the appearance of detectable levels of viral proteins and RNA. We also found, by using both immunofluorescence microscopy and cell fractionation, that E6AP is partially redistributed from the nucleus to the cytoplasm in EMCV-infected cells, thereby increasing its availability to participate in cytoplasmic virus replication processes.
Synthesis and antiviral evaluation of novel peptidomimetics as Norovirus protease inhibitorsAbstract
A series of tripeptidyl transition state inhibitors with new P1 and warhead moieties were synthesized and evaluated in a GI-1 norovirus replicon system and against GII-4 and GI-1 norovirus proteases. Compound 19, containing a 6-membered ring at the P1 position and a reactive aldehyde warhead exhibited sub-micromolar replicon inhibition. Retaining the same peptidyl scaffold, several reactive warheads were tested for protease inhibition and norovirus replicon inhibition. Of the six that were synthesized and tested, compounds 42, 43, and 45 potently inhibited the protease in biochemical assay and GI-1 norovirus replicon in the nanomolar range.