Blind Passage Research Articles

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

The Significance of the 98th Amino Acid in GP2a for Porcine Reproductive and Respiratory Syndrome Virus Adaptation in Marc-145 Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the pig industry. Marc-145 cells are widely used for PRRSV isolation, vaccine production, and investigations into virus biological characteristics. Despite their significance in PRRSV research, Marc-145 cells struggle to isolate specific strains of the North American virus genotype (PRRSV-2). The involvement of viral GP2a, GP2b, and GP3 in this phenomenon has been noted. However, the vital amino acids have not yet been identified. In this study, we increased the number of blind passages and successfully isolated two strains that were previously difficult to isolate with Marc-145 cells. Both strains carried an amino acid substitution in GP2a, specifically phenylalanine to leucine at the 98th amino acid position. Through a phylogenetic and epidemiologic analysis of 32 strains, those that were not amenable to isolation widely exhibited this mutation. Then, by using the PRRSV reverse genetics system, IFA, and Western blotting, we identified the mutation that could affect the tropism of PRRSV-2 for Marc-145 cells. Furthermore, an animal experiment was conducted. Through comparisons of clinical signs, mortality rates, and viral load in the organs and sera, we found that mutation did not affect the pathogenicity of PRRSV-2. In conclusion, our study firmly establishes the 98th amino acid in GP2a as a key determinant of PRRSV-2 tropism for Marc-145 cells.

Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Molecular Detection and Isolation of Lumpy Skin Disease Virus during an Outbreak in West Hararghe Zone, Eastern Ethiopia.

Lumpy skin disease (LSD) is a highly contagious viral disease that causes significant economic losses in cattle populations globally. This study aimed to isolate and detect the LSD virus responsible for an outbreak in selected areas (Daaroo Labuu, Hawwii Guddina, and Gumbi Bordede district) of the West Hararghe Zone in Ethiopia between January 2020 and December 2021. Out of the 625 animals examined for the presence of LSD, only 73 animals showed clinical signs, and skin scrapes were collected from these animals for further analysis. Among those, 12 animals (1.9%) succumbed to the disease. Skin biopsy samples from 45 animals displaying clinical signs of LSD were inoculated in Vero cell lines because of limited equipment. After three blind passages, all samples developed cytopathic effects (CPEs). The presence of the LSD virus was confirmed using real-time PCR. Conventional PCR detected LSDV in 47 (64.4%) of the skin scrap samples, while high-resolution melt qPCR detected it in 49 (67.1%) samples. The study revealed a morbidity rate of 11.68%, a mortality rate of 1.92%, and a case fatality rate of 16.44% based on clinical data. The findings suggest that LSD causes significant economic losses, even in vaccinated animals prior to an outbreak. To effectively control and eradicate LSD, the government should develop new strategic policies. Community awareness campaigns are necessary to improve vector control measures and drainage systems. In addition, the present vaccination policy and strategy should be re-evaluated for effectiveness. This study focused on a specific region and timeframe, limiting generalizability. Factors such as environmental conditions and management practices were not extensively explored. Similar studies should be conducted in different regions to assess the prevalence and genetic diversity of LSDV. The effectiveness of control measures and vaccination strategies should be investigated. The impact of environmental factors and management practices on LSD transmission and disease severity warrants further exploration. This study provides insights into the detection and isolation of the LSD virus during an outbreak in the West Hararghe Zone of Ethiopia. The results highlight the need for continued surveillance and monitoring of emerging infectious diseases in the region. Furthermore, the importance of using molecular methods for detecting and characterizing viral outbreaks in livestock populations is emphasized.

Elucidating the biological characteristics and pathogenicity of the highly virulent G2a porcine epidemic diarrhea virus.

Since the large-scale outbreak of porcine epidemic diarrhoea (PED) in 2010, caused by the genotype 2 (G2) variant of the porcine epidemic diarrhoea virus (PEDV), pig farms in China, even those vaccinated with the G2b vaccine, have experienced infections from the G2a variant, leading to significant economic losses. This study successfully isolated the G2a strain DY2020 from positive small intestine contents (SICs) by blind passage on Vero cells for four generations. The SICs were taken from Daye, Hubei Province, China. The biological characteristics were identified by indirect immunofluorescence assay (IFA) and transmission electron microscopy (TEM). The growth kinetics of the strain on Vero cells were detected by TCID50, and the virus titre could reach 107.35 TCID50 ml-1 (SD: 5.07×106). The pathogenicity towards colostrum-deprived piglets was conducted by assessing faecal viral shedding, morphometric analysis of intestinal lesions, and immunohistochemical staining. The results showed that DY2020 was highly virulent to colostrum-deprived piglets, with severe watery diarrhoea and other clinical symptoms appeared at 6 h post-infection (h p.i.), and all died within 30 h. Pathological tissue examination results showed that the lesions mainly occurred in the intestines of piglets, causing pathological changes such as shortening of intestinal villi. In summary, the discovery of the G2a strain DY2020 in this study is of great significance for understanding Hubei PEDV and provides an important theoretical basis for the development of new efficient PEDV vaccines.

Unexpected thermal stability of two enveloped megaviruses, Emiliania huxleyi virus and African swine fever virus, as measured by viability PCR

BackgroundThe particle structure of Emiliania huxleyi virus (EhV), an algal infecting member of nucleocytoplasmic large DNA viruses (NCLDVs), contains an outer lipid membrane envelope similar to that found in animal viruses such as African swine fever virus (ASFV). Despite both being enveloped NCLDVs, EhV and ASFV are known for their stability outside their host environment.MethodHere we report for the first time, the application of a viability qPCR (V-qPCR) method to describe the unprecedented and similar virion thermal stability of both EhV and ASFV. This result contradicts the cell culture-based assay method that suggests that virus “infectivity” is lost in a matter of seconds (for EhV) and minutes (for ASFV) at temperature greater than 50 °C. Confocal microscopy and analytical flow cytometry methods was used to validate the V-qPCR data for EhV.ResultsWe observed that both EhV and ASFV particles has unprecedented thermal tolerances. These two NCLDVs are exceptions to the rule that having an enveloped virion anatomy is a predicted weakness, as is often observed in enveloped RNA viruses (i.e., the viruses causing Porcine Reproductive and Respiratory Syndrome (PRRS), COVID-19, Ebola, or seasonal influenza). Using the V-qPCR method, we confirm that no PRRSV particles were detectable after 20 min of exposure to temperatures up to 100 °C. We also show that the EhV particles that remain after 50 °C 20 min exposure was in fact still infectious only after the three blind passages in bioassay experiments.ConclusionsThis study raises the possibility that ASFV is not always eliminated or contained after applying time and temperature inactivation treatments in current decontamination or biosecurity protocols. This observation has practical implications for industries involved in animal health and food security. Finally, we propose that EhV could be used as a surrogate for ASFV under certain circumstances.

Cognitive models for mentally visualizing a sharp instrument in a blind procedure.

Our objective was to understand the cognitive strategies used by surgeons to mentally visualize navigation of a surgical instrument through blind space. We conducted semi-structured interviews with 15 expert and novice surgeons following simulated retropubic trocar passage on 3D-printed models of pelvises segmented from preop MRIs. Midurethral sling surgery involves blind passage of a trocar among the urethra, bladder, iliac vessels, and bowel while relying primarily on haptic feedback from the suprapubic bone (SPB) for guidance. Our conceptual foundation was based on Lahav's study on blind people's mental mapping of spaces using haptic cues. Participants detailed how they mentally pictured the trocar's location relative to vital anatomy. We coded all responses and used constant comparative analysis to generate themes, confirmed with member checking. Expert and novice participants utilized multiple cognitive strategies combined with haptic feedback to accomplish safe trocar passage. Some used a step-by-step route strategy, visualizing sequential 2D axial images of anatomy adjacent to the SPB. Others used a map strategy, forming global 3D pictures. Although these mental pictures vanished when they were "lost," a safe zone could be reestablished by touching the SPB. Experts were more likely to relate their body position to the trocar path and rely on minor variations in resistance. Novices were more inclined toward backtracking of the trocar. Our findings may be extended to any blind surgical procedure. Teaching visualization strategies and incorporating tactile feedback can be used intraoperatively to help learners navigate their instrument safely around vital organs.

Lack of antiviral activity of ivermectin against foot-and-mouth disease virus serotype O in BALB/c mice

Ivermectin is an FDA approved drug and showed in vitro antiviral activity against different serotypes of Foot-and-mouth disease virus (FMDV). We here assessed the effect of ivermectin in 12 day old female BALB/c mice infected with 50LD50 FMDV serotype O intraperitoneally. Initially FMDV was adopted on 3-day old BALB/c mice by blind passages. After successful adaptation of virus mice showed hind limb paralysis. Mice were divided in 6 different groups and each group has 6 mice. Ivermectin was given at clinically prescribed dose of 500 μg/kg subcutaneously at different time interval. Ivermectin was given at 0 h post infection (hpi) and 12 hpi. Moreover we compared commercially available ivermectin with purified ivermectin preparation in sterilized DMSO. Viral load was evaluated through RT-qPCR and ELISA in different groups. Results showed that positive control and negative control has CT-value 26.28 and 38 respectively. Treated groups at 0hpi, 12hpi, purified ivermectin and pre-post treatment group has CT values 24.89, 29.44, 27.26 and 26.69 respectively that showed there was no significant reduction in virus load in treated groups as compare to positive control. In histopathology of lung tissue perialveolar capillaries were congested and alveoli were altelactic. Some emphysema was seen in alveoli and mild thickening in the alveolar wall was observed. In the alveolar epithelium mononuclear cells infiltration was seen. There was discoloration haemorrhages and enlargement of heart. Degeneration, fragmentation and loss of sarcoplasm were seen in the cardiac muscle fibers. Above results showed that ivermectin did not lessen lung and heart viral load. This study contributes that ivermectin does not have a significant antiviral effect when used in mice against FMDV serotype O, according to a growing body of research.

(409) Low Transfascial Fixation of the IPP Reservoir

Abstract Introduction Inflatable penile prosthesis reservoir herniation or migration may occur with space of Retzius or submuscular placement. The dangers of high submuscular placement include bowel or bladder perforation (from blind instrument passage) and or bowel herniation (from unintentional peritoneal placement and stretching of small opening with reservoir filling). Even under direct vision submuscular placement, reservoirs can ultimately reside into the scrotum, retroperitoneum, and lateral abdominal wall. The Transfascial Fixation (TFF) technique was recently developed to prevent reservoir migration with placement either submuscularly or in the space of Retzius, but video of this technique has not been previously published. Objective To visually demonstrate the proper surgical technique of TFF during IPP reservoir placement. Methods An IPP surgical candidate at high risk of complications should reservoir migration occur was identified. Video of TFF technique performed during IPP surgery was recorded using an in-light overhead camera and a GoPro aseptically positioned on the lead surgeon. Results Successful demonstration of TFF technique during IPP placement. Conclusions TFF technique is a feasible option to aid prevention of penile implant reservoir herniation. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast, Boston Scientific, Metuchen Pharmaceuticals, Acerus Pharmaceuticals, Endo Pharmaceuticals, AbbVie Inc.

(408) THALIA Technique Fixation of the IPP Reservoir

Abstract Introduction Inflatable penile prosthesis reservoir herniation or migration may occur with space of Retzius or submuscular placement. The dangers of high submuscular placement include bowel or bladder perforation (from blind instrument passage) and or bowel herniation (from unintentional peritoneal placement and stretching of small opening with reservoir filling). Even under direct vision submuscular placement, reservoirs can ultimately reside into the scrotum, retroperitoneum, and lateral abdominal wall. The Tubing, Hitch And Lasso, Intussusception Anchor (THALIA) technique was recently developed to prevent reservoir migration with placement either submuscularly or in the space of Retzius, but video of this technique has not been previously published. Objective To visually demonstrate the proper surgical technique of THALIA during IPP reservoir placement. Methods An IPP surgical candidate at high risk of complications should reservoir migration occur was identified. Video of THALIA technique performed during IPP surgery was recorded using an in-light overhead camera and a GoPro aseptically positioned on the lead surgeon. Results Successful demonstration of THALIA technique during IPP placement. Conclusions THALIA technique is a feasible option to aid prevention of penile implant reservoir herniation. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast

Surgeon estimation of retropubic trocar position in blind 3D space.

Retropubic midurethral sling surgery involves the blind passage of trocars near vital organs. We quantified the proximity of surgeons' mental representation of trocar position relative to actual position using a pelvis simulation platform. We hypothesized that novice surgeons, compared with experts, would estimate the trocar's location to be further from the actual location. Novice and expert surgeons performed bilateral retropubic trocar passes of a Gynecare TVT trocar (#810041B-#810,051) on the simulation platform. We measured the trocar tip's position using a motion capture system, and recorded vocalizations when they perceived contacting the bone and crossing three landmark-oriented planes. We calculated differences (∆Bone, ∆Turn, ∆Top, ∆Pop) between vocalization times and when the trocar crossed the corresponding plane. We performed Mann-Whitney and Chi-squared tests to investigate differences between novices and experts and Levene's test to assess equality of variances for subject-level variation. A total of 34 trials, including 22 expert and 12 novice trials, were performed by six participants. ∆Bone was significantly smaller among novice surgeons (1.27 vs 2.81s, p=0.013). There were no significant differences in the remaining three deltas or in vocalizing early versus late. Levene's test revealed no significant differences in within-subject variability for any of the four deltas. Novices passed the trocar anterior to the pubic bone on three passes. Novices were similar to expert surgeons in their estimation of the trocar's location and may have relied more heavily on anticipatory mechanisms to compensate for lack of experience. Teaching surgeons should make sure the novice surgeon trocar pass starts posterior to the bone.

V11-07 THALIA TECHNIQUE FIXATION OF THE IPP RESERVOIR

You have accessJournal of UrologyCME1 Apr 2023V11-07 THALIA TECHNIQUE FIXATION OF THE IPP RESERVOIR Lexiaochuan Wen, Jayson Kemble, Eileen Byrne, Jonathan Warner, Sevann Helo, Matthew Ziegelmann, and Tobias Kohler Lexiaochuan WenLexiaochuan Wen More articles by this author , Jayson KembleJayson Kemble More articles by this author , Eileen ByrneEileen Byrne More articles by this author , Jonathan WarnerJonathan Warner More articles by this author , Sevann HeloSevann Helo More articles by this author , Matthew ZiegelmannMatthew Ziegelmann More articles by this author , and Tobias KohlerTobias Kohler More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003337.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Inflatable penile prosthesis reservoir herniation or migration may occur with space of Retzius or submuscular placement. The dangers of high submuscular placement include bowel or bladder perforation (from blind instrument passage) and or bowel herniation (from unintentional peritoneal placement and stretching of small opening with reservoir filling). Even under direct vision submuscular placement, reservoirs can ultimately reside into the scrotum, retroperitoneum, and lateral abdominal wall. The Tubing, Hitch And Lasso, Intussusception Anchor (THALIA) technique was recently developed to prevent reservoir migration with placement either submuscularly or in the space of Retzius, but video of this technique has not been previously published. The aim of this video is to demonstrate the proper surgical technique of THALIA during IPP reservoir placement. METHODS: An IPP surgical candidate at high risk of complications should reservoir migration occur was identified. Video of THALIA technique performed during IPP surgery was recorded using an in-light overhead camera and a GoPro aseptically positioned on the lead surgeon. RESULTS: Successful demonstration of THALIA technique during IPP placement. CONCLUSIONS: THALIA technique is a feasible option to aid prevention of penile implant reservoir herniation. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1000 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lexiaochuan Wen More articles by this author Jayson Kemble More articles by this author Eileen Byrne More articles by this author Jonathan Warner More articles by this author Sevann Helo More articles by this author Matthew Ziegelmann More articles by this author Tobias Kohler More articles by this author Expand All Advertisement PDF downloadLoading ...

V11-05 LOW TRANSFASCIAL FIXATION OF THE IPP RESERVOIR

You have accessJournal of UrologyCME1 Apr 2023V11-05 LOW TRANSFASCIAL FIXATION OF THE IPP RESERVOIR Jayson Kemble, Lexiaochuan Wen, Eileen Byrne, Mohit Khera, Jonathan Warner, Sevann Helo, Matthew Ziegelmann, and Tobias Kohler Jayson KembleJayson Kemble More articles by this author , Lexiaochuan WenLexiaochuan Wen More articles by this author , Eileen ByrneEileen Byrne More articles by this author , Mohit KheraMohit Khera More articles by this author , Jonathan WarnerJonathan Warner More articles by this author , Sevann HeloSevann Helo More articles by this author , Matthew ZiegelmannMatthew Ziegelmann More articles by this author , and Tobias KohlerTobias Kohler More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003337.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Inflatable penile prosthesis reservoir herniation or migration may occur with space of Retzius or submuscular placement. The dangers of high submuscular placement include bowel or bladder perforation (from blind instrument passage) and or bowel herniation (from unintentional peritoneal placement and stretching of small opening with reservoir filling). Even under direct vision submuscular placement, reservoirs can ultimately reside into the scrotum, retroperitoneum, and lateral abdominal wall. The Transfascial Fixation (TFF) technique was recently developed to prevent reservoir migration with placement either submuscularly or in the space of Retzius, but video of this technique has not been previously published. The aim of this video is to demonstrate the proper surgical technique of TFF during IPP reservoir placement. METHODS: An IPP surgical candidate at high risk of complications should reservoir migration occur was identified. Video of TFF technique performed during IPP surgery was recorded using an in-light overhead camera and a GoPro aseptically positioned on the lead surgeon. RESULTS: Successful demonstration of TFF technique during IPP placement. CONCLUSIONS: TFF technique is a feasible option to aid prevention of penile implant reservoir herniation. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1000 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jayson Kemble More articles by this author Lexiaochuan Wen More articles by this author Eileen Byrne More articles by this author Mohit Khera More articles by this author Jonathan Warner More articles by this author Sevann Helo More articles by this author Matthew Ziegelmann More articles by this author Tobias Kohler More articles by this author Expand All Advertisement PDF downloadLoading ...

Characterization and pathogenicity of the porcine epidemic diarrhea virus isolated in China.

Piglet diarrhea caused by the porcine epidemic diarrhea virus (PEDV) is a common problem on pig farms in China associated with high morbidity and mortality rates. In this study, three PEDV isolates were successfully detected after the fourth blind passage in Vero cells. The samples were obtained from infected piglet farms in Jilin (Changchun), and Shandong (Qingdao) Provinces of China and were designated as CH/CC-1/2018, CH/CC-2/2018, and CH/QD/2018. According to the analysis of the complete S protein gene sequence, the CH/CC-1/2018 and CH/CC-2/2018 were allocated to the G2b branch, while CH/QD/2018 was located in the G1a interval and was closer to the vaccine strain CV777. Successful detection and identification of the isolated strains were carried out using electron microscopy and indirect immunofluorescence. Meanwhile, animal challenge experiments and viral RNA copies determination were used to compare the pathogenicity. The results showed that CH/CC-1/2018 in Changchun was more pathogenic than CH/QD/2018 in Qingdao. In conclusion, the discovery of these new strains is conducive to the development of vaccines to prevent the pandemic of PEDV, especially that the CH/CC-1/2018, and CH/CC-2/2018 were not related to the classical vaccine strain CV777.

Evaluation of protective effects of heat-inactivated cyprinid herpesvirus-2 (CyHV-2) vaccine against herpesviral hematopoietic necrosis disease (HVHND) in goldfish (Carassius auratus)

Cyprinid herpesvirus-2 (CyHV-2) is an important virus that causes herpesviral hematopoietic necrosis disease (HVHND) leading to huge economic losses in goldfish (Carassius auratus). However, until now no proper prophylactic measure or treatment is available for CyHV-2 infection in goldfish. Hence, in this experiment, we developed a heat-inactivated CyHV-2 vaccine and evaluated its performance in goldfish. Initially, CyHV-2 was propagated in the fantail goldfish fin (FtGF) cell line and the titer of the viral inoculum was 107.8 TCID50/ml. Subsequently, various temperatures (40 °C, 50 °C, 60 °C, 70 °C, and 80 °C) were evaluated to achieve the complete inactivation of CyHV-2. Only the viral inoculum inactivated at 80 °C for 1 h did not show any cytopathic effect in the FtGF cell line after five blind passages. Hence the heat-inactivated CyHV-2 vaccine developed at 80 °C was further used for immunization trials in goldfish. The experimental goldfish were intraperitoneally immunized with 300 μL of the heat-inactivated CyHV-2 vaccine. Subsequently, the kidney and spleen tissues were sampled at various time points post-vaccination (6th hr, 2nd day, 4th day, 6th day, 10th day, 16th day, and 30th day) to evaluate the expression of immune genes (IL-12, IL-10, IFN-γ, CD8, and CD4). A significant upregulation of immune genes was observed at various time points in the kidney and spleen tissue of the vaccinated goldfish. Furthermore, in order to study the efficacy of the vaccine, the experimental fish were challenged with CyHV-2 (107.8 TCID50/ml) after the 30th day post-vaccination. The survival of the fish in the vaccine group (86.7%) was significantly higher compared to the non-vaccinated group (20%). Moreover, the relative percentage survival of the vaccinated group was 83.34%. In spite of the single dose, the heat-killed vaccine developed in the present study elicited the immune response and offered better protection in goldfish against CyHV-2. However, further large-scale field performance evaluation studies are necessary to develop this vaccine on a commercial scale.

Mouse adaptation of H6 avian influenza viruses and their molecular characteristics.

H6 avian influenza viruses (AIVs) not only continue to circulate in both domestic poultry and wild waterfowl, but also have occasionally caused spillovers infections in pigs and humans, posing a potential threat to public health. However, the molecular mechanism of H6 AIV adaptation to mammals remains largely unknown. In this study, two mouse-adapted (MA) H6 AIV strains, named as MA E-Teal/417 and MA GWF-Goose/740, were generated through blind passages in BALB/c mice. The two MA H6 strains replicated more efficiently and showed higher virulence than the corresponding wild type (WT) H6 strains in mice. Genome sequencing revealed that MA E-Teal/417 and MA GWF-Goose/740 carried six amino acid mutations (PB2-T224A/E627K, HA-G124R, NA-F167L/Y356H and M1-M92R), and four amino acid mutations (PB1-K577E, PA-T97I/D514E and HA-T276K), respectively, when compared to the corresponding WT virus. Receptor binding assay showed MA E-Teal/417 had stronger binding activity to α-2,3 SA than WT E-Teal/417. Moreover, the polymerase activity analysis found the RNP polymerase activity of both MA H6 viruses was significantly higher than that of the corresponding WT virus in 293T cells. All these demonstrate that H6 AIV can acquire limit amino acid substitutions to adapt to mammals and increase virulence, highlighting the significance of monitoring such mutations of H6 AIV in the field for alarming the potential of its cross-transmission and pathogenesis in mammals.

Live enteroviruses, but not other viruses, detected in human pancreas at the onset of type 1 diabetes in the DiViD study

Aims/hypothesisEnterovirus (EV) infection of pancreatic islet cells is one possible factor contributing to type 1 diabetes development. We have reported the presence of EV genome by PCR and of EV proteins by immunohistochemistry in pancreatic sections. Here we explore multiple human virus species in the Diabetes Virus Detection (DiViD) study cases using innovative methods, including virus passage in cell cultures.MethodsSix recent-onset type 1 diabetes patients (age 24–35) were included in the DiViD study. Minimal pancreatic tail resection was performed under sterile conditions. Eleven live cases (age 43–83) of pancreatic carcinoma without diabetes served as control cases. In the present study, we used EV detection methods that combine virus growth in cell culture, gene amplification and detection of virus-coded proteins by immunofluorescence. Pancreas homogenates in cell culture medium were incubated with EV-susceptible cell lines for 3 days. Two to three blind passages were performed. DNA and RNA were extracted from both pancreas tissue and cell cultures. Real-time PCR was used for detecting 20 different viral agents other than EVs (six herpesviruses, human polyomavirus [BK virus and JC virus], parvovirus B19, hepatitis B virus, hepatitis C virus, hepatitis A virus, mumps, rubella, influenza A/B, parainfluenza 1–4, respiratory syncytial virus, astrovirus, norovirus, rotavirus). EV genomes were detected by endpoint PCR using five primer pairs targeting the partially conserved 5′ untranslated region genome region of the A, B, C and D species. Amplicons were sequenced. The expression of EV capsid proteins was evaluated in cultured cells using a panel of EV antibodies.ResultsSamples from six of six individuals with type 1 diabetes (cases) and two of 11 individuals without diabetes (control cases) contained EV genomes (p<0.05). In contrast, genomes of 20 human viruses other than EVs could be detected only once in an individual with diabetes (Epstein–Barr virus) and once in an individual without diabetes (parvovirus B19). EV detection was confirmed by immunofluorescence of cultured cells incubated with pancreatic extracts: viral antigens were expressed in the cytoplasm of approximately 1% of cells. Notably, infection could be transmitted from EV-positive cell cultures to uninfected cell cultures using supernatants filtered through 100 nm membranes, indicating that infectious agents of less than 100 nm were present in pancreases. Due to the slow progression of infection in EV-carrying cell cultures, cytopathic effects were not observed by standard microscopy but were recognised by measuring cell viability. Sequences of 5′ untranslated region amplicons were compatible with EVs of the B, A and C species. Compared with control cell cultures exposed to EV-negative pancreatic extracts, EV-carrying cell cultures produced significantly higher levels of IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP1).Conclusions/interpretationSensitive assays confirm that the pancreases of all DiViD cases contain EVs but no other viruses. Analogous EV strains have been found in pancreases of two of 11 individuals without diabetes. The detected EV strains can be passaged in series from one cell culture to another in the form of poorly replicating live viruses encoding antigenic proteins recognised by multiple EV-specific antibodies. Thus, the early phase of type 1 diabetes is associated with a low-grade infection by EVs, but not by other viral agents. Graphical abstract

Isolation and molecular characterization of lumpy skin disease virus from hard ticks, Rhipicephalus (Boophilus) annulatus in Egypt

BackgroundLumpy skin disease (LSD), a disease of cattle and buffaloes, has recently become widely prevalent in Egypt. The aim of this study was to ascertain the potential role of Rhipicephalus (Boophilus) annulatus ticks in the transmission of this disease. Samples collected from suspected lumpy skin disease virus (LSDV) infected cows that had previously been vaccinated with the Romanian sheep pox virus (SPPV) in various Egyptian governorates were obtained between May to November over two consecutive years, namely 2018 and 2019. Ticks were morphologically identified and the partial cytochrome oxidase subunit I gene (COI) were sequenced, revealing that they were closely related to R. (Boophilus) annulatus. The G-protein-coupled chemokine receptor (GPCR) gene of the LSDV was used to test hard ticks.ResultsTwo positive samples from Kafr El-Sheikh province and one positive sample from Al-Behera province were reported. BLAST analysis revealed that the positive samples were closely related to the Kazakhstani Kubash/KAZ/16 strain (accession number MN642592). Phylogenetic analysis revealed that the GPCR gene of the LSDV recently circulating in Egypt belongs to a global cluster of field LSDV with a nucleotide identity of 98–100%. LSDV isolation was successfully performed four days after inoculation using 9 to 11-day-old embryonated chicken eggs showing characteristic focal white pock lesions dispersed on the choriallantoic membrane after three blind passages. Intracytoplasmic inclusion bodies, cell rupture, vacuoles in cells, and virus particles ovoid in shape were demonstrated by electron microscopy.ConclusionIn this study the role of hard ticks in the transmission of the LSDV to susceptible animals in Egypt was revealed and confirmed by various methods.

Скринінг вірусних захворювань у чорноморської камбали-калкана (Scophthalmus maeoticus, Pallas, 1814) у північному Причорномор’ї та Азовському морі

Purpose. The development of marine aquaculture in the Black Sea is relevant and promising for many reasons, as stated in the decisions and documents of national organizations in the region and the General Fisheries Commission for the Mediterranean (GFCM). Turbot is one of the most valuable fish marketed in seaside countries of the Black Sea as well as in the European Union. Various aspects of the biology of turbot have been investigated yet.Butunfortunately, surveillance procedures for listed diseasesandanalysis of the possibility its prevention and spread have not been provided. Therefore, in this study, in order to evaluate the data on viral diseases of turbot in Ukraine, in 2020 a field survey in free-living Black Sea turbot (Scophthalmus maeoticus) in the northern parts of Black Sea and Sea of Azov was carried out. Methodology. Before sampling all fish were observed for the external lesions, measured from snout to tail length and of total length and weight. Sampling included pooled internal organs, gills and brain. Cell culture and polymerase chain reaction (PCR) methods were used to identify viral diseases. Findings. An external fish examination did not reveal any sign of disease. The internal organs, as well as the gills, were in good condition and had appropriate color, shape and without any pathological changes. All tested samples were free of IPNV, VHSV and VNN viruses as was determined by the methods of cell culture and PCR. The cytopathic effect (CPE) on cells was not observed after the first and the second blind passages. Using the RT-PCR method, we did not identify any of the viruses we were looking for. Originality. This was the first attempt to screen turbot viral diseases in the Ukrainian waters of the Black and Azov Seas. Practical value. In future the permanent surveillance of viral diseases in turbotin accordance with the EU strategy on animal health allows to prevent the outbreaks and develop new approaches for the diagnostic tests in purpose to study the ecology of pathogens in different areas. Key words: turbot, the Black Sea, viral diseases, cell culture, PCR.

A Preliminary Study of SARS-CoV-2's Permanence and Potential Infective Capacity in Mineromedicinal Waters of Copahue, Neuquén, Argentina.

Copahue Thermal Center is characterized by the presence of mineromedicinal acidic waters with high temperatures, therapeutic peloids, and relevant consortia of extremophiles species, distributed in small natural pools which cannot be disinfected. The objective of this research was to investigate the survival of SARS-CoV-2 in Copahue’s waters and its remaining infective capacity. In a first assay, a decrease of more than 50% of the initially viral load compared to the initially inoculated positive sample was detected for all the water samples analyzed. After that, two of the Copahue springs, which are used as an immersion bath in closed environments without going through any disinfection treatment, was selected to determine the viral viability. VERO cell infections were performed, with no cytopathic effect detected, but a strikingly high resistance of the virus, detecting its genome by real time PCR, during the seven days of study under laboratory conditions. SARS-CoV-2 survival in acid media was reaffirmed, which is a peculiarity for a covered virus. A decrease in the detectable viral load of the positive sample was found as the infection time passed, becoming completely negative in the subsequent blind passages. More research is needed to further study the feasibility of SARS-CoV-2 in mineromedicinal waters, especially natural acidic waters that cannot disinfected, in order to expand information about the risk to populations that are exposed to them.