Increase In Number Of Plaques Research Articles

Re-emergence of Oropouche virus between 2023 and 2024 in Brazil: an observational epidemiological study

Oropouche virus is an arthropod-borne virus that has caused outbreaks of Oropouche fever in central and South America since the 1950s. This study investigates virological factors contributing to the re-emergence of Oropouche fever in Brazil between 2023 and 2024. In this observational epidemiological study, we combined multiple data sources for Oropouche virus infections in Brazil and conducted in-vitro and in-vivo characterisation. We collected serum samples obtained in Manaus City, Amazonas state, Brazil, from patients with acute febrile illnesses aged 18 years or older who tested negative for malaria and samples from people with previous Oropouche virus infection from Coari municipality, Amazonas state, Brazil. Basic clinical and demographic data were collected from the Brazilian Laboratory Environment Management System. We calculated the incidence of Oropouche fever cases with data from the Brazilian Ministry of Health and the 2022 Brazilian population census and conducted age-sex analyses. We used reverse transcription quantitative PCR to test for Oropouche virus RNA in samples and subsequently performed sequencing and phylogenetic analysis of viral isolates. We compared the phenotype of the 2023-24 epidemic isolate (AM0088) with the historical prototype strain BeAn19991 through assessment of titre, plaque number, and plaque size. We used a plaque reduction neutralisation test (PRNT50) to assess the susceptibility of the novel isolate and BeAn19991 isolate to antibody neutralisation, both in serum samples from people previously infected with Oropouche virus and in blood collected from mice that were inoculated with either of the strains. 8639 (81·8%) of 10 557 laboratory-confirmed Oropouche fever cases from Jan 4, 2015, to Aug 10, 2024, occurred in 2024, which is 58·8 times the annual median of 147 cases (IQR 73-325). Oropouche virus infections were reported in all 27 federal units, with 8182 (77·5%) of 10 557 infections occurring in North Brazil. We detected Oropouche virus RNA in ten (11%) of 93 patients with acute febrile illness between Jan 1 and Feb 4, 2024, in Amazonas state. AM0088 had a significantly higher replication at 12 h and 24 h after infection in mammalian cells than the prototype strain. AM0088 had a more virulent phenotype than the prototype in mammalian cells, characterised by earlier plaque formation, between 27% and 65% increase in plaque number, and plaques between 2·4-times and 2·6-times larger. Furthermore, serum collected on May 2 and May 20, 2016, from individuals previously infected with Oropouche virus showed at least a 32-fold reduction in neutralising capacity (ie, median PRNT50 titre of 640 [IQR 320-640] for BeAn19991 vs <20 [ie, below the limit of detection] for AM0088) against the reassortant strain compared with the prototype. These findings provide a comprehensive assessment of Oropouche fever in Brazil and contribute to an improved understanding of the 2023-24 Oropouche virus re-emergence. Our exploratory in-vitro data suggest that the increased incidence might be related to a higher replication efficiency of a new Oropouche virus reassortant for which previous immunity shows lower neutralising capacity. São Paulo Research Foundation, Burroughs Wellcome Fund, Wellcome Trust, US National Institutes of Health, and Brazilian National Council for Scientific and Technological Development. For the Portuguese translation of the abstract see Supplementary Materials section.

P173 Progression of subclinical cardiovascular disease in SLE: a five year follow up study

Abstract Background Patients with SLE have 5-10-fold increased risk of developing CVD compared to age and sex-matched controls. The average age of developing a first CVD event in patients with SLE is only 49. In this study we aimed to describe the rate and determinants of carotid plaque progression in a cohort of SLE patients who were asymptomatic of CVD at baseline. Methods We carried out vascular ultrasound studies of 100 patients with SLE asymptomatic of CVD at baseline. Sixty-nine patients were rescanned over a median of 5 years of follow up. Ninety-four percent who were re-scanned were women and the mean overall age was 46 years (SD 11). Clinical and CVD risk was assessed at baseline and follow up. The same protocol for assessment at baseline using B-mode Doppler ultrasound to measure intimal media thickness and plaque was used at follow up to assess progression. We also assessed total plaque area (TPA), a more sensitive measure of plaque, and echolucency expressed as gray scale median (GSM) which is linked to plaque lipid content. Results Of the 100 patients with a baseline scan, 69 patients had a second scan at a median of 5 years follow up. New plaque developed in 9% and 26% had an increase in plaque number. The mean overall IMT (0.111 vs 0.064, p &amp;lt; 0.01) and common carotid IMT (0.065 vs 0.055, p &amp;lt; 0.01) were significantly raised in plaque vs non-plaque patients. In a multi -variable analysis CIMT at follow-up was independently associated with age (beta 0.415, p &amp;lt; 0.001) and diastolic blood pressure (beta 0.285, p &amp;lt; 0.021). Independent predictors of plaque at follow-up scan on multi-variable analysis were age at scan&amp;gt;52 years (OR 10.41, CI 2.66-40.80) and systolic BP &amp;gt; 133 (OR 5.26, CI 1.396 - 19.862). In contrast, total cholesterol was negatively correlated with TPA (beta = -1.167, p = 0.002) and with GSM (beta = -0.513, p = 0.012). Conclusion Amongst these 69 patients, 26% had increased plaque and none had decreased plaque over a median of five years follow-up. Measurement of novel ultrasound variables such as TPA and echolucency may identify more modifiable risk factors that can be used to improve CVD outcomes in patients with SLE. Disclosures J. Bakshi: None. M. Griffin: None. S. Croca: None. F. Farhina: None. D. Isenberg: None. A. Nicolaides: None. A. Rahman: None.

Everolimus delayed and suppressed cytomegalovirus DNA synthesis, spread of the infection, and alleviated cytomegalovirus infection

Everolimus is an inhibitor of mammalian target of rapamycin (mTOR) and reduces the risk of cytomegalovirus (CMV) infection in transplant recipients. Everolimus inhibits mTOR complex 1, which regulates factors involved in several crucial cellular functions and is required for CMV replication. However, it is not clear how everolimus regulates CMV replication and prevents and alleviates CMV infection. Effects of everolimus on CMV infection, spread, and DNA synthesis and release from infected cells were assessed by plaque formation, infectious centre assay, real-time PCR of infected cells, and culture supernatant in CMV-infected cultures with and without everolimus. Everolimus enhanced plaque formation by 3.6 times, but the size of the plaques was reduced to 36.4% of untreated cultures in the absence of a pretreatment period. Everolimus reduced viral adsorption but enhanced the replication efficiency of inoculated virus, resulting in an increase in plaque number in the early phase of infection. Preinfection treatment of cells with everolimus efficiently exhibited its antiviral efficacy, and everolimus delayed and suppressed viral DNA synthesis and release from infected cells. Everolimus had suppressed the spread of infection and reduced the number of total infected cells to 40% of untreated cells on day 9, indicating reduction of the size of CMV lesions to one-sixth in 2–3 replication cycles. Preinfection treatment of the cells with everolimus augmented its suppressive effect on CMV infection and replication. Everolimus reduced the total number of infected cells and limited the CMV lesions, and this reduction in the spread of CMV infection would alleviate CMV infection in transplant recipients.

Abstract B132: Antitumor effect of oncolytic vaccinia virus in gastric cancer

Abstract Introduction: As the prognosis of metastatic gastric cancer patients is extremely poor, development of novel effective treatment is necessary. Pexa-Vec (pexastimogene devacirpvec; JX-594) engineered from the Wyeth vaccine strain has emerged as an attractive oncolytic virotherapy in cancer treatment. The aim of this study is to investigate the efficacy of Pexa-Vec against human gastric cancer cell lines and to identify potential biomarkers of oncolytic virotherapy. Method: Forty-nine human gastric cancer cell lines and normal human umbilical vein endothelial cell (HUVEC), FHs 173We, human embryonic kindey 293(HEK 293), and Hela cell line as control were used in this study. For evaluation of cytopathic effect, gastric cancer cells were infected with JX594 (M.O.I [multiplicity of infection]= 0.1-5), then incubated at 37°C. Cell viability was measured 72 hours postinfection using MTT assay. Viral plaque assay was used for quantification of Pexa-Vec infection. Copy number variation (CNV), single nucleotide variation (SNV), gene expression using whole exome sequencing (WES,) and RNA sequencing of 49 gastric cancer cell lines were analyzed. In addition, expression levels for TK and EGFR family were confirmed by Western blot, and secreted VEGF (vascular endothelial growth factor) levels in conditioned media of gastric cancer were measured using ELISA (enzyme-linked immunosorbent assay). Result: Cytopathic effect of Pexa-Vec was observed in all 49 gastric cancer cell lines. Median ED 50 (effective dose 50) was 0.37 pfu/cell (range 0.02-6.18) in gastric cancer cells and 0.30 pfu/cell (range 0.14-1.67) in normal control cells. Interestingly, EBV-infected cells (YCCEL1/YCC-10, SNU 719) showed relative resistance to Pexa-Vec when compared with other cells (ED 50; 4.34 [range 2.49-6.18]) vs 0.36 pfu/cell (range 0.02-5.00, p=0.003). Plaque assay was performed to quantify Pexa-Vec viral infection of gastric cancer cell lines, but the plaques were too small and diffuse to quantify. Increase in virus M.O.I. seemed to be correlated with increase in plaque numbers, yet this did not explain the association with the degree of TK expression in gastric cancer cells. In addition, EGFR, HER2, HER3, and secreted VEGF expressions were not related to the cytotoxicity of Pexa-Vec (R=-0.19, p=0.19; R=0.06, p=0.537; R=0.18, p=0.232; R=-0.067, p=0.69). From RNA sequencing data, VEGF B expression showed negative correlation with antitumor acitivity of Pexa-Vec (R=- 0.295, p=0.04). Conclusion: Pexa-Vec showed moderate antitumor effects in gastric cancer cell lines in vitro compared with other cancer types. Interestingly, EBV-infected gastric cancer cells showed relatively lower efficacy. In our study, correlation was not observed between TK and EGFR expression levels and cytopathic effects. Therefore, we need to further investigate the mechanisms of action of Pexa-Vec for development of metastatic gastric cancer treatment. Citation Format: Jee Hung Kim, Woo Sun Kwon, Tae Soo Kim, Kyu Hyun Park, Joong Bae Ahn, Namhee Lee, Ji Won Choi, Hyun Cheol Chung, Sun Young Rha. Antitumor effect of oncolytic vaccinia virus in gastric cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B132.

Characterization of amyloid deposition in the APPswe/PS1dE9 mouse model of Alzheimer disease

Transgenic mice carrying disease-linked forms of genes associated with Alzheimer disease often demonstrate deposition of the beta-amyloid as senile plaques and cerebral amyloid angiopathy. We have characterized the natural history of beta-amyloid deposition in APPswe/PS1dE9 mice, a particularly aggressive transgenic mouse model generated with mutant transgenes for APP (APPswe: KM594/5NL) and PS1 (dE9: deletion of exon 9). Ex vivo histochemistry showed Abeta deposition by 4 months with a progressive increase in plaque number up to 12 months and a similar increase of Abeta levels. In vivo multiphoton microscopy at weekly intervals showed increasing beta-amyloid deposition as CAA and plaques. Although first appearing at an early age, CAA progressed at a significantly slower rate than in the Tg2576 mice. The consistent and early onset of beta-amyloid accumulation in the APPswe/PS1dE9 model confirms its utility for studies of biochemical and pathological mechanisms underlying beta-amyloid deposition, as well as exploring new therapeutic treatments.

The Effect of Herpes Simplex Virus Type 1 L-Particles on Virus Entry, Replication, and the Infectivity of Naked Herpesvirus DNA

Herpes simplex virus type 1(HSV-1) L-particles are known to be composed mainly of envelope and tegument proteins, to lack the nucleocapsid, and to be noninfectious. Thus L-particles represent interesting vaccine candidates. L-particles at >1000/cell interfered with HSV-1 virion adsorption and penetration. While L-particles did not affect HSV-1 growth kinetics in resting or nonresting BHK cultures infected with purified virions, treatment with L-particles before, or after, transfection with HSV-1 DNA resulted in a progressive increase in plaque numbers (five- to sixfold at 1000 L-particles/cell). Transfection assays using HSV-1tsmutant DNA (ts1201) revealed that enhancement was due to induction of otherwise nonreplicating genomes. The enhancement obtained with L-particles produced by WT HSV-1 or by mutants that are either deleted, or defective, in certain gene products was compared. Most important were the Vmw110 (ICP0) and Vmw65 (α-TIF) proteins, but VP11/12, VP13/14, and vhs also have a role. The L-particle-associated Vmw175 (ICP 4) protein did not appear be involved. The effect of homologous and heterologous combinations of pseudorabies virus, equineherpesvirus-1, and HSV-1 DNA's and L-particles was investigated in transfection assays. The L-particles of each virus, to varying extent, enhanced the plaquing efficiency of their own DNA but were also effective in heterologous combinations.

Growth hormone secretion by human peripheral blood mononuclear cells detected by an enzyme-linked immunoplaque assay.

In order to evaluate the secretion of GH from human peripheral blood mononuclear cells (PBMC) and its possible role as a modulator of lymphoproliferation, we have developed a hormonal enzyme-linked immunoplaque assay. This assay captures GH between a monoclonal and polyclonal antibody. This is followed by adding substrate and a horseradish peroxidase-conjugated antibody against the polyclonal antibody which produces violet colored plaques where GH has been secreted. This assay is sensitive, specific, highly reproducible, and can detect picogram quantities of GH. Using this assay we have detected GH secretion from approximately 1% of human PBMC under unstimulated conditions. Regression analysis showed a linear relationship between the number of cells plated and the number of GH plaques formed. Therefore, GH plaques were formed by single cells or its progeny and did not represent aggregation of secreting cells. Preincubation of PBMC with cycloheximide, a protein synthesis inhibitor, completely abolished the formation of GH plaques which suggests that the PBMC were responsible for the synthesis of the secreted GH. In addition, we have also observed that stimulation of human PBMC with T-cell mitogens, Concanavalin A and PHA and the cytokine IL-2 led to significant increases in GH plaque area and number whereas stimulation with LPS, a B cell mitogen, was ineffective. The PHA and IL-2 induced increase in plaque number suggests that they can recruit noncommitted lymphocytes to actively secrete GH which raises the possibility that this secreted GH may serve as a growth factor in T cell proliferation. We conclude that this immunoplaque assay may be useful in evaluating the secretion of other peptides from human immune cells.

Neutralization and Enhancement of Infectivity of Non-salmonid Fish Rhabdoviruses by Rabbit and Pike Immune Sera

SUMMARY Plaque neutralization assays were carried out using the rhabdoviruses of pike fry (2 isolates), spring viraemia of carp and grass carp. When rabbit anti-pike fry rhabdovirus sera were used, no neutralizing activity was observed but plaque counts increased with heat-inactivated sera or sera that had been stored at -20 °C for prolonged periods. Antibody binding to the virus was demonstrated by gradient centrifugation experiments. The use of immune serum alone or in combination with anti-rabbit gamma globulin serum resulted in aggregation of radioactive virus. Aggregation was also observed when anti-rabbit gamma globulin serum was added under neutralization test conditions. Addition of non-inactivated guinea-pig serum to mixtures of pike fry rhabdovirus and rabbit antiserum to spring viraemia of carp virus resulted in strong neutralization, whereas this antiserum alone or in the presence of inactivated guinea-pig serum gave an increase in plaque numbers. The increase in plaque number and the complement-dependent neutralization phenomena were weak or absent in the homologous spring viraemia of carp virus system. Immunization of pike with pike fry rhabdovirus resulted in an increase in virus neutralizing activity which could be attributed to the macroglobulin fraction of the serum. After immunizations over a period of 6 months with virus antigens, no further increase in neutralizing activity was observed. Pike sera showed 50% plaque reduction at concentrations between 0.017 and 0.3%. Using these sera it could be demonstrated that the grass carp virus and the V 75/94 isolate from pike fry were indistinguishable from the original pike fry rhabdovirus, whereas spring viraemia of carp virus was different.

Effects of pyrrol-carboxylic acid derivatives on the growth of coliphages

Effects of 14 pyrrol-carboxylic acid derivatives and analogues (PY-compounds) on the growth of coliphage MS2 using E. coli E102 (Hfr) as the host were measured by the agar double-layer method. Enlargements of plaque size were observed with 7 PY-compounds but increase in plaque numbers was not induced. These enlargements of plaque size were specific to RNA coliphages MS2, GA and Qβ and not found with DNA coliphages δ AC and T4. Furthermore, the interaction between the PY-compound PY-10 and the coliphage MS2 was dependent on the host bacterium (indicator strain). When E102 (Hfr) was used, the enlargement was marked, in the case of substrain W1895 (Hfr) it was less, while in the case of substrain W6 (F +) it was undetectable. The one-step growth of the phage MS2 and the production of intracellular phage MS2 were little affected by the PY-compound PY-10. However, the rate of one-step growth was increased in the early stage after infection. Accordingly, the enlargements of plaque size by the PY-compounds might be correlated with an increase in rate of release of phage particles.

INFLUENCE OF ACID POLYSACCHARIDES ON PLAQUE FORMATION BY INFLUENZA A2 AND B VIRUSES.

Summary1. The inhibitory effect of the sulfated agar polysaccharide on strains of influenza A2 and B viruses was investigated. Complexing of the inhibitor from agar through the use of DEAE-dextran resulted in an improvement in plaque development as well as an increase in number of plaques formed. An optimum DEAE-dextran concentration of 1.0 mg/ml agar was found. 2. The inhibitor did not inactivate virus and had no effect on the hemagglutinating activity of both viruses, but it did interfere with virus adsorption to MK monolayers.