Human cytomegalovirus inhibits the proliferation and invasion of extravillous cytotrophoblasts via Hippo-YAP pathway

Background Human cytomegalovirus (HCMV) infection in utero is very common during pregnancy, which can lead to adverse outcomes in both pregnancy and progeny, but its pathogenesis has not been fully clarified. The decrease of extravillous cytotrophoblasts (EVT) invasion is an essential pathophysiological process of some pregnancy complications. Hippo-YAP signaling pathway plays an important role in regulating cell proliferation and apoptosis. However, whether YAP is involved in HCMV uterine infection remains to be studied. Methods The primary EVT was cultured and infected by the HCMV strain AD169 virus in vitro. Immunofluorescence staining of HCMVpp65 antigen was conducted afterward to confirm the establishment of an infection model. The optimal virus infection dose was determined by the EVT proliferation status in vitro. Real-time PCR was performed to examine the mRNA level of major genes involved in the Hippo pathway in EVT after HCMV infection. The effect of HCMV on the expression of YAP protein in EVT was evaluated by Immunofluorescence staining and Western blot. An in vitro cell invasion assay was carried out to analyze the influence of HCMV on EVT invasion. The changes of EVT invasion was accessed by establishing YAP silencing and over-expression models using YAP1 specific siRNA and plasmid pcDH. Results The optimal HCMV infection dose was 282.5TCID50/ml. Compared to the control group, the infection of HCMV significantly reduced the mRNA expression of Mst1, Mst2, SAV, Lats1, Lats2, Mob1, YAP1, TAZ, TEAD1-4 genes and YAP protein expression in the Hippo-YAP pathway. HCMV infection also decreased the EVT invasion. In non-infected EVT, the number of transmembrane EVT cells was significantly reduced when YAP1 gene was silenced, while it was significantly increased when YAP1 gene was over-expressed. In the HCMV-infected EVT, the number of transmembrane EVT cells significantly increased when over-expressed and eventually recovered to the level of NC. Conclusions HCMV may decrease EVT invasion by inhibiting the expression of mRNA and protein of YAP in the Hippo-YAP signaling pathway. HCMV eventually reduces the invasion ability of EVT by inhibiting multiple genes in the Hippo-YAP signaling pathway, especially inhibiting YAP which serves as the downstream effector.

Establishment and validation of a guinea pig model for human congenital toxoplasmosis

Background Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection in humans and animals may lead to severe symptoms in the offspring, especially in the brain. A suitable animal model for human congenital toxoplasmosis is currently lacking. The aim of this study is to establish and validate the guinea pig as a model for human congenital toxoplasmosis by investigating the impact of the T. gondii infection dose, the duration of infection and the gestational stage at infection on the seroconversion, survival rate of dams, fate of the offspring, T. gondii DNA loads in various offspring tissues and organs and the integrity of the offspring brain. Methods Pregnant guinea pigs were infected with three different doses (10, 100, 500 oocysts) of T. gondii strain ME49 at three different time points during gestation (15, 30, 48 days post-conception). Serum of dams was tested for the presence of T. gondii antibodies using immunoblotting. T. gondii DNA levels in the dam and offspring were determined by qPCR. Offspring brains were examined histologically. Results We found the survival rate of dams and fate of the offspring to be highly dependent on the T. gondii infection dose with an inoculation of 500 oocysts ending lethally for all respective offspring. Moreover, both parameters differ depending on the gestational stage at infection with infection in the first and third trimester of gestation resulting in a high offspring mortality rate. The duration of infection was found to substantially impact the seroconversion rate of dams with the probability of seroconversion exceeding 50% after day 20 post-infection. Furthermore, the infection duration of dams influenced the T. gondii DNA loads in the offspring and the integrity of offspring brain. Highest DNA levels were found in the offspring brain of dams infected for ≥ 34 days. Conclusion This study contributes to establishing the guinea pig as a suitable model for human congenital toxoplasmosis and thus lays the foundation for using the guinea pig as a suitable animal model to study scientific questions of high topicality and clinical significance, which address the pathogenesis, diagnosis, therapy and prognosis of congenital toxoplasmosis. Graphical abstract

THE EFFECT OF TOXOPLASMAS ON THE CHANGES IN THE EXPRESSION OF BIRC5, ERBB-2/HER2-NEU, GLI, VEGF PROTOONCOGENES AND THE ANTI-ONCOGENE TP53 IN RATS IN THE EXPERIMENT

Objectives. To study the effect of toxoplasmas on the changes in the expression of protooncogenes BIRC5, ERBB-2/HER2-NEU, GLI, VEGF and the anti-oncogene TP53 in rats in an experiment depending on the infection dose and the period of parasite development. Material and methods.The experiment was conducted on female Wistar rats to determine changes in the expression of the protooncogenes survivin (BIRC5), epidermal growth factor (ErbB-2/HER2-Neu), GLI 1, vascular endothelial growth factor (VEGF) and the anti-oncogene TP53 in comparison with the reference genes β-actin (ACTB) and GAPDH by means of PCR analysis in the tissues of 10 healthy and 120 animals invaded at different doses. Statistical comparison of the results of all groups was drawn with the data of the «control» series (healthy animals, biopsies of the lungs, liver, spleen, brain). The results obtained in the experimental groups were as follows: the infection dose of 25 toxoplasma tachyzoites per 1 g of the animal body weight (5000 tachyzoites per female) and the infection dose of 50 toxoplasma tachyzoites per 1 g of the animal body weight (10000 tachyzoites per female), then they were also compared with each other. Statistical processing of the obtained data was carried out using the program Statistica 10.0. The differences were considered to be reliable at a significance level of less than 0.05 (p<0.05). Results. Toxoplasma was found to cause an infection dose-dependent increase in the expression of the protooncogenes survivin (BIRC5), epidermal growth factor (ErbB-2/HER2-Neu), GLI, vascular endothelial growth factor (VEGF) and a change in the strength expression of the anti-oncogene TP53 at all stages of the parasite development. Conclusions. Experimental toxoplasmosis alters the expression of the protooncogenes survivin (BIRC5), epidermal growth factor (ErbB-2/HER2-Neu), GLI, vascular endothelial growth factor (VEGF), and the anti-oncogene TP53 in the tissues of the intermediate host.

Human Cytomegalovirus Inhibits the Proliferation and Invasion of Extravillous Cytotrophoblasts Via Hippo-YAP Pathway

Background: Human cytomegalovirus (HCMV) infection in utero is very common during pregnancy, which can lead to adverse outcomes in both pregnancy and progeny, but its pathogenesis has not been fully clear. The decrease of extracellular trophoblast (EVT) invasion is an essential pathophysiological process of some pregnancy complications. Hippo-YAP signaling pathway plays an important role in regulating cell proliferation and apoptosis. However, whether YAP is involved in HCMV uterine infection remains to be studied.Methods: The primary EVT was cultured and infected by the HCMVAD169 virus in vitro. Immunofluorescence staining of HCMVpp65 antigen was conducted afterward to confirm the establishment of an infection model. The optimal virus infection dose was determined by the EVT proliferation status in vitro. Real-time PCR was performed to examine the mRNA level of major genes involved in the Hippo pathway in EVT after HCMV infection. The effect of HCMV on the expression of YAP protein in EVT was evaluated by Immunofluorescence staining and Western blot. An in vitro cell invasion assay was carried out to analyze the influence of HCMV on EVT invasion. The changes of EVT invasion was accessed by establishing YAP silencing and over-expression models using YAP1 specific siRNA and plasmid pcDH.Results: The optimal HCMV infection dose was 30μl 100 TCID50 HCMV / 120μl medium. Compared to the control group, the infection of HCMV significantly reduced the mRNA expression of Mst1, Mst2, SAV, Lats1, Lats2, Mob1, YAP1, TAZ, TEAD1-4 genes and YAP protein expression in the Hippo-YAP pathway. HCMV infection also decreased the EVT invasion. In non-infected EVT, the number of transmembrane EVT cells was significantly reduced when YAP1 gene was silenced, while it was significantly increased when YAP1 gene was over-expressed. In the HCMV-infected EVT, the number of transmembrane EVT cells significantly increased when over-expressed and eventually recovered to the level of NC.Conclusions: HCMV may decrease EVT invasion by inhibiting the expression of mRNA and protein of YAP in the Hippo-YAP signaling pathway. HCMV eventually reduces the invasion ability of EVT by inhibiting multiple genes in the Hippo-YAP signaling pathway, especially inhibiting YAP which serves as the downstream effector.

Gambaran Jumlah dan Hitung Jenis Leukosit Ayam Petelur yang diinfeksi L2 Toxocara Cati

The aim of this study was to determine leukocytes and differential counting in chicken after infected with L2 Toxocra cati. In this study, it was used twenty chickens, wich were 14 weeks old. They were divided into 4 groups. Chickens were infected by orally with dose 0 eggs/ml L2 T. cati, 10 eggs/ml L2 T. cati, 100 eggs/ml L2 T. cati and 1000 eggs/ml L2 T. cati. Blood sampling were conducted on 2, 7 and 21 days after infection. Leukocyte value was determined by improve neubauer and differential counting stained with Wright’s stain then determined using microscope 1000x. The data was analyzed by Anova Factorial and then continued by BNJ Test at 5%. The result showed that increased of leukocytes value and eosinophil at 7 and 21 days after infection L2 T. cati with different dose, increased of leukocytes value and eosinophil on infection dose 10 eggs/ml L2 T. cati, 100 eggs/ml L2 T. cati and 1000 eggs/ml L2 T. cati. It shows that there was not intereraction between time of infection process and infection dose of L2 T. Cati.

Low and high infection dose transmissions of SARS-CoV-2 in the first COVID-19 clusters in Northern Germany

ObjectivesWe used viral genomics to deeply analyze the first SARS-CoV-2 infection clusters in the metropolitan region of Hamburg, Germany. Epidemiological analysis and contact tracing together with a thorough investigation of virus variant patterns revealed low and high infection dose transmissions to be involved in transmission events.MethodsInfection control measures were applied to follow up contract tracing. Metagenomic RNA- and SARS-CoV-2 amplicon sequencing was performed from 25 clinical samples for sequence analysis and variant calling.ResultsThe index patient acquired SARS-CoV-2 in Italy and after his return to Hamburg transmitted it to 2 out of 132 contacts. Virus genomics and variant pattern clearly confirms the initial local cluster. We identify frequent single nucleotide polymorphisms at positions 241, 3037, 14408, 23403 and 28881 previously described in Italian sequences and now considered as one major genotype in Europe. While the index patient showed a single nucleotide polymorphism only one variant was transmitted to the recipients. Different to the initial cluster, we observed in household clusters occurring at the time in Hamburg also intra-host viral species transmission events.ConclusionsSARS-CoV-2 variant tracing highlights both, low infection dose transmissions suggestive of fomites as route of infection in the initial cluster and high and low infection dose transmissions in family clusters indicative of fomites and droplets as infection routes. This suggests (1) single viral particle infection can be sufficient to initiate SARS-CoV-2 infection and (2) household/family members are exposed to high virus loads and therefore have a high risk to acquire SARS-CoV-2.

A meta-analysis reveals temperature, dose, life stage, and taxonomy influence host susceptibility to a fungal parasite

Complex ecological relationships, such as host-parasite interactions, are often modeled with laboratory experiments. However, some experimental laboratory conditions, such as temperature or infection dose, are regularly chosen based on convenience or convention and it is unclear how these decisions systematically affect experimental outcomes. Here, we conducted a meta-analysis of 58 laboratory studies that exposed amphibians to the pathogenic fungus Batrachochytrium dendrobatidis (Bd) to better understand how laboratory temperature, host life stage, infection dose, and host species affect host mortality. We found that host mortality was driven by thermal mismatches: hosts native to cooler environments experienced greater Bd-induced mortality at relatively warm experimental temperatures and vice versa. We also found that Bd dose positively predicted Bd-induced host mortality and that the superfamilies Bufonoidea and Hyloidea were especially susceptible to Bd. Finally, the effect of Bd on host mortality varied across host life stages, with larval amphibians experiencing lower risk of Bd-induced mortality than adults or metamorphs. Metamorphs were especially susceptible and experienced mortality when inoculated with much smaller Bd doses than the average dose used by researchers. Our results suggest that when designing experiments on species interactions, researchers should carefully consider the experimental temperature, and inoculum dose, and life stage and taxonomy of the host species.

Dynamical Analysis of the SEIB Model for Brucellosis Transmission to the Dairy Cows with Immunological Threshold

As we all know, bacteria is different from virus which with certain types can be killed by the immune cells in the body. The brucellosis, a bacterial disease, can invade the body by indirect transmission from environment, which has not been researched by combining with immune cells. Considering the effects of immune cells, we put a minimum infection dose of brucellosis invading into the dairy cows as an immunological threshold and get a switch model. In this paper, we accomplish a thorough dynamics analysis of a SEIB switch model. On the one hand, we can get a disease-free and bacteria-free steady state and up to three endemic steady states which may be thoroughly analyzed in different cases of a minimum infection dose in a switch model. On the other hand, we calculate the basic reproduction number R0 and know that the disease-free and bacteria-free steady state is a global stability when R0<1, and the one of the endemic steady state is a conditionally global stability when R0>1. We find that different amounts of R0 may lead to different steady states of brucellosis, and considering the effects of immunology is more serious in mathematics and biology.