High prevalence of HCMV and viral load in tumor tissues and peripheral blood of glioblastoma multiforme patients.

Glioblastoma multiforme is the most prevalent and malignant tumor of the central nervous system. In the last few years, accumulating evidence has suggested an association between human cytomegalovirus (HCMV) infection and glioblastoma multiforme. In this study, tumor tissues and peripheral blood of patients with glioblastoma multiforme were examined for the presence of HCMV DNA. Twenty-two fresh surgical brain specimens and 20 peripheral blood samples were analyzed by real-time PCR (qPCR) and hemi-nested PCR (nPCR) for the presence of pp65 and (glycoprotein B) gB viral genomic regions, respectively. HCMV DNA was detected in the majority of the tumor samples analyzed (95% by qPCR and 91% by nPCR). About half of the patients with tumors positive for HCMV also had detectable viral DNA in their peripheral blood (47% by qPCR and 61% by nPCR). Genome copy numbers were determined and in the majority of the tumor samples cellular DNA outnumbers viral DNA (average of 1 infected cell in 33 cells). The gB genotypes were determined in HCMV-positive samples and gB2 was the most prevalent genotype in the tumor and blood samples. The results show a high prevalence of HCMV in glioblastoma multiforme samples reinforcing a possible association between HCMV infection and tumor development.

Correction: Molecular modelling of the HCMV IL-10 protein isoforms and analysis of their interaction with the human IL-10 receptor.

[This corrects the article DOI: 10.1371/journal.pone.0277953.].

Human embryonic kidney (HEK-293) cell line: An alternative for rabies virus diagnosis and research.

Rabies is a serious public health problem in developing countries and is caused by Rabies lyssavirus (RABV), a neurotropic RNA virus. The gold standard test for rabies diagnosis is the direct fluorescent antibody test (DFAT). Nevertheless, a confirmatory method is recommended, such as rabies tissue culture infection test (RTCIT). Several cell lines have been tested for RTCIT, and the murine neuroblastoma (Neuro-2a) cell line has been shown to be the most permissive for infection. The human embryonic kidney (HEK-293) cell line was recently thought as an option, due to neuronal protein expression and easy maintenance. In the present work, we evaluated the susceptibility of HEK-293 cell line to RTCIT compared to Neuro-2a. We used a total of 93 brain samples, 48 negatives and 45 positives for RABV previously tested by DFAT or RT-PCR and by RTCIT in Neuro-2a. Of the positive samples, 43 were positive in the traditional RTCIT using Neuro-2a. Two protocols of HEK-293 cell line to RTCIT were tested (with and without virus adsorption) with different incubations times: 24, 48 and 72 h. The highest positive rate in HEK-293 (41 positive samples) resulted from the adsorption protocol with 72 h incubation period, in contrast to 43 positive samples with the traditional RTCIT with Neuro-2a. No satisfactory results were observed using the protocol without adsorption, regardless of the incubation time. Despite the slightly higher sensitivity of Neuro-2a cells, the use of the HEK-293 cells still offers positive aspects, such as, more rapid results, with the advantage of fast and easy growth over Neuro-2a cell line. Therefore, our findings confirm that HEK-293 cells are susceptible to RABV and can be an alternative for RTCIT.

Human Cytomegalovirus Interleukin 10 Homologs: Facing the Immune System.

Human Cytomegalovirus (HCMV) can cause a variety of health disorders that can lead to death in immunocompromised individuals and neonates. The HCMV lifecycle comprises both a lytic (productive) and a latent (non-productive) phase. HCMV lytic infection occurs in a wide range of terminally differentiated cell types. HCMV latency has been less well-studied, but one characterized site of latency is in precursor cells of the myeloid lineage. All known viral genes are expressed during a lytic infection and a subset of these are also transcribed during latency. The UL111A gene which encodes the viral IL-10, a homolog of the human IL-10, is one of these genes. During infection, different transcript isoforms of UL111A are generated by alternative splicing. The most studied of the UL111A isoforms are cmvIL-10 (also termed the "A" transcript) and LAcmvIL-10 (also termed the "B" transcript), the latter being a well-characterized latency associated transcript. Both isoforms can downregulate MHC class II, however they differ in a number of other immunomodulatory properties, such as the ability to bind the IL10 receptor and induce signaling through STAT3. There are also a number of other isoforms which have been identified which are expressed by differential splicing during lytic infection termed C, D, E, F, and G, although these have been less extensively studied. HCMV uses the viral IL-10 proteins to manipulate the immune system during lytic and latent phases of infection. In this review, we will discuss the literature on the viral IL-10 transcripts identified to date, their encoded proteins and the structures of these proteins as well as the functional properties of all the different isoforms of viral IL-10.

CRISPR, Prime Editing, Optogenetics, and DREADDs: New Therapeutic Approaches Provided by Emerging Technologies in the Treatment of Spinal Cord Injury.

Spinal cord injury (SCI) causes temporary disabilities or permanent effects including neuropathic pain and spastiscity. The damage often results from mechanical trauma, which in turn triggers the neuroinflammatory process. Neuroinflammation plays essential roles in the structural, biochemical, and cellular changes that take place in the spinal cord after the injury. Indeed, SCI activates many different signaling pathways that coordinate the resulting cellular responses. While neuroinflammation serves as a physiological reaction to harmful stimuli, it is clear that long-lasting inflammatory response leads to aggravation of the neurodegenerative processes, becoming detrimental to recovery post-injury. In this context, we present some possible therapeutic targets in these activated signaling pathways and provide new perspectives for SCI treatment based on recently developed technologies, including clustered regularly interspaced short palindromic repeats (CRISPR)-based methods (including prime editing), optogenetics, and designer receptor exclusively activated by designer drugs (DREADDs). We critically analyze the recent advances in the deployment of these methods focusing on the control of the initial neuroinflammatory response. We then propose alternatives and provide new avenues for SCI treatment based on these emerging technologies.

Cynomolgus monkeys are successfully and persistently infected with hepatitis E virus genotype 3 (HEV-3) after long-term immunosuppressive therapy.

Epidemiological studies found that hepatitis E virus genotype 3 (HEV-3) infection was associated with chronic hepatitis and cirrhosis in immunocompromised patients. Our study aimed to investigate the relationship between the host immunosuppressive status and the occurrence of HEV-related chronic hepatitis. Here we describe a successful experimental study, using cynomolgus monkeys previously treated with tacrolimus, a potent calcineurin inhibitor immunosuppressant, and infected with a Brazilian HEV-3 strain isolated from naturally infected pigs. HEV infected monkeys were followed up during 160 days post infection (dpi) by clinical signs; virological, biochemical and haematological parameters; and liver histopathology. The tacrolimus blood levels were monitored throughout the experiment. Immunosuppression was confirmed by clinical and laboratorial findings, such as: moderate weight loss, alopecia, and herpes virus opportunistic infection. In this study, chronic HEV infection was characterized by the mild increase of liver enzymes serum levels; persistent RNA viremia and viral faecal shedding; and liver histopathology. Three out of four immunosuppressed monkeys showed recurrent HEV RNA detection in liver samples, evident hepatocellular ballooning degeneration, mild to severe macro and microvesicular steatosis (zone 1), scattered hepatocellular apoptosis, and lobular focal inflammation. At 69 dpi, liver biopsies of all infected monkeys revealed evident ballooning degeneration (zone 3), discrete hepatocellular apoptosis, and at most mild portal and intra-acinar focal inflammation. At 160 dpi, the three chronically HEV infected monkeys showed microscopic features (piecemeal necrosis) corresponding to chronic hepatitis in absence of fibrosis and cirrhosis in liver parenchyma. Within 4-months follow up, the tacrolimus-immunosuppressed cynomolgus monkeys infected with a Brazilian swine HEV-3 strain exhibited more severe hepatic lesions progressing to chronic hepatitis without liver fibrosis, similarly as shown in tacrolimus-immunosuppressed solid organ transplant (SOT) recipients. The cause-effect relationship between HEV infection and tacrolimus treatment was confirmed in this experiment.