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The clinical manifestations of neurological complications associated with varicella zoster virus (VZV) are non-specific and indistinguishable from those of other viral infections. Therefore, the definite diagnosis requires evidence of VZV infection in cerebrospinal fluid (CSF). The aim of this study was to determine the frequency of VZV DNA detection in CSF of patients with neurological diseases in order to obtain information concerning involvement of VZV infection in neuropathology in the country. This study is a retrospective survey of test results obtained from January 2015 to October 2019. During this period, 411 CSF specimens were tested for the presence of VZV DNA by nested PCR. Fisher's exact test was used to test for statistically significant difference in the frequency of VZV DNA positivity of CSF specimens from different groups. Of all 411 tested CSF samples, 11.2% were positive for VZV DNA. The highest VZV prevalence was detected in CFS from patients with meningitis-18.2%, followed by patients with cranial neuritis (15.4%), encephalitis (12.2%), Guillain-Barre syndrome (11.1%), myelitis (10%), and with other neurological syndromes (8.2%). The difference of VZV prevalence in CSF of patients according to the gender and age was not statistically significant. Our results indicated that VZV is a frequent causative agent of neurological diseases, suggesting an important role of VZV infection for neuropathology in the country. Therefore, efforts for wider application of VZV identification in CSF to facilitate faster onset of antiviral treatment and further strategies concerning varicella zoster virus vaccines in the country are needed.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a disabling multisystem chronic disease. The etiology and pathogenesis of ME/CFS are unknown. Infections of cytomegalovirus (CMV), Epstein‐Barr virus (EBV), and human herpesvirus‐6 (HHV‐6) are suspected as etiological agents for ME/CFS. This study aims to estimate prevalence and type (active/latent) of EBV, CMV, and HHV‐6 infections in Bulgarian ME/CFS patients. In the study were included 58 patients with ME/CFS and 50 healthy controls. Virus‐specific antibodies were detected by enzyme‐linked immunosorbent assay and viral genomic sequences in peripheral blood mononuclear cell (PBMCs) and plasma samples by nested polymerase chain reaction (PCR). We did not observe any significant differences in virus‐specific immunoglobulin G and immunoglobulin M positivity rates between patients with ME/CFS and control group. In ME/CFS plasma samples, EBV DNA was found in 24.1%, CMV DNA in 3.4%, and HHV‐6 DNA in 1.7% of samples. EBV DNA was detected in 4%, and CMV and HHV‐6 DNA were not found in plasma samples of controls. The frequency of viral genome detection in PBMCs of patients and controls was 74% vs 78% for CMV, 81% vs 84% for EBV, and 82.8% vs 82% for HHV‐6. The difference in frequency of EBV active infection in ME/CFS and control group was statistically significant (P = .0027). No ME/CFS and control individuals with active CMV and HHV‐6 infection were observed. In conclusion, this study using both serological and PCR‐based techniques for distinguishing between active and latent infection showed high rate of active EBV infection among patients with ME/CFS indicating that at least in a subset of cases, EBV is important factor for the development of disease.

This review aimed at determining the prevalence and incidence of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) in Europe. We conducted a primary search in Scopus, PubMed and Web of Science for publications between 1994 and 15 June 2019 (PROSPERO: CRD42017078688). Additionally, we performed a backward-(reference lists) and forward-(citations) search of the works included in this review. Grey literature was addressed by contacting all members of the European Network on ME/CFS (EUROMENE). Independent reviewers searched, screened and selected studies, extracted data and evaluated the methodological and reporting quality. For prevalence, two studies in adults and one study in adolescents were included. Prevalence ranged from 0.1% to 2.2%. Two studies also included incidence estimates. In conclusion, studies on the prevalence and incidence of ME/CFS in Europe were scarce. Our findings point to the pressing need for well-designed and statistically powered epidemiological studies. To overcome the shortcomings of the current state-of-the-art, EUROMENE recommends that future research is better conducted in the community, reviewing the clinical history of potential cases, obtaining additional objective information (when needed) and using adequate ME/CFS case definitions; namely, the Centers for Disease Control & Prevention−1994, Canadian Consensus Criteria, or Institute of Medicine criteria.

A possible association between high-risk human papillomaviruses (HPV) and lung cancer has been investigated for decades with discrepant results. The aim of this study was to determine the prevalence of HPV16 and 18 in Bulgarian patients with lung cancer. Two hundred and nine biopsy specimens from patients with histologically proven lung cancer and without cancer were analyzed. Each sample was subjected to three parallel PCRs using broad spectrum GP5+/6+ primers and type-specific (TS) primers for HPV types 16 and 18. Of the 132 lung carcinoma samples, 33 (25%) were positive for HPV16 and/or HPV18 by TS PCR whereas only five (3.8%) samples were HPV positive by consensus PCR. All non-malignant controls were HPV negative. HPV18 was the more prevalent, being found in 11.4% of samples, followed by HPV16 in 9.1% samples; 4.5% of lesions were positive for both HPV16 and HPV18. HPV16/18 were most prevalent in small cell carcinoma (29.2%) and least prevalent in squamous cell carcinoma (23.3%). HPV was only detected in squamous cell carcinoma and adenosquamous carcinoma by consensus PCR. This study revealed a high HPV16/18 prevalence in lung carcinoma samples from Bulgarian patients when TS PCR was used to detect them. The difference between HPV positivity as detected by consensus and by TS PCR was significant, indicating the importance of methodological issues in explaining the discrepancies between previous studies. HPV18 was more common than HPV16. No association between HPV16/18 status and histopathological diagnosis was identified.

Aim: Although sexually transmitted viral infections are significant and increasing public health concern, little is known about their prevalence among Bulgarian women. The aim of this study was to investigate cervical viral infections in asymptomatic women. Methods: The study group included 52 randomly selected asymptomatic female volunteers from Bulgarian border town Kardzhali. Cervical specimens were tested by real-time PCR for human papillomaviruses (HPV), herpes simplex virus (HSV) types 1 and 2, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human immunodeficiency virus (HIV). Results: The investigation demonstrated a high rate (61.5%) of infection with one or more viruses of uterine cervix of participating women considered at low risk. The most prevalent was HPV, found in 32.7% of all women. The genotyping of high-risk (HR) HPV positive specimens showed that HPV16 was the most prevalent HR type. HSV prevalence (30.8%) was almost as high as that of HPV and most women were HSV1 infected. 9.6% and 5.8% of all specimens were positive for EBV and CMV, respectively. Conclusions: Our results indicate that women from Kardzhali region, involved in this study, might be at risk for development of genital tract pathology, including cervical cancer, and for transmission of virus infection sexually and perinatally.

A possible association between high-risk human papillomaviruses (HPV) and lung cancer has been investigated for decades with discrepant results. The aim of this study was to determine the prevalence of HPV16 and 18 in Bulgarian patients with lung cancer. Two hundred and nine biopsy specimens from patients with histologically proven lung cancer and without cancer were analyzed. Each sample was subjected to three parallel PCRs using broad spectrum GP5+/6+ primers and type-specific (TS) primers for HPV types 16 and 18. Of the 132 lung carcinoma samples, 33 (25%) were positive for HPV16 and/or HPV18 by TS PCR whereas only five (3.8%) samples were HPV positive by consensus PCR. All non-malignant controls were HPV negative. HPV18 was the more prevalent, being found in 11.4% of samples, followed by HPV16 in 9.1% samples; 4.5% of lesions were positive for both HPV16 and HPV18. HPV16/18 were most prevalent in small cell carcinoma (29.2%) and least prevalent in squamous cell carcinoma (23.3%). HPV was only detected in squamous cell carcinoma and adenosquamous carcinoma by consensus PCR. This study revealed a high HPV16/18 prevalence in lung carcinoma samples from Bulgarian patients when TS PCR was used to detect them. The difference between HPV positivity as detected by consensus and by TS PCR was significant, indicating the importance of methodological issues in explaining the discrepancies between previous studies. HPV18 was more common than HPV16. No association between HPV16/18 status and histopathological diagnosis was identified.

Myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS) is a common and severe disease with a considerable social and economic impact. So far, the etiology is not known, and neither a diagnostic marker nor licensed treatments are available yet. The EUROMENE network of European researchers and clinicians aims to promote cooperation and advance research on ME/CFS. To improve diagnosis and facilitate the analysis of clinical trials surrogate markers are urgently needed. As a first step for developing such biomarkers for clinical use a database of active biomarker research in Europe was established called the ME/CFS EUROMENE Biomarker Landscape project and the results are presented in this review. Further we suggest strategies to improve biomarker development and encourage researchers to take these into consideration for designing and reporting biomarker studies.

Rabbits and rats were inoculated with material derived from FLK cells producing permanently bovine leukaemia virus (BLV). The viral presence in the inoculum was proved by transmission electron microscopy, immunofluorescence, immunogold labelling demonstrating viral Tax protein, and PCR analysis. About 30 % of the infected animals sustained BLV seropositivity during the experiment, and demonstrated symptoms of lympholeukaemia - clinical manifestation of an immunosuppressive condition, increased number of lymphocytes and lymphoblasts, and preneoplastic lymphoid cell accumulations in the liver, lungs, kidneys, and lymph nodes. BLV DNA, detected by PCR in diseased animals, indicates the role of BLV as an aetiological factor of lympholeukaemia, developed in these animals after BLV infection. The alterations in rats were more pronounced than those in rabbits. The results prove that these two species of laboratory animals, especially rats, are suitable models for the in vivo studies of leukaemogenesis caused by BLV/HTLV infections.

Cervical cancer is caused by a persistent infection with high-risk human papillomavirus (hr-HPV). Accurate grading of premalignant precursor lesions, referred to as cervical intraepithelial neoplasia (CIN) is important for clinical management of patients. A promising candidate marker to identify high-grade CIN lesions is the cellular protein p16INK4a, an indirect indicator of cell cycle dysregulation commonly expressed in cervical dysplasias and carcinomas associated with hr-HPV. Like other cell cycle regulatory proteins, the diagnostic role of p16INK4A is still under consideration.The aim of the current study was to investigate the expression of p16INK4A mRNA by a quantitative reverse transcriptase PCR (qRT-PCR) in cervical specimens and evaluate the correlation between the p16INK4A expression levels and the grade of CIN as well as hr-HPV genotypes. The qRT-PCR assay for the detection of p16INK4A mRNA was developed and optimized. As an internal control, integrin gene was selected. The levels of p...

Protein engineering provides an opportunity to generate new immunogens with desired features. Previously, we have demonstrated that hamster polyomavirus major capsid protein VP1-derived virus-like particles (VLPs) are highly immunogenic and can be employed for the insertion of foreign epitopes at certain surface-exposed positions. In the current study, we have designed pseudotype VLPs consisting of an intact VP1 protein and VP2 protein fused with the target antigen—cellular marker p16INK4A—at its N terminus. Both proteins coexpressed in yeast were self-assembled to pseudotype VLPs harbouring the inserted antigen on the surface. The pseudotype VLPs were used for generation of antibodies against p16INK4Athat represents a potential biomarker for cells transformed by high-risk human papillomavirus (HPV). The pseudotype VLPs induced in immunized mice a strong immune response against the target antigen. The antisera raised against pseudotype VLPs showed specific immunostaining of p16INK4Aprotein in malignant cervical tissue. Spleen cells of the immunized mice were used to generate monoclonal antibodies against p16INK4Aprotein. The specificity of antibodies was proven by the immunostaining of HPV-transformed cells. In conclusion, the current study demonstrates the potential of pseudotype VLPs with inserted target antigen as a new type of immunogens to generate antibodies of high diagnostic value.

Cervical carcinoma is a leading cause of mortality from cancer among women worldwide. Oncogenic types of human papillomavirus (HPV) are recognised as causative agents of cervical cancer and its precursor, cervical intraepithelial neoplasia (CIN). It was shown that successful treatment of CIN results in clearance of HPV infection and treatment failure is accompanied by the detection of viral persistence. Therefore, follow-up testing for HPV of CIN treated patients is now accepted as an option for monitoring for recurrent disease.The aim of this study was to investigate the prevalence of high risk HPV after treatment of Bulgarian women for high-grade CIN. Cervical specimens were obtained from 59 women treated for CIN by conization. They were subjected to Pap smear test and HPV DNA testing by PCR. Cytological abnormalities were found in 18.6% of all women. HPV DNA was detected in 91% of women with abnormal cytology. Only 8.3% of women with normal cytology were HPV positive. HPV genotyping showed that...

IntroductionMyalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disease involving central nervous system and immune system disorders, as well as cardiovascular abnormalities. ME/CFS is characterised by severe chronic fatigue lasting for at least 6 months, including clinical symptoms such as tender cervical or axillary lymph nodes, muscle pain, joint pain without swelling or redness, post-exertional malaise for more than 24 hours and unrefreshing sleep. Studies on the epidemiology of ME/CFS in Europe only include single countries and, therefore, the prevalence and incidence of ME/CFS in Europe (as a whole) is unknown. One of the purposes of the European Network on ME/CFS (EUROMENE; European Union-funded COST Action; Reference number: 15111) is to address this gap in knowledge. We will systematically review the literature reporting figures from European countries to provide a robust summary and identify new challenges.Methods and analysisWe will systematically search the literature databases Scopus, PubMed and Web of Science for studies published in the last 10 years (ie, after 2007). No language restriction will be applied. Two independent reviewers will search, screen and select studies as well as extract data about their main characteristics and evaluate their methodological and reporting quality. When disagreements emerge, the reviewers will discuss to reach a consensus. We plan to produce a narrative summary of our findings as we anticipate that studies are scarce and heterogeneous. The possibility of performing meta-analyses will be discussed in a EUROMENE meeting.Ethics and disseminationEthical approval is not required as only publicly available data will be included. Findings will be described in EUROMENE reports, published in peer-reviewed journal(s) and presented at conferences. The findings will be also communicated to policy-makers, healthcare providers, people with ME/CFS and other sections of society through regular channels including the mass-media.PROSPERO registration numberCRD42017078688

A series of hybrid viruses between murine leukemia virus (MuLV) and foamy virus (FV) have been constructed. The chimeric viruses bear the inner core of MuLV and differ in the composition of their outer glycoproteins, which were delivered from FV. Four of hybrid viruses were replication competent and their biological properties, including host cell range and cytopathogenicity, were characterized in vitro. Compared with wild-type MuLV the himeras have the extended host cell range of FV, which is mainly specified by the receptor binding domain of envelope. This portion of the hybrids was from FV. In cell cultures hybrids replicate to lower cell-free virus titters compared to the two parental viruses. A cytopathic effect similar to that of FV and characterized by the formation of multinucleated giant cells was observed in HT1080, M.dunni and NIH3T3 cells infected with hybrid viruses. The MuLV/FV himeras are a new model that will be useful for a variety of retrovirus pathogenesis and vaccine studies.

Human papillomavirus (HPV) infection is the leading risk factor for cervical intraepithelial neoplasia (CIN) and cervical cancer. Detection of high-risk HPV infections might identify women who are at increased risk of development or progression of a cervical lesion and may have prognostic significance. Therefore there is considerable interest in identifying and accurately typing the viruses. PCR amplification of HPV genomes is the most sensitive method for the detection of cervicovaginal HPV. The different PCR techniques used to detect HPV infection, however, affect the frequency with which viral DNA is identified. Using different PCR primer sets, we tried to optimize the detection and typing of HPVs. We tested both consensus and type-specific primers. PCR utilizing the two most commonly used consensus primer sets, MY09/MY11 and GP5+/GP6+, located within the L1 region of HPV genome, amplified a broad spectrum of HPV genotypes in a single reaction. To identify the HPV types, samples were also subje...

Replication-competent chimeric retroviruses constructed of members of the two subfamilies of Retroviridae , orthoretroviruses and spumaretroviruses, specifically murine leukemia viruses (MuLV) bearing hybrid MuLV-foamy virus (FV) envelope ( env ) genes, were characterized. All viruses had the cytoplasmic tail of the MuLV transmembrane protein. In ESL-1, a truncated MuLV leader peptide (LP) was fused to the complete extracellular portion of FV Env, and ESL-2 to -4 contained the complete MuLV-LP followed by N-terminally truncated FV Env decreasing in size. ESL-1 to -4 had an extended host cell range compared to MuLV, induced a cytopathology reminiscent of FVs, and exhibited an ultrastructure that combined the features of the condensed core of MuLV with the prominent surface knobs of FVs. Replication of ESL-2 to -4 resulted in the acquisition of a stop codon at the N terminus of the chimeric Env proteins. This mutation rendered the MuLV-LP nonfunctional and indicated that the truncated FV-LP was sufficient to direct Env synthesis into the secretory pathway. Compared to the parental viruses, the chimeras replicated with only moderate cell-free titers.

Background Although Bulgaria has one of the highest rates of cervical cancer incidence and mortality in the EU, data on HPV type distribution are limited. Aim To determine the prevalence of HPV types 16 and 18 in archival specimens from cervical cancer cases from Bulgaria. Methods A total of 145 formalin-fixed, paraffin-embedded tissue samples from women with histologically proven invasive squamous cell carcinoma of the uterine cervix were available for this study. Detection of HPV types 16 and 18 was based on two parallel PCRs from each sample using type-specific primers for these genotypes. The quality of the extracted DNA was evaluated by PCR using PC03/PC04 β-globin-specific primers. Sections from each block were H&E stained and assessed by the study pathologist to confirm the histological diagnosis. Results Of the 127 β-globin-positive squamous cell carcinoma cases, 98 (77.2%) were positive for HPV16 and/or HPV18 DNA. HPV16 was the more prevalent type and was found as a single infection in 86 (67.7%) cases. HPV type 18 as a single infection was detected in eight (6.3%) cases. Four specimens (3.2%) were double infected with HPV types 16 and 18. Conclusions HPV types 16 and 18 are important risk factors for cervical cancer in Bulgaria. These data are helpful for estimating the potential impact of HPV vaccines in Bulgaria and indicate that ∼77% of cervical cancer cases in the country are potentially preventable by vaccination with currently available prophylactic HPV16/18 vaccines.

In recent years viral serology based on virus-like particles (VLPs) in ELISA has become widely used. We have developed a polyomavirus-specific ELISA using purified VLPs containing the VP1 major capsid protein of JC virus (JCV). The JCV VLPs were produced in yeast cells from Saccharomyces cerevisiae and analyzed by SDS-PAGE, Western blot and electron microscopy. The optimum concentrations of JCV VLP protein and the respective reagents used to perform ELISA were determined. We have applied this VLPs-based ELISA to detect the presence of specific antibodies to JCV in serum samples from Bulgarian population. We found 32% and 90% JCV seropositivity among children 1–3 years of age and adults, respectively. The human polyomaviruses JCV commonly infect humans but viruses remain latent following primary infection. JCV reactivation occurres under conditions of severe immunosuppression and can induce progressive multifocal leukoencephalopathy. In recent years a possible association of JCV with human cancer h...

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The present study estimates the prevalence and risk factors of human papillomavirus (HPV) infection among female sex workers (FSWs) in Bulgaria. HPV DNA was detected by polymerase chain reaction (PCR) in 43.4% of FSWs. HPV16 was the most common type, found in 17.0% of samples, followed by HPV31 (9.4%). Age and smoking were associated with a significantly greater risk of being infected. The results of this study indicate that FSWs in Bulgaria are at increased risk for cervical cancer and represent an important source of HPV infection for the general population in the country. They should be considered as a priority group in cervical cancer control programmes.

The effect of timing of a local host-versus-graft reaction on the induction of acute experimental allergic encephalomyelitis (EAE) was studied in guinea pigs. 20 × 107 γ-irradiated allogeneic cells injected 4 days after encephalitogenic challenge resulted in the development of EAE with an earlier onset, an increased delayed-type hypersensitivity (DTH) response and an increase in lymphoid cell infiltration in the spinal cord. Challenge with allogeneic cells on days −4 and −7, however, produced a delay in onset and a protracted course of disease, with 30–40% of the animals recovering. Evidence of disease was confirmed histologically.

Type A oncornavirus-like particles (TAOLP) were found in the cytoplasm of turkey bone marrow cells transformed by strain Mc-31 avian leukemia virus. They resembled morphologically the cores of budding virions and the cores of immature extracellular Mc-31 virus particles. TAOLP were observed more often in T3Mc31 cells where cytopathological changes existed. TAOLP were not found in the materials from in vivo experiments. A relationship is likely to exist between TAOLP and Mc-31 virus and TAOLP could represent a side product of Mc-31 virus replication.