Search

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne viral infection caused by Crimean-Congo hemorrhagic fever virus (CCHFV). Serological screening of CCHF is important and current ELISA use antigens prepared from virus which is expensive due to requirement of high bio-containment facilities. in this study, we aimed to develop a new recombinant ELISA. For this purpose, CCHFV genome were expressed as 13 proteins in E. coli and among them abundantly purified recombinant Nucleocapsid protein (rNP) and Mucin-like variable domain (rMLD) were used as antigen in ELISA (Rec-ELISA). Rec-ELISA using rNP, rMLD and a combination of both (rNP/rMLD) were probed with acute (n=64; collected between days 1 and 7 after onset of symptoms), convalescent (n=35; collected 8 days after onset of symptoms), consecutive sera (n=25) of confirmed CCHF cases and control sera (n=43). The sensitivity and specificity of Rec-ELISA using rNP/rMLD were 73% and 98% in acute cases and 97% and 98% in convalescent cases. The median interquartile absorbance value to discriminate the acute and convalescent phases of CCHF was significantly higher with ELISA using rNP/rMLD (P0.05) and rMLD (P=0.001). These results indicate that the Rec-ELISA using rNP/rMLD may be very useful to diagnose convalescent CCHF cases especially in field studies., Turkish Scientific and Technological Research Council (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK), The authors would like to acknowledge Turkish Scientific and Technological Research Council (TUBITAK) for supporting Sultan Gulce-z by a 2219 post-doctoral research fellowship programme to conduct part of the studies at Department of Infectious Diseases, University of California, Irvine.

In 2019, the World Health Organization declared 3 billion to be at risk of developing Crimean Congo Hemorrhagic Fever (CCHF). The causative agent of this deadly infection is CCHFV. The data related to the biology and immunology of CCHFV are rather scarce. Due to its indispensable roles in the viral life cycle, NP becomes a logical target for detailed viral immunology studies. In this study, humoral immunity to NP was investigated in CCHF survivors, as well as in immunized mice and rabbits. Abundant antibody response against NP was demonstrated both during natural infection in humans and following experimental immunizations in mice and rabbits. Also, cellular immune responses to recombinant NP (rNP) was detected in multispecies. This study represents the most comprehensive investigation on NP as an inducer of both humoral and cellular immunity in multiple hosts and proves that rNP is an excellent candidate warranting further immunological studies specifically on vaccine investigations., Author summary Crimean Congo Hemorrhagic Fever Virus (CCHFV) is the most lethal human pathogen of medical importance after the dengue virus among arboviruses. The increasing geographic spread of Hyalomma ticks, which are responsible for viral transmission widespread, threatens billions of people. WHO currently declares the field of research on CCHFV as the second most urgently needed areas of investigations on emerging pathogens. About 10 to 40% of those infected with the virus lose their life due to the rapidly developing severe clinical manifestations. Pandemic potential and the lack of any approved treatment or vaccine make raise the studies on CCHFV as critical. The studies on CCHFV are challenging due to the necessities of BSL-4 facilities and the immunological characterization of individual structural proteins will lay the groundwork for the steps to be taken to treat and prevent this emerging disease. As is known from other RNA viruses, nucleoprotein (NP) has crucial roles in the viral life cycle, both in viral replication and transcription and in the formation of the virion structure. So far, detailed and comprehensive immunological characterizations on NP in multiple are not undertaken. Our study was set out to embark such detailed investigation. The strong humoral and cellular immune response to NP demonstrated by this study indicates that NP might be an excellent candidate for future scrutinies on vaccines and diagnostic reagents.

The COVID-19 pandemic has put global public health at high risk, rapidly spreading around the world. Although several COVID-19 vaccines are available for mass immunization, the world still urgently needs highly effective, reliable, cost-effective, and safe SARS-CoV-2 coronavirus vaccines, as well as antiviral and therapeutic drugs, to control the COVID-19 pandemic given the emerging variant strains of the virus. Recently, we successfully produced receptor-binding domain (RBD) variants in the Nicotiana benthamiana plant as promising vaccine candidates against COVID-19 and demonstrated that mice immunized with these antigens elicited a high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2. In this study, we engineered the nucleocapsid (N) protein and co-expressed it with RBD of SARS-CoV-2 in Nicotiana benthamiana plant to produce an antigen cocktail. The purification yields were about 22 or 24 mg of pure protein/kg of plant biomass for N or N+RBD antigens, respectively. The purified plant produced N protein was recognized by N protein-specific monoclonal and polyclonal antibodies demonstrating specific reactivity of mAb to plant-produced N protein. In this study, for the first time, we report the co-expression of RBD with N protein to produce a cocktail antigen of SARS-CoV-2, which elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. Thus, obtained data support that a plant-produced antigen cocktail, developed in this study, is a promising vaccine candidate against COVID-19.

The rapid spread of SARS-CoV-2 with its mutating strains has posed a global threat to safety during this COVID-19 pandemic. Thus far, there are 123 candidate vaccines in human clinical trials and more than 190 candidates in preclinical development worldwide as per the WHO on 1 October 2021. The various types of vaccines that are currently approved for emergency use include viral vectors (e.g., adenovirus, University of Oxford/AstraZeneca, Gamaleya Sputnik V, and Johnson &amp, Johnson), mRNA (Moderna and Pfizer-BioNTech), and whole inactivated (Sinovac Biotech and Sinopharm) vaccines. Amidst the emerging cases and shortages of vaccines for global distribution, it is vital to develop a vaccine candidate that recapitulates the severe and fatal progression of COVID-19 and further helps to cope with the current outbreak. Hence, we present the preclinical immunogenicity, protective efficacy, and safety evaluation of a whole-virion inactivated SARS-CoV-2 vaccine candidate (ERUCoV-VAC) formulated in aluminium hydroxide, in three animal models, BALB/c mice, transgenic mice (K18-hACE2), and ferrets. The hCoV-19/Turkey/ERAGEM-001/2020 strain was used for the safety evaluation of ERUCoV-VAC. It was found that ERUCoV-VAC was highly immunogenic and elicited a strong immune response in BALB/c mice. The protective efficacy of the vaccine in K18-hACE2 showed that ERUCoV-VAC induced complete protection of the mice from a lethal SARS-CoV-2 challenge. Similar viral clearance rates with the safety evaluation of the vaccine in upper respiratory tracts were also positively appreciable in the ferret models. ERUCoV-VAC has been authorized by the Turkish Medicines and Medical Devices Agency and has now entered phase 3 clinical development (NCT04942405). The name of ERUCoV-VAC has been changed to TURKOVAC in the phase 3 clinical trial.

Crimean–Congo hemorrhagic fever (CCHF) is an emerging zoonotic infectious disease caused by Crimean–Congo hemorrhagic fever virus (CCHFV). The first clinical CCHF infection was described in 1944 in the Crimean Peninsula, exclusively in humans, with case-fatality rates exceeding 30%. The increasing number of cases, high mortality rate, and lack of effective therapy make CCHF a serious threat to public health and a potential bioterrorism agent. The present study evaluated the development, immunogenicity, and immune response durations for cell-culture-derived inactivated vaccine (CCVax) formulations in comparison with those of mouse-brain-derived vaccine (MBVax) formulations. In this study, the Kelkit06 CCHF virus strain was propagated in both suckling mice and Vero E6 cells, and purified with a sucrose gradient. Formalin-inactivated vaccine candidates were formulated at various doses [low dose (LD), 5 μg; medium dose (MD), 10 μg; high dose (HD), 20 μg)] and mixed with an alum adjuvant. BALB/c mice received the same doses of the vaccine formulations three times at 3-week intervals. The humoral endpoint IgG responses were evaluated and compared for the MBVax and CCVax treatments. The duration of the presence of IgG and neutralizing antibody (Ab) titers was evaluated and compared until up to 1 year after immunization. The humoral IgG responses indicated that the CCVax and MBVax candidates enhanced the IgG endpoint titers in a dose-dependent manner, which were induced more strongly in all the CCVax groups than in the MBVax mice. The fold changes in neutralizing Ab levels were also found to be higher in the CCVax groups: between 2- and 7.6-fold after the second week of the last immunization. The neutralization titers peaked 4 months after immunization in all the vaccine-receiving groups, but these were still comparable at the end of the first year. The CCVax formulations induced higher IgG and neutralizing Ab titers at all the measured time points. In this study, we showed that cell-culture-purified and formalin-inactivated vaccine candidates induced strong and robust immunity in vaccinated mice dose-dependently, more so than mouse-brain-derived vaccines., Crimean–Congo hemorrhagic fever virus; Bunyavirus; Hemorrhagic fever; Inactivated vaccine; Protective response; Long-term immunity; Humoral immune response.

Copyright: © 2020 Pavel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the Nairoviridae family within the Bunyavirales order of viruses. Crimean-Congo hemorrhagic fever (CCHF) is the most widespread among tick-borne human viral diseases. It is endemic in many areas of Africa, Asia, the Middle East, in the Balkans, Russia and countries of the former Soviet Union. The confirmed CCHF cases were seen in Spain in 2016 to signify expansion of the virus into new geographical areas. CCHFV causes a viral human disease characterized by sudden onset of fever, headache, abdominal pain, nausea, hypotension, hemorrhage, and hepatic dysfunction with fatality rates up to 30%. Currently, there are no spesific treatments or licensed vaccines available for CCHFV. The absence of a susceptible animal model for CCHFV infection was severely hindered work on the development of vaccines. However, several animal models of CCHFV infection have been recently developed and used to assess vaccine efficacy. In this study, we have used the transiently immune-suppressed (IS) mouse model that MAb-5A3 was used to block IFN-I signaling in immune intact, wild-type mice at the time of CCHFV infection to evaluate the immune response and efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection but the cell culture based vaccine more effectively induced to CCFHV spesific antibodies and T cell responses. This is the first comparison of the cell culture based and the mouse brain derived vaccines for assessing the protective efficacy and the immunogenicity in the IS mouse CCHFV model., Author summary Crimean-Congo hemorrhagic fever is one of the most medically important tick-borne disease. CCHFV causes severe clinical signs in humans but not in its vertebrate animal hosts. Transmission of the virus to humans could be through the bite of infected ticks or by exposure to the tissues or blood of infected animals. Nosocomial infections have also been reported in endemic countries and are considered to be significant problems for health-care workers and family members. With the increasing number of the cases and expanding new foci of the CCHF endemic area, there is an urgent need to prioritize control strategies on the consideration of disease reduction. Recently, different approaches have been attempted towards the development of a CCHFV vaccine. We previously developed a cell culture based inactivated CCHFV vaccine that demonstrated protective efficacy in IFNAR-/- mice. To date, the only CCHFV vaccine tested in humans is the suckling mouse brain derived vaccine used only in Bulgaria. However, there are no experimental data to show whether this vaccine confers to protective efficacy in a mouse model. We have used the IS mouse model to explore the immunogenicity and vaccine efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection after challenge studies but the cell culture based vaccine more effectively induced to CCFHV spesific antibody and T cell responses.

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.

Coronaviruses are positive-strand, RNA-dependent RNA polymerase-utilizing viruses that require a polymerase template switch, characterized as discontinuous transcription, to place a 5′-terminal genomic leader onto subgenomic mRNAs (sgmRNAs). The usually precise switch is thought to occur during the synthesis of negative-strand templates for sgmRNA production and to be directed by heptameric core donor sequences within the genome that match an acceptor core (UCUAAAC in the case of bovine coronavirus) near the 3′ end of the 5′-terminal genomic leader. Here it is shown that a 22-nucleotide (nt) donor sequence engineered into a packageable bovine coronavirus defective interfering (DI) RNA and made to match a sequence within the 65-nt virus genomic leader caused a template switch yielding an sgmRNA with only a 33-nt minileader. By changing the donor sequence, acceptor sites between genomic nt 33 and 97 (identical between the DI RNA and the viral genome) could be used to generate sgmRNAs detectable by Northern analysis (∼2 to 32 molecules per cell) by 24 h postinfection. Whether the switch was intramolecular only was not determined since a potentially distinguishing acceptor region in the DI RNA rapidly conformed to that in the helper virus genome through a previously described template switch known as leader switching. These results show that crossover acceptor sites for discontinuous transcription (i) need not include the UCUAAAC core and (ii) rest within a surprisingly wide 5′-proximal “hotspot.” Overlap of this hotspot with that for leader switching and with elements required for RNA replication suggests that it is part of a larger 5′-proximal multifunctional structure.

Objective: To determine the presence of Helicobacter pylori in the middle ear effusion of patients with otitis media with effusion (OME) by polymerase chain reaction (PCR). Study Design: A prospective study in patients with OME. Methods: The study was performed in 38 patients with OME who were admitted to the ENT Clinic, Firat University from June 2003 to April 2004. In all cases, a myringotomy operation (with or without placement of a ventilation tube) was carried out. The effusion samples aspirated from the middle ear were analyzed with PCR assay. Results: A total of 55 aspiration samples collected from 38 children ranging in age from 2 to 12 were included in the study. Fifteen of the subjects were girls, and 23 were boys. In 17 patients, both ears demonstrated effusions, whereas in 21 patients, only one ear had effusions. Nine (16.3%) of 55 the middle ear effusion samples were shown to be H. pylori positive by PCR. Conclusions:H. pylori was detected in the middle ear effusion of some patients with OME. These results may have interesting implications for a possible role of H. pylori in OME. In addition, these results suggest that further studies are needed to investigate the role of H. pylori in the etiology of OME.

Hemorrhagic shock causes mucosal damage in intestine and it results in translocation of bacteria to distant organs. In this study, effects of various doses of L-Tryptophan on the prevention of bacterial translocation in hemorrhagic shock induced rabbits were investigated. This study was carried out on six groups, each was consisting of 10 rabbits. While any procedure was conducted on the rabbits in group 1 (as a control group), 1 x 10(10)Escherichia coli isolate were administered rabbits in the other groups by gavage. In groups 3, 4, 5, and 6, hemorrhagic shock was induced. After induction of hemorrhagic shock, 10, 50, and 200 mg/kg L-Tryptophan were intragastrically administered to animals in groups 4, 5, and 6, respectively. Blood and terminal ileum samples were taken to detect bacterial translocation by polymerase chain reaction and mucosal damage by histopathological examination at 24 h after hemorrhagic shock. The occurrence of bacterial translocation increased as well when intestinal bacterial intensity was increased (P < 0.05). The most intensive bacterial translocation was formed in group 3 as a result of the additive effect of hemorrhagic shock to bacterial augmentation. It was observed that bacterial translocation was significantly reduced in groups 5 and 6 that are 50 and 200 mg/kg L-Tryptophan were administered (P < 0.01). Histopathological changes on mucosa and submucosa support these results. As a result, we concluded that augmentation of intestinal bacterial intensity induces bacterial translocation, the addition of hemorrhagic shock to bacterial augmentation makes more excessive translocation and mucosal changes have effective roles in these events. L-Tryptophan decreased the intestinal mucosal damage and bacterial translocation induced by hemorrhagic shock, in a dose-dependent manner.

Human toxocariasis is commonly seen in places where stray and Toxocara canis-infected dog population is high. There is a strong correlation between frequency of Toxocara infection, life style, and infection risk. Institutionalization of mental retarded patients increases to risk of toxocariasis. In this study, we aimed at investigating the frequency of Toxocara infection among children with mental retardation not requiring institutionalization. The study included 96 cases, who had educatable mental retardation and 85 healthy subjects who comprised the control group. Anti-Toxocara IgG or IgM antibodies were investigated in all serum samples, using ELISA method. The frequency of Toxocara infection was found significantly higher in mental retarded cases than in those in the control group (18.8% and 7.1% respectively) (p < 0.05). There was no significant difference between mental retarded children and the control group in terms of mean age, age groups, gender, owning dogs and cats and duration of their ownership, socio-economic level and behavioural factors, and personal hygiene (p > 0.05). We did not find any significant difference between Toxocara seropositive and seronegative mental retarded children in terms of demographic factors and epidemiological factors that could increase the risk of Toxocara infection (p > 0.05). The present study is the first seroprevalence study carried out with a mental retarded group not requiring institutionalization. Determination of high frequency of Toxocara infection suggests that these subjects constitute a risk factor for Toxocara infection, which may be attributed to their behavioural patterns.

Mumps is a disease caused by a virus that can infect many parts of the body, especially the parotid salivary glands. A case of epidemic parotitis in an adult female affected by bilateral conjunctivitis is presented. Mumps virus was detected in tear samples by reverse transcription nested polymerase chain reaction (RT-n-PCR). The conjunctivitis resolved completely within 11 d.

Background Molluscum contagiosum has a worldwide occurrence and its primary mode of transmission is via direct human contact including sexual means. The aim of the study was to implement a polymerase chain reaction-based assay for detection and subtyping of Molluscum contagiosum virus (MCV) in skin lesions diagnosed with molluscum contagiosum in a large regional teaching hospital in Turkey. Methods For this purpose, a total of 61 patients were included in the study. Randomly selected single lesion from each patient was used to extract DNA material and a specific PCR reaction amplifying 393-bp- and 575-bp-long regions from MCV genome was used in the detection. Subtyping was carried out by digestion of the amplified 575-bp product with restriction endonuclease enzyme Bam HI. Both amplified and restriction enzyme digested products visualized on agarose gel electrophoresis. Results All 61 molluscum cases (100%) included in the study contained MCV genetic material as demonstrated by the presence of 393- and 575-bp-long PCR amplified products. Restriction enzyme Bam HI digestion of the 575-bp-long amplicon indicated that the infecting subtype in all the cases (100%) was MCV subtype I. Conclusions Results of this study demonstrate that subtype I is the only infecting strain dominant in our region. Because the only consecutive molluscum patients admitted to our hospital were included in the study, our data do not rule out the possibility that other genotypes might be present in the Turkish population. However, it is not unreasonable to conclude that similar trends exist in the rest of the country. Results also show that a molecular-based diagnostic assay would be feasible in cases where diagnosis was deemed necessary.

Objective: To investigate etiological role of Epstein-Barr virus (EBV) DNA in breast cancer. Materials and Methods: The presence of EBV DNA in 57 breast cancer tissues was investigated with a sensitive PCR assay. The breast cancer tissues were from invasive ductular (n = 28), lobular (n = 20) and other miscellaneous carcinomas (n = 9). Tissues from normal breasts and patients with various benign breast diseases (n = 55): fibrocystic disease (n = 34), fibroadenoma (n = 16), hyperplasia, and granulomatous mastitis (n = 5), were used as control samples. Results: EBV DNA was detected in 13 (23%) cancerous tissues (7 ductular, 4 lobular, 2 other carcinoma) and 19 (35%) in the control tissues. The difference between EBV presence in malignant and benign tissues was not statistically significant (p > 0.05). Conclusion: The presence of EBV DNA was detected almost equally in both breast cancer and normal tissues, which indicates no etiological role for EBV in breast cancer. We suggest further etiological studies.

Objectives: To study the prevalence and genotype distribution of hepatitis G virus (HGV) in hemodialysis patients in East Anatolia, Turkey. Subjects and Materials: Eighty-nine hemodialysis patients and 30 healthy individuals were analyzed by using reverse-transcriptase nested polymerase chain reaction with primers specific for 5′ untranslated region. HGV genotyping was performed by PCR and three randomly selected HGV-positive samples were sequenced. Results: Of the 89 hemodialysis patients, HGV RNA was detected in 9 (10.2%). All of our isolates were assigned to genotype 2. Conclusion: Our results showed that hemodialysis patients carry the risk for HGV infection in East Anatolia, Turkey.

Background: Simian virus 40 (SV40) has been a model experimental system for the study of cell transformation and tumorigenesis for many years. The study of SV40 in humans has aroused interest in the related BK virus (BKV) and JC virus (JCV) and their role in human disease. Objectives: SV40 has been found in a variety of human samples, both malignant and normal. Many independent studies have suggested that SV40 plays a role for some cancers. However, in most cases the role of SV40 remains unclear. Study design: The subject of this study consisted of 99 patients with thyroid nodules. Both thyroid nodule and normal thyroid tissue were taken from each patient to test whether they contained SV40 sequences. Results: We detected SV40 sequences by polymerase chain reaction (PCR) in four of 99 thyroid nodules. Two of them were papillary thyroid carcinomas and the others were benign thyroid nodules. No SV40 was detected in 99 of normal tyhroid tissues of the same patients. DNA sequence analysis, performed in four positive samples, confirmed that PCR products belong to the SV40 T antigen (Tag) region. Conclusion: The possible role of SV40 in the development of thyroid nodules and the spread of SV40 by horizontal infection in the human population are discussed.

The clinical features, therapy and outcome of anthrax cases from the Elazig province (the eastern Anatolian region) of Turkey seen in our clinic over an 8-year period were reviewed. The records of 25 anthrax cases observed in our clinic during the period January 1994 to April 2002 were examined. All cases were cutaneous; 18 (72%) patients exhibited malignant pustules and seven (28%) malignant edema. Three of the patients with a malignant pustule developed anthrax sepsis when admitted to our clinic. All cases were treated with penicillin. One patient who had penicillin allergy was treated with ciprofloxacin. In addition, patients with malignant edema were also treated with systemic corticosteroids (methylprednisolone or dexamethasone). Two patients died due to anthrax sepsis; one case with anthrax sepsis recovered. The mortality rate was 8%. Anthrax is still a reality in Turkey. Cutaneous anthrax should be considered in any patient with a painless ulcer with vesicles, edema and a history of exposure to animals or animal products. In our series, penicillin and ciprofloxacin were effective in treatment of anthrax. Our anthrax sepsis case demonstrates that anthrax sepsis is not always fatal if antibiotic treatment is given early after diagnosis.