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© 2022 The Author(s)Background: Development of safe and effective vaccine options is crucial to the success of fight against COVID-19 pandemic. Herein, we report interim safety and immunogenicity findings of the phase 1&2 trials of ERUCoV-VAC, an inactivated whole virion SARS-CoV-2 vaccine. Methods: Double-blind, randomised, single centre, phase 1 and 2 trials included SARS-CoV-2 seronegative healthy adults aged 18–55 years (18–64 in phase 2). All participants, except the first 4 in phase 1 who received ERUCoV-VAC 3 μg or 6 μg unblinded and monitored for 7 days for safety purposes, were assigned to receive two intramuscular doses of ERUCoV-VAC 3 μg or 6 μg (an inactivated vaccine containing alhydrogel as adjuvant) or placebo 21 days apart (28 days in phase 2) according to computer-generated randomisation schemes. Both trials are registered at ClinicalTrials.gov (phase 1, NCT04691947 and phase 2, NCT04824391). Results: Forty-four participants (3 μg [n:17], 6 μg [n:17], placebo [n:10]) in phase 1 and 250 (3 μg [n:100], 6 μg [n:100], placebo [n:50]) in phase 2 received ≥1 dose. In phase 1 trial, 25 adverse events AEs (80 % mild) occured in 15 participants (34.1 %) until day 43. There was no dose-response relationship noted in safety events in ERUCoV-VAC recipients (p = 0.4905). Pain at injection site was the most common AE (9/44;20.5 %). Both doses of ERUCoV-VAC 3 μg and 6 μg groups were comparable in inducing SARS-CoV-2 wild-type neutralising antibody (MNT50): GMTs (95 %CI) were 8.3 (6.4–10.3) vs. 8.6 (7.0–10.2) at day 43 (p = 0.7357) and 9.7 (6.0–13.4) vs. 10.8 (8.8–12.8) at day 60 (p = 0.8644), respectively. FRNT50 confirmed MNT50 results: SARS-CoV-2 wild-type neutralising antibody GMTs (95 %CI) were 8.4 (6.3–10.5) vs. 9.0 (7.2–10.8) at day 43 (p = 0.5393) and 11.0 (7.0–14.9) vs. 12.3 (10.3–14.5) at day 60 (p = 0.8578). Neutralising antibody seroconversion rates (95 %CI) were 86.7 % (59.5–98.3) vs 94.1 % (71.3–99.8) at day 43 (p = 0.8727) and 92.8 % (66.1–99.8) vs. 100 % (79.4–100.0) at day 60 (p = 0.8873), in ERUCoV-VAC 3 μg and 6 μg groups, respectively. In phase 2 trial, 268 AEs, (67.2 % moderate in severity) occured in 153 (61.2 %) participants. The most common local and systemic AEs were pain at injection site (23 events in 21 [8.4 %] subjects) and headache (56 events in 47 [18.8 %] subjects), respectively. Pain at injection site was the only AE with a significantly higher frequency in the ERUCoV-VAC groups than in the placebo arm in the phase 2 study (p = 0.0322). ERUCoV-VAC groups were comparable in frequency of AEs (p = 0.4587). ERUCoV-VAC 3 μg and 6 μg groups were comparable neutralising antibody (MNT50): GMTs (95 %CI) were 30.0 (37.9–22.0) vs. 34.9 (47.6–22.1) at day 43 (p = 0.0666) and 34.2 (23.8–44.5) and 39.6 (22.7–58.0) at day 60, (p = 0.2166), respectively. FRNT50 confirmed MNT50 results: SARS-CoV-2 wildtype neutralising antibody GMTs were 28.9 (20.0–37.7) and 30.1 (18.5–41.6) at day 43 (p = 0.3366) and 34.2 (23.8–44.5) and 39.6 (22.7–58.0) at day 60 (p = 0.8777). Neutralising antibody seroconversion rates (95 %CI) were 95.7 % (91.4–99.8) vs. 98.9 % (96.9–100.0) at day 43 (p = 0.8710) and 96.6 % (92.8–100.0) vs 98.9 % (96.7–100.0) at day 60 (p = 0.9129) in ERUCoV-VAC 3 μg and 6 μg groups, respectively. Conclusions: Two-dose regimens of ERUCoV-VAC 3 μg and 6 μg 28 days both had an acceptable safety and tolerability profile and elicited comparable neutralising antibody responses and seroconversion rates exceeding 95 % at day 43 and 60 after the first vaccination. Data availability Data will be made available on request.

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.

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.

Zoonotic infections are globally important diseases and lead to huge economic losses in both low- and middle-income and high-income countries. Global warming, environmental and ecological changes, illegal movement of animals and humans, regional civil wars, and poverty are predisposing factors for the emergence of zoonotic infections and their distribution worldwide; they are also a big threat for the future. In addition, environmental pollution and antimicrobial resistance are immense serious threats and dangers to prevent and control zoonotic infections. The natural location of Turkey allows many emerged or re-emerged infections with zoonotic characteristics by animal movements, such as bird immigrations, and by human movements due to civil wars as seen with regional refugees. Numerous zoonotic diseases, including 37 bacterial, 13 fungal, 29 viral, 28 parasitic (3 trematodes, 7 cestodes, 10 nematodes, and 8 protozoan), and totally 107 infections, have been reported from Turkey to date. Additionally, many ectoparasitic zoonoses within 15 different arthropod groups and one leech infestation have been reported from Turkey to date. The "One Health" initiative is particularly relevant for developing strategies to combat zoonotic diseases. In this article, we review the occurrence of zoonotic diseases in man and animals in Turkey in the light of the "One Health" perspective.

In Turkey, arthropod-borne diseases such as human babesiosis, dirofilariasis, leishmaniasis, malaria, lymphatic filariasis, onchocerciasis, anthrax, bartonellosis, louse-borne relapsing fever, lyme disease, plague, trench fever, tularemia, epidemic typhus, tick-borne typhus, Crimean-Congo hemorrhagic fever, phlebovirus infections, tick-borne encephalitis and west nile virus in humans; animal trypanosomiasis, avian haemosporidiasis, babesiosis, canine hepatozoonosis, canine lesihmaniosis, dipetalonemiasis, dirofilariosis, habronemiasis, onchocerciasis, parafilariasis, setariasis, theileriosis, thelaziasis, anaplasmosis, ehrlichiosis, feline infectious anemia, akabane, bluetongue, bovine ephemeral fever, equine infectious anemia, louping ill, west nile virus, colony collapse disorders (CCD) were reported in domestic animals and Wolbachia endobacteria infection was also reported in mosquitos so far. On the other hand, aphids (Aphididae), leafhoppers (Cicadellidae), white flies (Aleyrodidae), beetles (Coleoptera), trips (Thysanoptera), gal mites (Eriophyidae), and nematodes are also very important vectors of plant diseases in Turkey. Some of the most harmful plant viruses in the world such as tristeza, plum pox virus, and tomato ringspot viruses are also transmitted by arthropod vectors. In recent years, in spite of changes in ecological balance caused by abandoning agricultural land, use of natural areas to farm, release of rabbits, preventing water flooding, leaving the fields uncultivated, migration from urban to rural areas, hunting of wild animals, the dramatic drop in the number of farm animals, global warming and the strengthening of wild life was probably responsible for the increase of tick populations. The construction of ponds and dams, changes in riverbeds, drying out of swamps and marshes, dense ad-hoc and haphazard construction, changes in picnicking habits, wild irrigation in agriculture, and the use of unsuitable control measures by local authorities, resulted in an increase of flies. This led to an increase in infected vector populations and vector-borne diseases and a decrease in the potential for economic development. In conclusion, it is very important to initiate and develop integrated control programs targeting arthropod vectors, disease agents of human, animal and plants and natural reservoirs. These diseases have been caused high economic losses in Turkey, and therefore they should be further investigated, Türkiye’de insanlarda babesiosis, dirofilariasis, şark çıbanı, sıtma, lenfatik filariasis, onchocerciasis, şarbon, bartonellosis, bitle bulaşan dönemli ateş (louse-borne relapsing fever), lyme, veba, siper ateşi, tularemia, epidemik tifüs, tick-borne tifus, Kırım-Kongo kanamalı ateşi, phlebovirus enfeksiyonu, tick-borne encephalitis ve batı nil virusu; evcil hayvanlarda trypanasomiasis, kanatlı haemosporidiasisi, babesiosis, köpek hepatozoonosisi, köpek leishmaniosisi, dipetalonemiasis, dirofilariosis, yaz yarası, onchocerciasis, yaz kanaması, seteriasis, theileriosis, thelaziasis, anaplasmosis, ehrlichiosis, kedi enfeksiyöz anemisi, akabane, mavidil, üçgün hastalığı, loupingill, enfeksiyöz at anemisi, arıların colony collapse hastalığı (CCD) ve sivrisineklerde Wolbachia endobakteri enfeksiyonu şimdiye kadar rapor edilen artropod kaynaklı hastalıklardır. Diğer taraftan, Türkiye’de yaprak bitleri (Aphididae), yaprak zararlıları (Cicadellidae), beyazsinekler (Aleyrodidae), kın kanatlılar (Coleoptera), kirpik kanatlılar (Thysanoptera), gal akarları (Eriophyidae) ve nematodlar da bitki hastalıklarının önemli vektörleridirler. Tristeza, şarka, domates halkalı leke virüsleri gibi dünyadaki en zararlı bitki patojenlerinin de uygun vektörleri olmadan epidemi yapamadıkları bilinmektedir. Tarım alanlarının terk edilmesi, doğal alanların tarıma açılması, doğaya tavşan salınması, su taşkınlarının önlenmesi, yayla yasağı, av yasağı, tarlaların işlenmemesi, kırsal hayattan şehirlere göç, yırtıcı hayvanların öldürülmesi, çiftlik hayvanlarının sayısındaki dramatik düşüşe karşın yaban hayatının güçlenmesi ve küresel ısınma gibi son yıllarda ekolojik dengelerin değişmesine yol açan faktörler kene sayısında artışa yol açan olası nedenlerdir. Fizibilite yapılmadan gölet ve baraj inşası, nehir yataklarının değiştirilmesi, bataklık ve sazlıkların kurutulması, plansız ve gelişigüzel yoğun yapılaşma, piknik kültüründeki değişiklik, tarımda vahşi sulama, yerel yöneticilerin bilimsel yaklaşımdan yoksun, bilinçsiz vektör mücadelesi de sinek populasyonlarında artışa sebep olmuştur. Bu durum da vektör populasyonunda, vektörlerle bulaştırılan hastalıklarda artışa ve dolayısıyla ekonomik gelişmelerde azalmalara veya yavaşlamalara yol açmaktadır. Sonuç olarak, Türkiye’de insan, hayvan ve bitki sağlığını tehdit ederek ekonomik kayıplara yol açan vektörlere ve bu vektörlerin bulaştırdıkları hastalıklara karşı mücadele programları geliştirmek ve başlatmak, bu konuda daha ileri araştırmalar yapmak oldukça önemlidir

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.

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.

Background Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV. Methods Sixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies. Results Pseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed. Conclusion The pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies. This novel assay could serve as useful tools for CCHF research in epidemiology, vaccine development and other studies of immunity. It also provides an alternative to PRNT when viruses with no or poor CPE in cell culture.

A pseudo-plaque assay was developed for detection and quantitation of Crimean-Congo hemorrhagic fever virus Turkey-Kelkit06. Enzyme-catalyzed color development of infected cells probed with anti-Crimean-Congo hemorrhagic fever virus antibodies was used for determining the titer of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 and for its detection in samples from persons infected with the Crimean-Congo hemorrhagic fever virus. The pseudo-plaque assay accuracy was confirmed by comparing pseudo-plaque assay titers with fluorescent immunofocus assay and focus formation assay titers using three stocks of virus. No significant difference in virus titers of Crimean-Congo hemorrhagic fever Turkey-Kelkit06 among the three methods was observed. The pseudo-plaque assay is more sensitive than the fluorescent immunofocus assay for detecting the virus in primary isolates of Crimean-Congo hemorrhagic fever virus collected from humans, but no difference in sensitivity between the two methods was observed in the cell-adapted strain of Crimean-Congo hemorrhagic fever Turkey-Kelkit06. The pseudo-plaque assay is suitable for titration of Crimean-Congo hemorrhagic fever Turkey-Kelkit06, which does not develop plaques, suggesting it may also be suitable for the detection of other viruses. (C) 2012 Elsevier B.V. All rights reserved.

The parasitic trematode Fasciola hepatica is the causative agent of fasciolosis that is common in ruminants especially sheep and cattle and is occasionally found in humans. Fasciolosis has a worldwide distribution including Turkey and causes major economic losses in agricultural industry. Cathepsin L1 is one of the major molecules in the excretory-secretory products of F. hepatica and is involved in tissue penetration, immune evasion and feeding and therefore may be used in vaccination and serological diagnosis. The aim of this study was to evaluate cloning and expression of the cathepsin L1 gene of F. hepatica eucaryotic cells. For this purpose, total RNA was extracted from adult F. hepatica. Cathepsin L1 DNA amplicons were obtained with the reverse transcription polymerase chain reaction (RT-PCR). The 981 base-coding gene region of cathepsin L1 was amplified using specific primers to the cathepsin L1 gene. Then, the cathepsin L1 gene was cloned into the pCI-neo mammalian expression vector. The presence of the cathepsin L1 gene was confirmed by PCR screening and enzyme digestion assays. So, the resulting recombinant plasmid was named pFhCL1. Afterwards, the pFhCL1 vector was transiently transfected into Vero cells. The presence of the cathepsin L1 proteins was shown by Western immunoblotting.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the family Bunyaviridae , genus Nairovirus . The virus is transmitted to humans through infected tick bites or from direct contact with viremic animals or humans. In the present study, a total of 1,015 adult ticks were collected from cattle (603 specimens), sheep (17 specimens), and goats (395 specimens) in the Kelkit Valley in Turkey. Four tick species were recognized on the animals in the surveyed region. The most abundant species were Rhipicephalus bursa and Hyalomma marginatum marginatum , at 47.68% (484/1,015) and 46.40% (471/1,015), respectively. Reverse transcriptase PCR was used to recover partial sequences of the CCHFV small (S) genome segment. The presence of CCHFV was determined in 3 of 33 (9.09%) R. bursa pools and in 1 of 31 (3.22%) H. m. marginatum pools. Virus sequences from R. bursa were extremely different from those of the Greek CCHFV strain (U04958) isolated from an R. bursa tick. Phylogenetic analysis indicated that the CCHFV isolates obtained in this study clustered in group 5, whose range encompasses southwestern Russian and Kosovo. This is the first evidence of CCHFV in ticks from Turkey. Even though Hyalomma is the main vector for CCHFV, R. bursa may play a role in CCHFV transmission.

We investigated the prevalence and genotypic distribution of GB virus-C/hepatitis G virus (GBV-C/HGV) and TT virus (TTV) in blood donors, mentally retarded children and four groups of patients living in Eastern Anatolia, Turkey. The prevalence and genetic analysis of TTV were determined by using the primers of the UTR and ORF1 regions of TTV, respectively. Reverse transcription nested (RT-n)-PCR was used to amplify 5' UTR of GBV-C/HGV. Genotyping of HGV was carried out by PCR-based genotyping assay while RFLP was conducted to determine the genotypes of TTV. TTV DNA was detected in 118 of 410 sera tested, giving an overall prevalence of 28.7%; GBV-C/HGV-RNA was detected in only 17 cases, giving an overall prevalence of 4.1%. No significant differences were observed in the number of positive or negative tests for GBV-C/HGV and TTV according to duration of illness or mean duration of institutionalization in any of the groups studied. Although all samples from the study population belonged to genotypes 1 and 4, the most common TTV genotype is G2. In conclusion, our results indicate a low endemicity of GBV-C/HGV and TTV infection in Eastern Anatolia, Turkey. The presence of G2 strains reveals the limited genetic diversity of the GBV-C/HGV circulating in Turkey. We suggest that TTV infection of genotypes 1 and 4 is prevalent in the same region.

In this study, 78 middle ear aspiration samples from 59 children between the ages of 2 14 who have undergone myringotomy under general anesthesia were included. The presence of DNA genome of Chlamydia trachomatis was analyzed with polymerase chain reaction in the aspiration material. The relationship between the nature of the effusion fluid and the presence of Chlamydia trachomatis was investigated. Of the seventy eight middle ear effusions, 26 (33.3%) were serous, 31 were (39.7%) mucoid and 21 (26.9%) were sero mucoid in nature. Chlamydia trachomatis DNA genome was identified in seven (8.9%) effusion samples. Of these, three were mucoid (42.8%), three were serous (42.8%) and one was sero mucoid (14.2%) in character. We think that during the bacteriological analyses carried out for the cases of otitis media with effusion, Chlamydia trachomatis should be taken into consideration both at the time of diagnosis and the treatment.

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.

In the present study, we have investigated the seropositivity of Epstein-Barr virus (EBV) and the epidemiological factors affecting this seropositivity. A total of 540 subjects living in central Elazig Province and its surroundings were enrolled in the study. IgG antibodies against capsid antigen of EBV (anti-VCA IgG) were determined by ELISA. Seropositivity was found to be 99.4%. There was a significant relationship between increased anti-VCA IgG levels and age, living in crowded families, exposure in public places and low income (p0.05). However, no significant alterations in serum anti-VCA IgG antibody were observed in terms of gender, blood transfusion and educational status (p0.05). In conclusion, we have demonstrated the commonly presence of EBV Infections in the public and contamination with the infection at early stages of life in the Elazig region. An overpopulated family environment, low income status and living in shared accommodation facilities play important roles in acquiring EBV infection.

Mechanisms leading to subgenomic mRNA (sgmRNA) synthesis in coronaviruses are poorly understood but are known to involve a heptameric signaling motif, originally called the intergenic sequence. The intergenic sequence is the presumed crossover region (fusion site) for RNA-dependent RNA polymerase (RdRp) during discontinuous transcription, a process leading to sgmRNAs that are both 5′ and 3′ coterminal. In the bovine coronavirus, the major fusion site for synthesis of mRNA 5 ( GG UA G AC) does not conform to the canonical motif (UC[U,C]AAAC) at three positions (underlined), yet it lies just 14 nucleotides downstream from such a sequence (UCCAAAC). The infrequently used canonical sequence, by computer prediction, is buried within the stem of a stable hairpin (−17.2 kcal/mol). Here we document the existence of this stem by enzyme probing and examine its influence and that of neighboring sequences on the unusual choice of fusion sites by analyzing transcripts made in vivo from mutated defective interfering RNA constructs. We learned that (i) mutations that were predicted to unfold the stem-loop in various ways did not switch RdRp crossover to the upstream canonical site, (ii) a totally nonconforming downstream motif resulted in no measurable transcription from either site, (iii) the canonical upstream site does not function ectopically to lend competence to the downstream noncanonical site, and (iv) altering flanking sequences downstream of the downstream noncanonical motif in ways that diminish sequence similarity with the virus genome 5′ end caused a dramatic switch to the upstream canonical site. These results show that sequence elements downstream of the noncanonical site can dramatically influence the choice of fusion sites for synthesis of mRNA 5 and are interpreted as being most consistent with a mechanism of similarity-assisted RdRp strand switching during minus-strand synthesis.

8 páginas, 3 figuras, We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2., We gratefully acknowledge the authors, originating and submitting laboratories of the sequences from GISAID’s EpiCoV Database used in the phylogenetic analysis. We gratefully acknowledge all the staff working with sample collection, sample preparation, sequencing, data analysis and data sharing in all laboratories in the WHO European Region for making this work possible.