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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.

SARS-CoV-2 is a novel and highly pathogenic coronavirus, which has caused an outbreak in Wuhan City, China, in 2019 and then spread rapidly throughout the world. Although several COVID-19 vaccines are currently available for mass immunization, they are less effective against emerging SARS-CoV-2 variants, especially the Omicron (B.1.1.529). Recently, we successfully produced receptor-binding domain (RBD) variants of spike (S) protein of SARC-CoV-2 and an antigen cocktail in Nicotiana benthamiana, which are highly produced in plants and elicited high-titer antibodies with potent neutralizing activity against SARS-CoV-2. In this study, we demonstrate that these protein-based vaccine candidates are highly effective against Delta and Omicron variants. These data support that plant produced RBD and cocktail-based antigens are most promising vaccine candidates and may protect against Delta and Omicron-mediated COVID-19. Based on the neutralization ability, plant produced RBD and cocktail-based vaccine candidates are highly effective against SARS-CoV-2, independently of its emerging variants.

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

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a tick-borne disease with high mortality Fates in humans. The distribution of CCHFV includes over 30 countries in Asia, the Middle East, southeastern Europe, and Africa. It was first recognized in Turkey in 2002, with an increasing number of cases reported between 2002 and 2009. Recent analysis of complete genome sequences of CCHFV isolates has revealed that the genomic plasticity of the virus is surprisingly high for an arthropod-borne virus. We have determined the complete nucleotide and deduced amino acid sequences of strain CCHFV Turkey-Kelkit06 isolated from the blood of a patient in an endemic region of Turkey in 2006. The complete sequence length of the CCHFV Turkey-Kelkit06 strain is 19,186 nt, consisting of a 1673 nt S segment, a 5364 nt M segment, and a 12,149 nt L segment. Based on the analysis of S, M, and L segments, CCHFV Turkey-Kelkit06 clustered in Group V, which represents the Europe/Turkey geographic lineage. Although glycoproteins encoded by the M gene are the most variable part of the CCHFV Turkey-Kelkit06 strain, some functional domains of the glycoproteins are well conserved. Here, we report the complete sequence and genome organization of the CCHFV Turkey-Kelkit06 strain and its phylogenetic relationship to other strains of CCHFV. Collecting data on viral sequences among isolates from CCHF epidemics may provide valuable information regarding the molecular basis of the epidemic potential of the virus. (C) 2009 Elsevier B.V. All rights reserved.

Crimean-Congo hemorrhagic fever (CCHF) virus is member of the genus Nairovirus of the family Bunyaviridae. All members of the family Bunyaviridae 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. During recent years, outbreaks have been reported in Turkey. However, little information is available on the genetic diversity of CCHF virus in Turkey. In this study, a total of 1227 adult ticks were collected from domestic ruminants (796 specimens from cattle, 399 specimens from goats and 32 specimens from sheep). The presence of the M segment of CCHF virus was determined in 4 of 40 (10%) Hyalomma marginatum marginatum pools, in 2 of 38 (7.89%) Rhipicephalus bursa pools, and in 1 of 7 (7%) Boophylus annulatus pools. Hyalomma anatolicum anatolicum pools gave negative RT-PCR result against CCHF virus. Serum samples from seven patients infected with CCHF were selected and subjected to RT-PCR to amplify partial M segment of CCHF virus. This report introduces the first data on partial nucleotide sequences of M RNA segments of CCHF virus strains circulating in Turkey, isolated from ticks.

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 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.

Hepatitis B is one of the most common infectious diseases in the world, and 350 million people have been estimated to be chronic hepatitis B virus carriers world-wide. Hepatitis B virus (HBV) has been classified into 8 genotypes (A-H) based on an intergroup divergence of 8% or more in the complete nucleotide sequence. Different genotypes of the hepatitis B virus may influence the clinical outcome of the disease. HBV genotyping method using restriction fragment length polymorphism (RFLP) can reliably identify genotypes. HBV genotyping with S gene sequence is consistent with genetic analysis using the full genomic sequences. The aim of this study was to determine the genotypes of HBV by using restriction fragment length polymorphism (RFLP) method in the region of Elazig. A total of 127 HBV-DNA positive patients (74 male, 53 female) were included in the study. Semi-nested polymerase chain reaction (PCR) was performed to amplify the specific parts of HBV S gene. In the first step, 685 base paired (bp) region was amplified by sense primer HBMF1 and anti-sense primer HBMR2, while in the second step 485 bp region was amplified by using inner-sense primer HBMF2 and anti-sense primer HBMR2. PCR products were then digested by the restriction enzymes, Alwl, Earl, Hphl, Ncil and NlalV. The RFLP assay indicated that genotype D was the only detected type in our samples. In conclusion, genotype D is the predominant type among hepatitis B patients in our region. RFLP is considered to be an easy and useful method for genotyping HBV strains.

Monitoring of HBV replication level is very useful for the management of patients with chronic HBV. However, the use of the correct tools to quantify HBV-DNA levels in serum and monitor the replication of HBV is of paramount importance in terms of diagnosis, and antiviral treatment of patients with chronic HBV infection. The aim of this study was to combine the bDNA assay and HBV PCR to improve detection of viremia the patients with HBeAg-positive chronic hepatitis B infection.In this study, 67 HBeAg-positive chronic hepatitis B patients were analyzed to determine viremia level using bDNA and HBV PCR assays.Sixty-four patients with HBeAg-positive chronic hepatitis B showed positivity by conventional HBV PCR, whereas 56 subjects with HBeAg-positive chronic hepatitis B showed HBV-DNA levels by bDNA.The results indicated that it is reasonable to use the bDNA assay to determine HBV replicative activity first, and use conventional HBV-PCR for HBeAg-positive chronic hepatitis B patient samples that are negative in bDNA assay.

Candida dubliniensis is a recently identified opportunistic pathogen, which has close phylogenetic relation with Candida albicans. The aim of this study was the genotypic differentiation of 55 germ tube-positive Candida strains isolated from clinical specimens (30 blood, 25 throat swab specimens). The isolates were phenotypically identified by API ID 32C system, and genotypically identified by using polymerase chain reaction-restriction fragment lenght polymorphism (PCR-RFLP) method. Initially ITS2 region has been amplified by using universal fungal primers with PCR. After amplification and purification of approximately 340 base pair products, they were treated with species-specific restriction enzymes (MspA1 I and BsmAI for C. albicans and C. dubliniensis, respectively). As a result, API ID 32C system identified 52 (94.5%) isolates as C. albicans and 3 (5.5%) isolates as C. dubliniensis, whereas PCR-RFLP analysis yielded 50 (90.9%) C. albicans and 5 (9.1%) C. dubliniensis. It can be concluded that PCR-RFLP method may be used for the differentiation of C. dubliniensis and C. albicans isolates in clinical samples.

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.

We investigated the incidence of Epstein-Barr virus (EBV) infection in surgical specimens of squamous cell carcinoma of the larynx and vocal cord nodules.Formalin-fixed, paraffin-embedded tissue samples randomly selected from 22 patients with squamous cell carcinoma of the larynx (20 males, 2 females; mean age 52 years) and from 17 patients with vocal cord nodules (14 males, 3 females; mean age 38 years) were examined by the polymerase chain reaction. The patients were assessed in terms of localization of the disease, smoking habits, duration of smoking, and the presence or absence of EBV DNA.Twenty-one patients with laryngeal carcinoma and nine patients with vocal cord nodules were smokers. Polymerase chain reaction showed EBV DNA in 11 patients (50%) with laryngeal carcinoma and in seven patients (41.2%) with vocal cord nodules. No significant differences were found with respect to EBV DNA positivity, smoking habits, smoking period, the number of cigarettes consumed daily, localization of disease, and tumor stage (p0.05).Epstein-Barr virus does not seem to be directly associated with the pathogenesis of laryngeal squamous cell carcinoma, but its role in the etiology of laryngeal proliferative diseases needs more elucidation.

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.