Novel Tick Phlebovirus Genotypes Lacking Evidence for Vertebrate Infections in Anatolia and Thrace, Turkey

We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and pathogenicity in vertebrates. Ticks were collected at locations in Istanbul (Northwest Anatolia, Thrace), Edirne, Kirklareli, and Tekirdag (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Sanliurfa (Southeastern Anatolia) provinces from 2013-2018 and were analyzed following morphological identification and pooling. Specimens from individuals with febrile disease or meningoencephalitic symptoms of an unknown etiology were also evaluated. The pools were screened via generic tick phlebovirus amplification assays and products were sequenced. Selected pools were used for cell culture and suckling mice inoculations and next generation sequencing (NGS). A total of 7492 ticks were screened in 609 pools where 4.2% were positive. A phylogenetic sequence clustering according to tick species was observed. No human samples were positive. NGS provided near-complete viral replicase coding sequences in three pools. A comprehensive analysis revealed three distinct, monophyletic virus genotypes, comprised of previously-described viruses from Anatolia and the Balkans, with unique fingerprints in conserved amino acid motifs in viral replicase. A novel tick phlebovirus group was discovered circulating in the Balkans and Turkey, with at least three genotypes or species. No evidence for replication in vertebrates or infections in clinical cases could be demonstrated.Armed Forces Health Surveillance Board, Global Emerging Infections Surveillance and Response System (AFHSB-GEIS), United States of America [P0034_18_WR]; US ArmyUnited States Department of Defense [W911QY-16-C-0160]The study was supported in part by the Armed Forces Health Surveillance Board, Global Emerging Infections Surveillance and Response System (AFHSB-GEIS), United States of America (FY18 award P0034_18_WR (PI: Yvonne-Marie Linton) under US Army subcontract W911QY-16-C-0160)

Differential Growth Characteristics of Crimean-Congo Hemorrhagic Fever Virus in Kidney Cells of Human and Bovine Origin

Crimean-Congo hemorrhagic fever virus (CCHFV) causes a lethal tick-borne zoonotic disease with severe clinical manifestation in humans but does not produce symptomatic disease in wild or domestic animals. The factors contributing to differential outcomes of infection between species are not yet understood. Since CCHFV is known to have tropism to kidney tissue and cattle play an important role as an amplifying host for CCHFV, in this study, we assessed in vitro cell susceptibility to CCHFV infection in immortalized and primary kidney and adrenal gland cell lines of human and bovine origin. Based on our indirect fluorescent focus assay (IFFA), we suggest a cell-to-cell CCHF viral spread process in bovine kidney cells but not in human cells. Over the course of seven days post-infection (dpi), infected bovine kidney cells are found in restricted islet-like areas. In contrast, three dpi infected human kidney or adrenal cells were noted in areas distant from one another yet progressed to up to 100% infection of the monolayer. Pronounced CCHFV replication, measured by quantitative real-time RT-PCR (qRT-PCR) of both intra- and extracellular viral RNA, was documented only in human kidney cells, supporting restrictive infection in cells of bovine origin. To further investigate the differences, lactate dehydrogenase activity and cytopathic effects were measured at different time points in all mentioned cells. In vitro assays indicated that CCHFV infection affects human and bovine kidney cells differently, where human cell lines seem to be markedly permissive. This is the initial reporting of CCHFV susceptibility and replication patterns in bovine cells and the first report to compare human and animal cell permissiveness in vitro. Further investigations will help to understand the impact of different cell types of various origins on the virus–host interaction

Immunological Analysis Of A Cchfv Mrna Vaccine Candidate In Mouse Models

Development of new vaccine platforms against viral diseases is considered urgent. In recent years, mRNA constructs have attracted great interest in this field due to unique advantages over conventional gene transfer platforms. In the present study, we developed a new naked conventional mRNA vaccine expressing the non-optimized small (S) segment of the Ank-2 strain of Crimean-Congo Hemorrhagic Fever virus (CCHFV). We then analyzed its single and booster dose immunogenicity and protection potential in the challenge assay in two mice models, including IFNα/β/γR−/− and C57BL/6. The results obtained from the immunological assays, namely IL-4 and IFN-gamma ELISPOT, intracellular IFN-gamma staining, in-house sandwich ELISA, and survival data, demonstrated that our construct elicited the production of anti-nucleocapsid (N) specific immune responses in both mice models. A 100% protection rate was only obtained in the booster dose group of IFNα/β/γR−/− mice, indicating that this platform needs further optimization in future studies. In conclusion, we assessed a novel approach in CCHFV vaccination by introducing a conventional mRNA platform which can be considered in future experiments as an efficient and safe way to battle this disease.PubMedWoSScopu

Assessment of the Immunogenicity and Protective Aspects of a DNA Vaccine Targeting Crimean Congo Hemorrhagic Fever Virus Glycoprotein Gc

Aim: Crimean Congo Hemorrhagic Fever (CCHF) is a lethal, endemic infectious disease inhuman. For the preventive measures of the disease, there is currently no safe and efficient vaccine,widely for human use. Vaccine development for CCHF virus is an actively researched subject. Inthis study, we aimed to investigate the immunizing and protective potentials of the CCHF virussurface glycoprotein Gc that is delivered as a single antigen via a DNA based vaccine vector.Material and Methods: A DNA based vaccine targeting the immunogenic envelope glycoproteinGc of a CCHF virus isolate with Turkey origin (Ank2) was generated and its immunogenicity andprotective capability against lethal challenge in IFN?/?R-/- receptor knock out mice was assessed.Results: The developed vaccine candidate (pGc) elicited a considerable amount of neutralizingantibody responses in the vaccinated mice. The vaccine candidate significantly induced bothantiviral Th1 and B cell activating Th2 immune responses deduced from the cytokineproduction profiles in the vaccinated mice. However, despite the immune responses elicitedpost-immunization, the vaccine failed to confer protection against lethal CCHF virus infection.Conclusion: To the best of our knowledge, this is the first report of a DNA vaccine candidategenerated against CCHF virus based on the glycoprotein Gc. The pGc vaccine candidate exhibitedantigen-specific immunity in IFN/?/?R-/- mice, but was unable to produce a protection upon lethalchallenge with the homologous CCHF virus. Once we comprehensively understand the immunecorrelates of protection, we will be more eligible to significantly improve the efficacy of vaccines

Assessment of the immunogenicity and protective aspects of a dna vaccine targeting crimean congo hemorrhagic fever virus glycoprotein gc Kırım kongo kanamalı ateşi virüsü glikoprotein gc’yi hedef alan bir dna aşısının bağışıklık ve koruyuculuk sağlama özelliklerinin değerlendirilmesi

Aim: Crimean Congo Hemorrhagic Fever (CCHF) is a lethal, endemic infectious disease inhuman. For the preventive measures of the disease, there is currently no safe and efficient vaccine,widely for human use. Vaccine development for CCHF virus is an actively researched subject. Inthis study, we aimed to investigate the immunizing and protective potentials of the CCHF virussurface glycoprotein Gc that is delivered as a single antigen via a DNA based vaccine vector.Material and Methods: A DNA based vaccine targeting the immunogenic envelope glycoproteinGc of a CCHF virus isolate with Turkey origin (Ank2) was generated and its immunogenicity andprotective capability against lethal challenge in IFN?/?R-/- receptor knock out mice was assessed.Results: The developed vaccine candidate (pGc) elicited a considerable amount of neutralizingantibody responses in the vaccinated mice. The vaccine candidate significantly induced bothantiviral Th1 and B cell activating Th2 immune responses deduced from the cytokineproduction profiles in the vaccinated mice. However, despite the immune responses elicitedpost-immunization, the vaccine failed to confer protection against lethal CCHF virus infection.Conclusion: To the best of our knowledge, this is the first report of a DNA vaccine candidategenerated against CCHF virus based on the glycoprotein Gc. The pGc vaccine candidate exhibitedantigen-specific immunity in IFN/?/?R-/- mice, but was unable to produce a protection upon lethalchallenge with the homologous CCHF virus. Once we comprehensively understand the immunecorrelates of protection, we will be more eligible to significantly improve the efficacy of vaccines

Molecular investigation of Feline Herpesvirus 1 (FHV-1) and Feline Calicivirus in cats with respiratory system problem

Amaç: Bu çalışmada FHV (Feline Herpesvirus) ve FCV (Feline Calicivirus) şüpheli örneklerde söz konusu enfeksiyonların varlığı/yaygınlığının araştırılması, bildirilen semptomlarla ilişkilendirilmesi ve bu virusların moleküler karakterizasyonu amaçlandı. Gereç ve Yöntem: Bu amaçla klinik olarak solunum sistemi problemi gösteren toplam 70 kediden 31 nasal, 30 konjunktival, 8 oral, 7 orafarengeal ve 11 rektal swap ile 32 EDTA’lı kan örneği olmak üzere toplam 119 örnek alınmıştır. Alınan örneklerden viral nükleik asit ekstraksiyonu yapıldı ve söz konusu enfeksiyonların varlığı PCR ile araştırıldı. Pozitif bulunan örnekler moleküler karakterizasyon için dizin analizi işlemine tabi tutuldu. Bulgular: Örneklenen kedilerin toplam %45,71’i (32/70) FHV-1 ve %10’u (7/70) FCV nükleik asit varlığı yönünden pozitif olarak tespit edildi. Toplam pozitifliği bildirilen bu kedilerin, %4,29’i (3/70) her iki etken için birlikte pozitif olarak değerlendirildi. Örneklenen solunum sistemi bulgulu kedilerde FHV-1 enfeksiyonun daha yaygın olduğu görüldü; yaş bilgisi olan kedilerde enfeksiyonların her yaş grubunda oluştuğu gözlenirken; özellikle oral ve/veya orafarengeal swap örneklerinin her iki enfeksiyonun teşhisinde de duyarlı örnekler olduğu tespit edildi. Öneri: Bu enfeksiyonların, enfekte kedilerde görülmesinin yanı sıra sağlıklı görünümlü hayvanlarda da tespit edilebildiği ve söz konusu enfeksiyonların bulaşında bu durumun göz ardı edilmemesi gerekliliği ortaya çıkmaktadır. Bu nedenle klinik belirtilerin şiddetinin azaltılması, saçılımın ve persistansın en aza indirgenmesi adına özellikle toplu yaşayan, dışarıda bir arada bulunan kedilerin düzenli aşılanması üzerinde durulmalıdır. Ayrıca FHV ve FCV dışındaki etkenlerin de (M.felis, C.felis, B.bronchoseptica gibi bakterilerIer) kedilerde solunum, göz ve oral lezyonlarda bulunabileceği unutulmamalı ve bu etkenler de enfekte kedilerde kontrol edilmelidir.Aim: The aim of this study was to investigate the presence / prevalence of these infections in FHV (Feline Herpesvirus) and FCV (Feline Calicivirus) suspected samples, to correlate them with reported symptoms and molecular characterization of these viruses. Materials and Methods: For this purpose, a total of 119 specimens were collected from 70 from cats clinically showing respiratory system problem, 31 nasal, 30 conjunctival, 8 oral, 7 orafarengeal and 11 rectal swaps and 32 EDTA blood samples. Viral nucleic acid extraction was performed from the samples and the presence of these infections was investigated by PCR. Samples with positive results were subjected to sequence analysis for molecular characterization. Results: Sampled cats were evaluated as positive for 45.71% (32/70) FHV-1 and 10% (7/70) FCV. 4,29% (3/70) of these cats were positive for both infections. FHV-1 infection was more common in cats with respiratory system findings. While age-conscious cats were observed in all age groups; especially oral and / or orafarengeal swap samples were found to be sensitive samples in the diagnosis of both infections. Conclusion: These infections, as well as being seen in infected cats, can also be detected in healthy-looking animals and it should be noted that this should not be ignored in the transmission of these infections. Therefore, in order to reduce the severity of the clinical symptoms, to minimize the scattering and persistence, it is necessary to focus on the regular vaccination of the cats that live together, especially in the community. In addition, factors other than FHV and FCV (bacteria such as M.felis, C.felis, B.bronchoseptica) can be found in cats with respiratory, eye and oral lesions and these factors should be checked in infected cats

Co-Delivery Effect of CD24 on the Immunogenicity and Lethal Challenge Protection of a DNA Vector Expressing Nucleocapsid Protein of Crimean Congo Hemorrhagic Fever Virus

Crimean Congo hemorrhagic fever virus (CCHFV) is the causative agent of a globally-spread tick-borne zoonotic infection, with an eminent risk of fatal human disease. The imminent public health threat posed by the disseminated virus activity and lack of an approved therapeutic make CCHFV an urgent target for vaccine development. We described the construction of a DNA vector expressing a nucleocapsid protein (N) of CCHFV (pV-N13), and investigated its potential to stimulate the cytokine and total/specific antibody responses in BALB/c and a challenge experiment in IFNAR−/− mice. Because of a lack of sufficient antibody stimulation towards the N protein, we have selected cluster of differentiation 24 (CD24) protein as a potential adjuvant, which has a proliferative effect on B and T cells. Overall, our N expressing construct, when administered solely or in combination with the pCD24 vector, elicited significant cellular and humoral responses in BALB/c, despite variations in the particular cytokines and total antibodies. However, the stimulated antibodies produced as a result of the N protein expression have shown no neutralizing ability in the virus neutralization assay. Furthermore, the challenge experiments revealed the protection potential of the N expressing construct in an IFNAR −/− mice model. The cytokine analysis in the IFNAR−/− mice showed an elevation in the IL-6 and TNF-alpha levels. In conclusion, we have shown that targeting the S segment of CCHFV can be considered for a practical way to develop a vaccine against this virus, because of its ability to induce an immune response, which leads to protection in the challenge assays in the interferon (IFN)-gamma defective mice models. Moreover, CD24 has a prominent immunologic effect when it co-delivers with a suitable foreign gene expressing vector

Engineered cell-based therapies in ex vivo ready-made CellDex capsules have therapeutic efficacy in solid tumors

Encapsulated cell-based therapies for solid tumors have shown promising results in pre-clinical settings. However, the inability to culture encapsulated therapeutic cells prior to their transplantation has limited their translation into clinical settings. In this study, we created a wide variety of engineered therapeutic cells (ThC) loaded in micropore-forming gelatin methacryloyl (GelMA) hydrogel (CellDex) capsules that can be cultured in vitro prior to their transplantation in surgically debulked solid tumors. We show that both allogeneic and autologous engineered cells, such as stem cells (SCs), macrophages, NK cells, and T cells, proliferate within CellDex capsules and migrate out of the gel in vitro and in vivo. Furthermore, tumor cell specific therapeutic proteins and oncolytic viruses released from CellDex capsules retain and prolong their anti-tumor effects. In vivo, ThCs in pre-manufactured Celldex capsules persist long-term and track tumor cells. Moreover, chimeric antigen receptor (CAR) T cell bearing CellDex (T-CellDex) and human SC releasing therapeutic proteins (hSC-CellDex) capsules show therapeutic efficacy in metastatic and primary brain tumor resection models that mimic standard of care of tumor resection in patients. Overall, this unique approach of pre-manufactured micropore-forming CellDex capsules offers an effective off-the-shelf clinically viable strategy to treat solid tumors locally

Novel Tick Phlebovirus Genotypes Lacking Evidence for Vertebrate Infections in Anatolia and Thrace, Turkey

We screened ticks and human clinical specimens to detect and characterize tick phleboviruses and pathogenicity in vertebrates. Ticks were collected at locations in Istanbul (Northwest Anatolia, Thrace), Edirne, Kırklareli, and Tekirdağ (Thrace), Mersin (Mediterranean Anatolia), Adiyaman and Şanlıurfa (Southeastern Anatolia) provinces from 2013–2018 and were analyzed following morphological identification and pooling. Specimens from individuals with febrile disease or meningoencephalitic symptoms of an unknown etiology were also evaluated. The pools were screened via generic tick phlebovirus amplification assays and products were sequenced. Selected pools were used for cell culture and suckling mice inoculations and next generation sequencing (NGS). A total of 7492 ticks were screened in 609 pools where 4.2% were positive. A phylogenetic sequence clustering according to tick species was observed. No human samples were positive. NGS provided near-complete viral replicase coding sequences in three pools. A comprehensive analysis revealed three distinct, monophyletic virus genotypes, comprised of previously-described viruses from Anatolia and the Balkans, with unique fingerprints in conserved amino acid motifs in viral replicase. A novel tick phlebovirus group was discovered circulating in the Balkans and Turkey, with at least three genotypes or species. No evidence for replication in vertebrates or infections in clinical cases could be demonstrated.Peer Reviewe

Bovine Herpesvirus Type 4 (BoHV-4) Vector Delivering Nucleocapsid Protein of Crimean-Congo Hemorrhagic Fever Virus Induces Comparable Protective Immunity against Lethal Challenge in IFNα/β/γR−/− Mice Models

Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of a tick-borne infection with a significant mortality rate of up to 40% in endemic areas, with evidence of geographical expansion. Due to a lack of effective therapeutics and control measures, the development of a protective CCHFV vaccine remains a crucial public health task. This paper describes, for the first time, a Bovine herpesvirus type 4 (BoHV-4)-based viral vector (BoHV4-∆TK-CCHFV-N) and its immunogenicity in BALB/c and protection potential in IFNα/β/γR−/− mice models in comparison with two routinely used vaccine platforms, namely, Adenovirus type 5 and a DNA vector (pCDNA3.1 myc/His A), expressing the same antigen. All vaccine constructs successfully elicited significantly elevated cytokine levels and specific antibody responses in immunized BALB/c and IFNα/β/γR−/− mice. However, despite highly specific antibody responses in both animal models, the antibodies produced were unable to neutralize the virus in vitro. In the challenge experiment, only the BoHV4-∆TK-CCHFV-N and Ad5-N constructs produced 100% protection against lethal doses of the CCHFV Ank-2 strain in IFNα/β/γR−/− mice. The delivery platforms could not be compared due to similar protection rates in IFNα/β/γR−/− mice. However, during the challenge experiment in the T cell and passive antibody transfer assay, BoHV4-∆TK-CCHFV-N was dominant, with a protection rate of 75% compared to others. In conclusion, vector-based CCHFV N protein expression constitutes an effective approach for vaccine development and BoHV-4 emerged as a strong alternative to previously used viral vectors