Concurrent occurrence of human and equine West Nile virus infections in Central Anatolia, Turkey: the first evidence for circulation of lineage 1 viruses

SummaryBackgroundWest Nile fever is an important zoonotic infection caused by West Nile virus (WNV), a member of the Flaviviridae. Previous serological data from Turkey suggest widespread WNV circulation. This report includes cases of human and equine WNV infections occurring concurrently, and manifesting as central nervous system infections, in two neighboring provinces of Central Anatolia, Turkey. A partial phylogenetic analysis of the causative virus is given for the first time.MethodsThe cases were reported in February (horses) and March (human). Symptoms of the disease were similar in the two species, characterized by neurological manifestations suggesting meningoencephalitis. Real-time/nested PCRs and commercial immunoassays and a plaque reduction neutralization assay were employed for the detection of viral RNA and specific antibodies, respectively.ResultsWNV RNAs were detected in buffy coat (horses) and cerebrospinal fluid (human) samples. Partial nucleotide sequences of the E-gene coding region revealed that the strains are closely related to viruses of lineage 1, clade 1a. Accompanying equine serosurveillance demonstrated WNV-specific antibodies in 31.6% of the samples.ConclusionsThis is the first report of acute WNV infections caused by lineage 1 strains from Turkey, in concordance with previous reports from some European and North African countries

Multi-assay investigation of viral etiology in pediatric central nervous system infections

Introduction: In an attempt to identify a wide spectrum of viral infections, cerebrospinal fluid (CSF) specimens were collected from pediatric cases with the preliminary diagnosis of viral encephalitis/meningoencephalitis in two reference hospitals, from October 2011 to December 2015. Methodology: A combination of nucleic acid-based assays, including in house generic polymerase chain reaction (PCR) assays for enteroviruses, flaviviruses and phleboviruses, a commercial real-time PCR assay for herpesviruses and a commercial real time multiplex PCR, enabling detection of frequently-observed viral, bacterial and fungal agents were employed for screening. Results: The microbial agent could be characterized in 10 (10%) of the 100 specimens. Viral etiology could be demonstrated in 7 (70%) specimens, which comprises Human Herpesvirus 6 (4/7), Herpes Simplex virus type1 (2/7) and Enteroviruses (1/7). In 3 specimens (30%), Streptococcus pneumoniae, Listeria monocytogenes and Staphylococcus aureus were detected via the multiplex PCR, which were also isolated in bacteriological media. All specimens with detectable viral nucleic acids, as well as unreactive specimens via nucleic acid testing remained negative in bacteriological cultures. Conclusions: Herpes and enteroviruses were identified as the primary causative agents of central nervous system infections in children. Enterovirus testing must be included in the diagnostic work-up of relevant cases

Co-circulation of West Nile virus and distinct insect-specific flaviviruses in Turkey

Background: Active vector surveillance provides an efficient tool for monitoring the presence or spread of emerging or re-emerging vector-borne viruses. This study was undertaken to investigate the circulation of flaviviruses. Mosquitoes were collected from 58 locations in 10 provinces across the Aegean, Thrace and Mediterranean Anatolian regions of Turkey in 2014 and 2015. Following morphological identification, mosquitoes were pooled and screened by nested and real-time PCR assays. Detected viruses were further characterised by sequencing. Positive pools were inoculated onto cell lines for virus isolation. Next generation sequencing was employed for genomic characterisation of the isolates. Results: A total of 12,711 mosquito specimens representing 15 species were screened in 594 pools. Eleven pools (2%) were reactive in the virus screening assays. Sequencing revealed West Nile virus (WNV) in one Culex pipiens (s.l.) pool from Thrace. WNV sequence corresponded to lineage one clade 1a but clustered distinctly from the Turkish prototype isolate. In 10 pools, insect-specific flaviviruses were characterised as Culex theileri flavivirus in 5 pools of Culex theileri and one pool of Cx. pipiens (s.l.), Ochlerotatus caspius flavivirus in two pools of Aedes (Ochlerotatus) caspius, Flavivirus AV-2011 in one pool of Culiseta annulata, and an undetermined flavivirus in one pool of Uranotaenia unguiculata from the Aegean and Thrace regions. DNA forms or integration of the detected insect-specific flaviviruses were not observed. A virus strain, tentatively named as “Ochlerotatus caspius flavivirus Turkey”, was isolated from an Ae. caspius pool in C6/36 cells. The viral genome comprised 10,370 nucleotides with a putative polyprotein of 3,385 amino acids that follows the canonical flavivirus polyprotein organisation. Sequence comparisons and phylogenetic analyses revealed the close relationship of this strain with Ochlerotatus caspius flavivirus from Portugal and Hanko virus from Finland. Several conserved structural and amino acid motifs were identified. Conclusions: We identified WNV and several distinct insect-specific flaviviruses during an extensive biosurveillance study of mosquitoes in various regions of Turkey in 2014 and 2015. Ongoing circulation of WNV is revealed, with an unprecedented genetic diversity. A probable replicating form of an insect flavivirus identified only in DNA form was detected

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

Evaluation of the effects of acyclovir and/or human amniotic membrane on herpes virus culture and quantitative virus inactivity by real-time polymerase chain reaction

AIM: To investigate the permeability of amniotic membrane in herpes virus cell culture to acyclovir with real time polymerase chain reaction (RT-PCR).METHODS: Madin-Darby Bovine Kidney (MDBK) cell culture and Bovine Herpes Virus (BHV1) type 1 were used in the study. Cell cultures were grouped into two on the basis of herpes virus inoculation. Each group was sub-grouped into three. Amniotic membrane (V-HAM), acyclovir (V-A), and amniotic membrane and acyclovir (V-HAM-A) were applied to these subgroup cultures, respectively. After the application of the membrane and the drug, the cultures were evaluated at 24 and 48h for cytopathic effect positive (CPE+) with a tissue culture microscope. In the CPE (+) samples, the DNA was extracted for viral DNA analysis by RT-PCR.RESULTS: In control cultures without herpes virus CPE was not detected. Besides, amniotic membrane and acyclovir did not have cytotoxic effect on cell cultures. CPE were detected in Bovine Herpesvirus type-1 inoculated cell cultures after amniotic membrane and/or acyclovir application. DNA analysis with RT-PCR indicated that Cycle threshold (Ct) values were lower in the BHV1 and membrane applied group (amniotic membrane group< acyclovir group< membrane and acyclovir group). This showed that membrane did not have antiviral effect. The membrane and acyclovir cell culture groups with high Ct values indicated that membrane was permeable and had a low barrier effect to drug,CONCLUSION: In our in-vitro study, we found that amniotic membrane, which can be used in the treatment of corneal diseases, did not have antiviral effect. Besides, we detected that amniotic membrane was permeable to acyclovir in BHV-1 inoculated MDBK cell culture. However, more studies are necessary to investigate the quantitative effects of amniotic membrane and acyclovir

The Longest Infectious Virus Shedding in a Child Infected With the G614 Strain of SARS-CoV-2

COVID-19 spread globally and caused over 97 million cases with more than 2 million deaths. There is still ongoing discussion on the duration of infectious interval SARS-CoV-2 infection. Symptomatic children had longer virus shedding and there are some reports of prolonged infectious virus shedding in adults particularly patients having an immunocompromised status. A missense mutation, D614G, in the spike protein of SARSCoV-2, which has emerged as a predominant clade in Europe and is spreading worldwide that can result in higher viral loads in patients. Herein, we described the longest infectious virus shedding in a previously healthy child infected with SARS-CoV-2 expressing spike D614G substitution

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

Close Relationship Between West Nile Virus From Turkey And Lineage 1 Strain From Central African Republic

We sequenced West Nile viruses (WNVs) from Turkey and found close relationships to WNV lineage 1 strain ArB310/67 from the Central African Republic, distinct from other WNVs circulating in the Mediterranean Basin, eastern Europe, and the Middle East. These findings suggest independent introductions of WNV strains from Africa to the Middle East.PubMedWoSScopu

Molecular epidemiology of Crimean-Congo hemorrhagic fever virus in Turkey: Occurrence of local topotype

The goal of this study was to investigate the molecular epidemiology of Crimean-Congo hemorrhagic fever virus (CCHFV) in Turkey. The study was performed on a total of 48 confirmed human CCHF cases from 2006 to 2008. The majority of the CCHF viral strains in Turkey were found to belong to the European lineage. Local CCHF viral strains are grouped into two main clusters, which can be further divided into two sub-groups. We also identified an AP92-like virus causing clinical disease in Corum (a mid-Anatolian province). Phylogenetic analysis revealed that the most recent CCHFV infections were caused by intrinsic (or native) CCHF viral strains, which we identified as the local topotype. Comparison of deduced amino acid sequences of S-segment RNAs indicated that the local topotype was derived from viruses of previous years, most likely by a low rate recombination. No genetic differences, based on S- and M-segment RNA sequences, were found between human and tick viral isolates. This data suggest that replication of CCHFV in the tick vector, whether Rhiphicephalus spp. or Hyalomma spp., has no effect on the viral genomic structure. (C) 2010 Elsevier B.V. All rights reserved