Semi-automated diagnostic RT-PCR as a screening assay for antiviral compounds in a 96-well format against highly pathogenic RNA viruses.

Background and Aim: The CCHF (Crimean-Congo hemorrhagic fever) virus causes a severe disease in human with a case fatality rate of up to 50%. Since, there is no specific treatment or approved vaccine against CCHF viral infections, an accurate and early detection as well as a reliable surveillance and quantitative determination of viral load is necessary for patient improvement and case management. In this research, our aim was to develop a probe based one-step real-time reverse-transcription polymerase chain reaction (rRT-PCR) assay for in-house quantitative detection of CCHF virus. Methods: At first, the highly conserved S-fragment sequence of CCHF virus genome was adapted from GenBank and the specific probe and primers targeting this region were designed. Then, viral RNAs were extracted from 37 blood samples of different patients from east of Iran (Zahedan). The specificity and sensitivity of the probe and primers were also evaluated in positive blood samples, confirmed to have CCHF virus. A standard (PTG19-T vector containing S-fragment) for quantization was also constructed and the viral load was determined in some of positive samples. Results: From a total of 37 suspicious blood samples, 15 samples were confirmed to be positive for CCHF virus by this probe based one-step rRT-PCR assay and no false-positive result was detected according to sequencing data. The predicted fragment of 176 bp was also confirmed in all positive samples by gel-based electrophoresis analysis. The assay was linear between 10 to 103 copy numbers per each microliter of extracted plasmid for this technique and the viral load determined in one of patient blood samples was 55,000 viral particles per each milliliter, for example. Bioinformatics and experimental evaluations approved the specificity of this assay. The LOD of the assay was 10 (or fewer) copy numbers of viral genome per each microliter of the extracted genome. Conclusions: This research showed that the developed probe based one-step rRT-PCR assay is a specific, rapid, sensitive and the simple tool for detection and viral load determination of the CCHF virus. Keywords: CCHF Virus, Real-time RT-PCR, Quantitative, viral load, S-region.

The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques.

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.

Seroprevalence of four zoonotic viral diseases in camels in Qatar.

Emerging and re-emerging infectiousdiseases due to RNA virusescause major negative consequences for the quality of life, publichealth, and overall economic development. Most of the RNA virusescausing illnesses in humans are of zoonotic origin. Zoonotic virusescan directly be transferred from animals to humans through adaptation,followed by human-to-human transmission, such as in human immunodeficiencyvirus (HIV), severe acute respiratory syndrome coronavirus (SARS-CoV),Middle East respiratory syndrome coronavirus (MERS-CoV), and, morerecently, SARS coronavirus 2 (SARS-CoV-2), or they can be transferredthrough insects or vectors, as in the case of Crimean-Congo hemorrhagicfever virus (CCHFV), Zika virus (ZIKV), and dengue virus (DENV). Atthe present, there are no vaccines or antiviral compounds againstmost of these viruses. Because proteins possess a vast array of functionsin all known biological systems, proteomics-based strategies can provideimportant insights into the investigation of disease pathogenesisand the identification of promising antiviral drug targets duringan epidemic or pandemic. Mass spectrometry technology has providedthe capacity required for the precise identification and the sensitiveand high-throughput analysis of proteins on a large scale and hascontributed greatly to unravelling key protein–protein interactions,discovering signaling networks, and understanding disease mechanisms.In this Review, we present an account of quantitative proteomics andits application in some prominent recent examples of emerging andre-emerging RNA virus diseases like HIV-1, CCHFV, ZIKV, and DENV,with more detail with respect to coronaviruses (MERS-CoV and SARS-CoV)as well as the recent SARS-CoV-2 pandemic.

Objective To construct the recombinant Crimean-Congo hemorrhagic fever virus (CCHFV) which can express the secreted nanoluciferase (NanoLuc) and investigate its potential application for rapid antiviral compounds screening. Methods The ORF of NanoLuc and the mucin encoded by the M segment of CCHFV were merged, and the recombinant CCHFV (rCCHFV) was rescued through reverse genetic system. Then rCCHFV was used to evaluate the antiviral effect for ribavirin and Furin inhibitor in vitro. Results The rCCHFV_mucin_NLuc with NanoLuc reporter was obtained, and the relative light unit (RLU) which can reflect NanoLuc activity was positively correlated with median tissue culture infective dose (TCID50) in the infected cell supernatant (cor=0.998, P=0.001). When the concentration for the compounds was 10 μmol/L, there was no significant difference for the NanoLuc activity in the infected cell supernatant between Furin inhibitor and ribavirin (P > 0.1) from day 1 to 3 after treatment. But at day 4, the NanoLuc activity in Fruin inhibitor treated group was significantly higher than that of ribavirin treated group (P=0.001), and no significant difference was found between the Furin inhibitor and untreated group (P > 0.1). Conclusions The rCCHFV with NanoLuc reporter was recovered successfully and it could be used for the primary rapid screening of antiviral compounds in future. Key words: Luciferase; Crimean-Congo hemorrhagic fever virus; Antiviral compound; Screening

Middle East respiratory syndrome coronavirus on inanimate surfaces: A risk for health care transmission

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family BUNYAVIRIDAE: The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family BUNYAVIRIDAE: This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.

The Indian subcontinent with its population density, climatic conditions, means of subsistence, socioeconomic factors as well as travel and tourism presents a fertile ground for thriving of RNA viruses. Despite being pathogens of huge significance, there is very little focus on research into the biology and pathogenesis of RNA viruses in India. Studies on epidemiology and disease burden, risk factors, the immune response to RNA viruses, circulating virus strains and virus evolution, animal models of disease, antivirals and vaccines are strikingly absent. Emerging RNA viruses such as Zika virus, Nipah virus and Crimean-Congo haemorrhagic fever virus are a matter of grave concern to India. Here we summarize the outcome of the India|EMBO symposium on “RNA viruses: immunology, pathogenesis and translational opportunities” organized at Faridabad, National Capital Region, India, on March 28–30, 2018. The meeting focused on RNA viruses (non-HIV), and both national and international experts on RNA viruses covered topics ranging from epidemiology, immune response, virus evolution and vaccine trials concerning RNA viruses. The aim of the symposium was to create a road map for RNA virus research in India. Both concrete and tentative ideas pointing towards short-term and long-term goals were presented with recommendations for follow-up at government level.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a high-priority pathogen in pandemic preparedness research. Reverse genetics systems are a valuable tool to study viral replication and pathogenesis, design attenuated vaccines and create defined viral assay systems for applications such as antiviral screening. Here we present a novel reverse genetics system for MERS-CoV that involves maintenance of the full-length viral genome as a cDNA copy inserted in a bacterial artificial chromosome amenable to manipulation by homologue recombination, based on the bacteriophage λ Red recombination system. Based on a full-length infectious MERS-CoV cDNA clone, optimal genomic insertion sites and expression strategies for GFP were identified and used to generate a reporter MERS-CoV expressing GFP in addition to the complete set of viral proteins. GFP was genetically fused to the N-terminal part of protein 4a, from which it is released during translation via porcine teschovirus 2A peptide activity. The resulting reporter virus achieved titres nearly identical to the wild-type virus 48 h after infection of Vero cells at m.o.i. 0.001 (1×105 p.f.u. ml-1 and 3×105 p.f.u. ml-1, respectively), and allowed determination of the 50 % inhibitory concentration for the known MERS-CoV inhibitor cyclosporine A based on fluorescence readout. The resulting value was 2.41 µM, which corresponds to values based on wild-type virus. The reverse genetics system described herein can be efficiently mutated by Red-mediated recombination. The GFP-expressing reporter virus contains the full set of MERS-CoV proteins and achieves wild-type titres in cell culture.

Crimean-Congo hemorrhagic fever (CCHF) is a viral disease transmitted to humans mainly by bite of Ixodid ticks, mainly those of the Hyalomma genus. CCHFV belongs to the genus Nairovirus in the family Bunyaviridae. CCHF virus is a segmented, single stranded, negative sense and RNA viruses. The onset of the disease is very sudden, with symptoms such as fever, rigors, intense headache, chills, and backache or leg pains, myalgia, nausea, and vomiting. CCHF originally identified in the former Soviet Union and the Congo, has rapidly spread across large sections of Europe, Asia, and Africa, and has been reported in more than 30 countries. The climatic changes may affect the life cycle of ticks and the routes of migratory birds, leading to tick abundance and virus distribution in CCHF-free areas. Extended use of land for agriculture and farming and changes in hunting activities play also a role in CCHF incidence, while livestock trade and movement may influence host-tick-virus dynamics resulting in transfer of CCHFV-infected ticks in non-endemic areas. Recent years, the epidemiology of CCHF is changing in Balkans and Turkey. Balkan Peninsula is a known endemic CCHF area, and sporadic cases and even outbreaks are being reported every year. The annual number of human CCHF cases is increasing in Balkans and Turkey. While Bulgaria, Kosovo and Albania were ZET Krm-Kongo Kanamal Atei (KKKA), zellikle Ixodid cinsi kene sr (esas olarak Hyalomma cinsi)

Coronavirus Disease 2019: Coronaviruses and Kidney Injury.

BackgroundThe Crimean-Congo hemorrhagic fever (CCHF) virus causes severe disease in humans, with a high mortality rate. Since, there is no approved vaccine or specific treatment for CCHF, an early and accurate diagnosis, as well as reliable surveillance, is essential for case management and patient improvement.ObjectivesFor this research, our aim was to evaluate the application of a novel SYBR Green based one-step real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay for the in-house diagnosis of the CCHF virus.Patients and MethodsIn this experimental study, the highly conserved S-region sequence of the CCHF viral genome was first adapted from GenBank, and the specific primers targeting this region were designed. Then, the viral RNA was extracted from 75 serum samples from different patients in eastern Iran. The sensitivity and specificity of the primers were also evaluated in positive serum samples previously confirmed to have the CCHF virus, by this one-step rRT-PCR assay, as well as a DNA sequencing analysis.ResultsFrom a total of 75 suspected serum samples, 42 were confirmed to be positive for CCHF virus, with no false-positives detected by the sequencing results. After 40 amplification cycles, the melting curve analysis revealed a mean melting temperature (Tm) of 86.5 ± 0.6°C (quite different from those of the primer-dimers), and the positive samples showed only a small variation in the parameters. In all of the positive samples, the predicted length of 420 bp was confirmed by electrophoresis. Moreover, the sensitivity test showed that this assay can detect less than 20 copies of viral RNA per reaction.ConclusionsThis study showed that this novel one-step rRT-PCR assay is a rapid, reliable, repeatable, specific, sensitive, and simple tool for the detection of the CCHF virus.

Objective To establish a plaque assay for detection of Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Methods Vero and Huh7.5 cells were infected with MERS-CoV (EMC/2012) strains for different days (two or three days) and covered with different overlays (agarose or Avicel). The optimal host cells, time of infection and overlay were screened out by analyzing plague formation to establish the plaque assay for detection of MERS-CoV infection. Neutralizing antibodies against MERS-CoV were detected by the established assay. Results Clear plaques were observed on Vero cells infected with MERS-CoV at 3 days post-infection (p.i.), while only tip-size plaques were observed at 2 days p. i.. Clearer and bigger sizes of plaques were observed under the overlay of Avicel than those under the agarose. The Avicel-based plaque assay was suitable for detecting neutralizing antibodies against MERS-CoV. Conclusion We develop a modified plaque assay for detecting MERS-CoV based on Avicel-crystal violet staining. It is easier to operate the modified plaque assay in biological safety level 3 (BSL-3) laboratories. This study paves a way for quantification of MERS-CoV and detection of neutralizing antibodies against MERS-CoV. Key words: Middle East respiratory syndrome coronavirus (MERS-CoV); Biological safety level 3 (BSL-3) laboratory; Plaque assay; Vero cell; Avicel; Neutralization test

Taking forward a 'One Health' approach for turning the tide against the Middle East respiratory syndrome coronavirus and other zoonotic pathogens with epidemic potential.