Publications by authors named "Ali Mirazimi"

97 Publications

Cell-type-resolved quantitative proteomics map of interferon response against SARS-CoV-2.

iScience 2021 May 20;24(5):102420. Epub 2021 Apr 20.

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, ANA Futura, Campus Flemingsberg, 14152 Stockholm, Sweden.

The commonly used laboratory cell lines are the first line of experimental models to study the pathogenicity and performing antiviral assays for emerging viruses. Here, we assessed the tropism and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different human cell lines, compared their growth characteristics, and performed quantitative proteomics for the susceptible cell lines. Overall, Calu-3, Caco2, Huh7, and 293FT cell lines showed a high-to-moderate level of susceptibility to SARS-CoV-2. In Caco2 cells, the virus can achieve high titers in the absence of any prominent cytopathic effect. The protein abundance profile during SARS-CoV-2 infection revealed cell-type-specific regulation of cellular pathways. Type-I interferon signaling was identified as the common dysregulated cellular response in Caco2, Calu-3, and Huh7 cells. Together, our data show cell-type specific variability for cytopathogenicity, susceptibility, and cellular response to SARS-CoV-2 and provide important clues to guide future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.isci.2021.102420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056843PMC
May 2021

Organoid modeling of Zika and herpes simplex virus 1 infections reveals virus-specific responses leading to microcephaly.

Cell Stem Cell 2021 Apr 7. Epub 2021 Apr 7.

Institute of Molecular Biotechnology (IMBA), Austrian Academy of Sciences, Vienna BioCenter (VBC), Vienna 1030, Austria; Medical University of Vienna, Vienna 1030, Austria. Electronic address:

Viral infection in early pregnancy is a major cause of microcephaly. However, how distinct viruses impair human brain development remains poorly understood. Here we use human brain organoids to study the mechanisms underlying microcephaly caused by Zika virus (ZIKV) and herpes simplex virus (HSV-1). We find that both viruses efficiently replicate in brain organoids and attenuate their growth by causing cell death. However, transcriptional profiling reveals that ZIKV and HSV-1 elicit distinct cellular responses and that HSV-1 uniquely impairs neuroepithelial identity. Furthermore, we demonstrate that, although both viruses fail to potently induce the type I interferon system, the organoid defects caused by their infection can be rescued by distinct type I interferons. These phenotypes are not seen in 2D cultures, highlighting the superiority of brain organoids in modeling viral infections. These results uncover virus-specific mechanisms and complex cellular immune defenses associated with virus-induced microcephaly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2021.03.004DOI Listing
April 2021

Experimental Challenge of Sheep and Cattle with Dugbe Orthonairovirus, a Neglected African Arbovirus Distantly Related to CCHFV.

Viruses 2021 02 26;13(3). Epub 2021 Feb 26.

Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Suedufer 10, 17489 Greifswald-Insel Riems, Germany.

Dugbe orthonairovirus (DUGV) is a tick-borne arbovirus within the order . DUGV was first isolated in Nigeria, but virus isolations in ten further African countries indicate that DUGV is widespread throughout Africa. Humans can suffer from a mild febrile illness, hence, DUGV is classified as a biosafety level (BSL) 3 agent. In contrast, no disease has been described in animals, albeit serological evidence exists that ruminants are common hosts and may play an important role in the transmission cycle of this neglected arbovirus. In this study, young sheep and calves were experimentally inoculated with DUGV in order to determine their susceptibility and to study the course of infection. Moreover, potential antibody cross-reactivities in currently available diagnostic assays for Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) were assessed as DUGV is distantly related to CCHFV. Following subcutaneous inoculation, none of the animals developed clinical signs or viremia. However, all ruminants seroconverted, as demonstrated by two DUGV neutralization test formats (micro-virus neutralization test (mVNT), plaque reduction (PRNT)), by indirect immunofluorescence assays and in bovines by a newly developed DUGV recombinant N protein ELISA. Sera did not react in commercial CCHFV ELISAs, whereas cross-reactivities were observed by immunofluorescence and immunoblot assays.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v13030372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996740PMC
February 2021

Serological and molecular study of Crimean-Congo Hemorrhagic Fever Virus in cattle from selected districts in Uganda.

J Virol Methods 2021 04 27;290:114075. Epub 2021 Jan 27.

Section of Virology, Department of Biomedical Sciences and Veterinary Public Health, Swedish University of Agricultural Sciences, Box 7028, Uppsala, 750 07, Sweden; SLU Global Bioinformatics Centre, Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Box 7023, Uppsala, 750 07, Sweden. Electronic address:

Background: Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne viral hemorrhagic disease caused by Crimean-Congo Hemorrhagic Fever Virus (CCHFV) that poses serious public health challenges in many parts of Africa, Europe and Asia.

Methods: We examined 500 cattle sera samples from five districts for CCHFV antibodies using in-house and commercially available (IDVet) ELISA, Immunofluorescent assay (IFA) and Real-time polymerase chain reaction (RT-PCR).

Results: 500 cattle (73.8 % females) were analyzed; CCHFV seropositivity was 12.6 % (n = 63) and 75.0 % (n = 375) with the in-house and IDVet ELISAs, respectively. Seropositivity was associated with geographical location, increasing age, being female, and having a higher tick burden. Twenty four out of the 37 (64.8 %) were seropositive for CCHFV using IFA and all were negative for virus on RT-PCR. The IFA results were more comparable to IDVet (κ = 0.88, p = <0.01) than to in-house (κ = 0.32, p = 0.02).

Conclusions: Our study confirmed the presence and high prevalence of anti-CCHF antibodies in cattle based on three methods from all the five study districts, confirming presence and exposure of CCHFV. Given the zoonotic potential for CCHFV, we recommend a multidisciplinary public health surveillance and epidemiology of CCHFV in both animals and humans throughout the country.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jviromet.2021.114075DOI Listing
April 2021

Generation of enzymatically competent SARS-CoV-2 decoy receptor ACE2-Fc in glycoengineered Nicotiana benthamiana.

Biotechnol J 2021 Jan 22:e2000566. Epub 2021 Jan 22.

Department of Applied Genetics and Cell Biology, Institute of Plant Biotechnology and Cell Biology, University of Natural Resources and Life Sciences Vienna, Vienna, Austria.

Human angiotensin-converting enzyme 2 (ACE2) is the primary host cell receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binding and cell entry. Administration of high concentrations of soluble ACE2 can be utilized as a decoy to block the interaction of the virus with cellular ACE2 receptors and potentially be used as a strategy for treatment or prevention of coronavirus disease 2019. Human ACE2 is heavily glycosylated and its glycans impact on binding to the SARS-CoV-2 spike protein and virus infectivity. Here, we describe the production of a recombinant soluble ACE2-fragment crystallizable (Fc) variant in glycoengineered Nicotiana benthamiana. Our data reveal that the produced dimeric ACE2-Fc variant is glycosylated with mainly complex human-type N-glycans and functional with regard to enzyme activity, affinity to the SARS-CoV-2 receptor-binding domain, and wild-type virus neutralization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/biot.202000566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995010PMC
January 2021

Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids.

Viruses 2020 12 29;13(1). Epub 2020 Dec 29.

Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, 171 65 Stockholm, Sweden.

RNA viruses have gained plenty of attention during recent outbreaks of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus (ZIKV), and Ebola virus. ZIKV is a vector borne Flavivirus that is spread by mosquitoes and it mainly infects neuronal progenitor cells. One hallmark of congenital ZIKV disease is a reduced brain size in fetuses, leading to severe neurological defects. The World Health Organization (WHO) is urging the development of new antiviral treatments against ZIKV, as there are no efficient countermeasures against ZIKV disease. Previously, we presented a new class of host-targeting antivirals active against a number of pathogenic RNA viruses, such as SARS-CoV-2. Here, we show the transfer of the image-based phenotypic antiviral assay to ZIKV-infected brain cells, followed by mechanism-of-action studies and a proof-of-concept study in a three-dimensional (3D) organoid model. The novel antiviral compounds showed a therapeutic window against ZIKV in several cell models and rescued ZIKV-induced neurotoxicity in brain organoids. The compound's mechanism-of-action was pinpointed to late steps in the virus life cycle, impairing the formation of new virus particles. Collectively, in this study, we expand the antiviral activity of new small molecule inhibitors to a new virus class of Flaviviruses, but also uncover compounds' mechanism of action, which are important for the further development of antivirals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v13010037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823652PMC
December 2020

Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses.

Viruses 2020 12 10;12(12). Epub 2020 Dec 10.

Science for Life Laboratory, Department of Oncology-Pathology, Karolinska Institutet, 171 65 Stockholm, Sweden.

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v12121423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762994PMC
December 2020

Sheep and Cattle Are Not Susceptible to Experimental Inoculation with Hazara Orthonairovirus, a Tick-Borne Arbovirus Closely Related to CCHFV.

Microorganisms 2020 Dec 4;8(12). Epub 2020 Dec 4.

Institute of Novel and Emerging Infectious Diseases, Friedrich-Loeffler-Institut, Suedufer 10, 17489 Greifswald-Insel Riems, Germany.

Hazara orthonairovirus (HAZV) is a tick-borne arbovirus closely related to Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV). Whereas CCHFV is a biosafety level (BSL) 4 agent, HAZV is classified as BSL 2, as it is not known to cause any disease in humans. Belonging to the same serogroup as CCHFV, HAZV might act as a model which can provide a better understanding of this important zoonosis. Furthermore, the serological relatedness may cause diagnostic problems if antibodies against HAZV interfere with current CCHFV serological assays. Therefore, sheep and cattle-important natural hosts for CCHFV-were experimentally infected with HAZV to prove their susceptibility and evaluate potential antibody cross-reactivities. According to this study, neither sheep nor cattle are susceptible to experimental HAZV infections. Consequently, the HAZV infection in ruminants is clearly distinct from CCHFV infections. Sera of immunized animals weakly cross-reacted between HAZV and CCHFV in immunofluorescence and immunoblot assays, but not in commercial CCHFV ELISAs commonly used for field studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/microorganisms8121927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761912PMC
December 2020

Development of a Multivalent Kunjin Virus Reporter Virus-Like Particle System Inducing Seroconversion for Ebola and West Nile Virus Proteins in Mice.

Microorganisms 2020 Nov 29;8(12). Epub 2020 Nov 29.

School of Medical Science, Inflammatory Response and Infection Susceptibility Centre (iRiSC), Örebro University, 703 62 Örebro, Sweden.

Kunjin virus (KUNV) is an attenuated strain of the severe neurotropic West Nile virus (WNV). The virus has a single-strand positive-sense RNA genome that encodes a polyprotein. Following gene expression, the polyprotein is cleaved into structural proteins for viral packaging and nonstructural proteins for viral replication and expression. Removal of the structural genes generate subgenomic replicons that maintain replication capacity. Co-expression of these replicons with the viral structural genes produces reporter virus-like particles (RVPs) which infect cells in a single round. In this study, we aimed to develop a system to generate multivalent RVPs based on KUNV to elicit an immune response against different viruses. We selected the Ebola virus (EBOV) glycoprotein (GP) and the matrix protein (VP40) genes, as candidates to be delivered by KUNV RVPs. Initially, we enhanced the production of KUNV RVPs by generating a stable cell line expressing the KUNV packaging system comprising capsid, precursor membrane, and envelope. Transfection of the DNA-based KUNV replicon into this cell line resulted in an enhanced RVP production. The replicon was expressed in the stable cell line to produce the RVPs that allowed the delivery of EBOV GP and VP40 genes into other cells. Finally, we immunized BALB/cN mice with RVPs, resulting in seroconversion for EBOV GP, EBOV VP40, WNV nonstructural protein 1, and WNV E protein. Thus, our study shows that KUNV RVPs may function as a WNV vaccine candidate and RVPs can be used as a gene delivery system in the development of future EBOV vaccines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/microorganisms8121890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760487PMC
November 2020

A DNA-based vaccine protects against Crimean-Congo haemorrhagic fever virus disease in a Cynomolgus macaque model.

Nat Microbiol 2021 02 30;6(2):187-195. Epub 2020 Nov 30.

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden.

There is currently no specific prophylaxis or vaccine against Crimean-Congo haemorrhagic fever virus (CCHFV). Crimean-Congo haemorrhagic fever (CCHF) is a severe febrile illness transmitted by Hyalomma ticks in endemic areas, handling of infected livestock or care of infected patients. We report here the successful protection against CCHFV-mediated disease in a non-human primate disease model. Cynomolgus macaques were vaccinated with a DNA-based vaccine using in vivo electroporation-assisted delivery. The vaccine contained two plasmids encoding the glycoprotein precursor (GPC) and the nucleoprotein (NP) of CCHFV. Animals received three vaccinations and we recorded potent antibody and T cell responses after vaccination. While all sham-vaccinated animals developed viraemia, high tissue viral loads and CCHF-induced disease, the NP + GPC vaccinated animals were significantly protected. In conclusion, this is evidence of a vaccine that can protect against CCHFV-induced disease in a non-human primate model. This supports clinical development of the vaccine to protect groups at risk for contracting the infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-020-00815-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854975PMC
February 2021

Hazara virus and Crimean-Congo Hemorrhagic Fever Virus show a different pattern of entry in fully-polarized Caco-2 cell line.

PLoS Negl Trop Dis 2020 11 24;14(11):e0008863. Epub 2020 Nov 24.

Department of Laboratory medicine, Karolinska Institutet, Stockholm, Sweden.

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) and Hazara virus (HAZV) belong to the same viral serotype and family. HAZV has lately been used as a model system and surrogate to CCHFV. However, virus-host cell interaction and level of pathogenicity for these viruses are not well investigated nor compared. In this study, we compared HAZV and CCHFV infection of human polarized epithelial cells to shed light on similarities and differences in virus-host cell interaction between these two viruses. We investigated the pattern of infection of CCHFV and HAZV in fully polarized human cells, the Caco-2 cell line. Polarization of Caco-2 cells lead to difference in expression level and pattern of proteins between the apical and the basolateral membranes. We found that CCHFV virus, in contrast to HAZV, is more likely infecting polarized cells basolaterally. In addition, we found that cytokines/pro-inflammatory factors or other viral factors secreted from CCHFV infected moDC cells enhance the entry of CCHFV contrary to HAZV. We have shown that CCHFV and HAZV early in infection use different strategies for entry. The data presented in this study also highlight the important role of cytokines in CCHFV-host cell interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0008863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723249PMC
November 2020

and studies reveal complement system drives coagulation cascade in SARS-CoV-2 pathogenesis.

Comput Struct Biotechnol J 2020 11;18:3734-3744. Epub 2020 Nov 11.

Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Indore, MP, India.

The emergence and continued spread of SARS-CoV-2 have resulted in a public health emergency across the globe. The lack of knowledge on the precise mechanism of viral pathogenesis is impeding medical intervention. In this study, we have taken both and experimental approaches to unravel the mechanism of viral pathogenesis associated with complement and coagulation pathways. Based on the structural similarities of viral and host proteins, we initially generated a protein-protein interactome profile. Further computational analysis combined with Gene Ontology (GO) analysis and KEGG pathway analysis predicted key annotated pathways associated with viral pathogenesis. These include MAPK signaling, complement, and coagulation cascades, endocytosis, PD-L1 expression, PD-1 checkpoint pathway in cancer and C-type lectin receptor signaling pathways. Degree centrality analysis pinned down to MAPK1, MAPK3, AKT1, and SRC are crucial drivers of signaling pathways and often overlap with the associated pathways. Most strikingly, the complement and coagulation cascade and platelet activation pathways are interconnected, presumably directing thrombotic activity observed in severe or critical cases of COVID-19. This is complemented by studies of Huh7 cell infection and analysis of the transcriptome and proteomic profile of gene candidates during viral infection. The most known candidates associated with complement and coagulation cascade signaling by KEGG pathway analysis showed significant up-regulated fold change during viral infection. Collectively both and studies suggest complement and coagulation cascade signaling are a mechanism for intravascular coagulation, thrombotic changes, and associated complications in severe COVID-19 patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.csbj.2020.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7657020PMC
November 2020

JAK inhibition reduces SARS-CoV-2 liver infectivity and modulates inflammatory responses to reduce morbidity and mortality.

Sci Adv 2021 01 1;7(1). Epub 2021 Jan 1.

Department of Hematology, School of Medicine, University of Ioannina, Ioannina, Greece.

Using AI, we identified baricitinib as having antiviral and anticytokine efficacy. We now show a 71% (95% CI 0.15 to 0.58) mortality benefit in 83 patients with moderate-severe SARS-CoV-2 pneumonia with few drug-induced adverse events, including a large elderly cohort (median age, 81 years). An additional 48 cases with mild-moderate pneumonia recovered uneventfully. Using organotypic 3D cultures of primary human liver cells, we demonstrate that interferon-α2 increases ACE2 expression and SARS-CoV-2 infectivity in parenchymal cells by greater than fivefold. RNA-seq reveals gene response signatures associated with platelet activation, fully inhibited by baricitinib. Using viral load quantifications and superresolution microscopy, we found that baricitinib exerts activity rapidly through the inhibition of host proteins (numb-associated kinases), uniquely among antivirals. This reveals mechanistic actions of a Janus kinase-1/2 inhibitor targeting viral entry, replication, and the cytokine storm and is associated with beneficial outcomes including in severely ill elderly patients, data that incentivize further randomized controlled trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.abe4724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775747PMC
January 2021

Human soluble ACE2 improves the effect of remdesivir in SARS-CoV-2 infection.

EMBO Mol Med 2021 01 14;13(1):e13426. Epub 2020 Dec 14.

Department of Laboratory Medicine, Unit of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden.

There is a critical need for safe and effective drugs for COVID-19. Only remdesivir has received authorization for COVID-19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS-CoV-2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS-CoV-2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS-CoV-2 in both models. By using single amino-acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub-toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID-19 clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.202013426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799356PMC
January 2021

Human recombinant soluble ACE2 in severe COVID-19.

Lancet Respir Med 2020 11 24;8(11):1154-1158. Epub 2020 Sep 24.

Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada; Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S2213-2600(20)30418-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515587PMC
November 2020

ISG15 overexpression compensates the defect of Crimean-Congo hemorrhagic fever virus polymerase bearing a protease-inactive ovarian tumor domain.

PLoS Negl Trop Dis 2020 09 15;14(9):e0008610. Epub 2020 Sep 15.

Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University, Giessen, Germany.

Crimean-Congo Hemorrhagic Fever virus (CCHFV; family Nairoviridae) is an extremely pathogenic member of the Bunyavirales order. Previous studies have shown that the N-terminal domain of the CCHFV polymerase (L) contains an ovarian tumor-type protease (OTU) domain with the capability to remove both ubiquitin and ISG15 molecules from proteins. The approximately 200 amino acids-long OTU domain, if ectopically expressed, can interfere with both the induction of antiviral type I interferons (IFN) as well as the IFN-stimulated signaling. A OTU protease mutant (C40A), by contrast, was inactive in that respect. However, the effect of the OTU protease activity in the context of the full-length L protein (approximately 4000 amino acids) is only poorly characterized, and recombinant CCHFV with the C40A mutation could not be rescued. Here, we employed transcriptionally active virus-like particles (tc-VLPs) to investigate the interaction between the L-embedded OTU protease and the IFN system. Our data show a cis requirement of the OTU protease for optimal CCHFV polymerase activity in human HuH-7 cells. The L-embedded OTU did not influence IFN signaling, the sensitivity to IFN, or IFN induction. Moreover, the attenuation of OTU C40A-mutated L could not be relieved by inactivating the IFN response, but after overexpression of conjugation-competent ISG15 the polymerase activity recovered to wild-type levels. Consequently, ISG15 was used to produce OTU-deficient tc-VLPs, a potential vaccine candidate. Our data thus indicate that in the context of full-length L the OTU domain is important for the regulation of CCHFV polymerase by ISG15.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pntd.0008610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7518590PMC
September 2020

2020 taxonomic update for phylum Negarnaviricota (Riboviria: Orthornavirae), including the large orders Bunyavirales and Mononegavirales.

Arch Virol 2020 Dec 4;165(12):3023-3072. Epub 2020 Sep 4.

Colorado State University, Fort Collins, CO, USA.

In March 2020, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. At the genus rank, 20 new genera were added, two were deleted, one was moved, and three were renamed. At the species rank, 160 species were added, four were deleted, ten were moved and renamed, and 30 species were renamed. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-020-04731-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606449PMC
December 2020

ICTV Virus Taxonomy Profile: .

J Gen Virol 2020 08 24;101(8):798-799. Epub 2020 Aug 24.

USAMRIID, Fort Detrick, Frederick, Maryland, USA.

Members of the family produce enveloped virions with three single-stranded RNA segments comprising 17.1 to 22.8 kb in total. These viruses are maintained in arthropods and transmitted by ticks to mammals or birds. Crimean-Congo hemorrhagic fever virus is tick-borne and is endemic in most of Asia, Africa, Southern and Eastern Europe whereas Nairobi sheep disease virus, which is also tick-borne, causes lethal haemorrhagic gastroenteritis in small ruminants in Africa and India. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family , which is available at ictv.global/report/nairoviridae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.001485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641396PMC
August 2020

Comparison of Zaire ebolavirus realtime RT-PCRs targeting the nucleoprotein gene.

J Virol Methods 2020 10 22;284:113941. Epub 2020 Jul 22.

HUS Diagnostic Center, HUSLAB, Clinical Microbiology, University of Helsinki and Helsinki University Hospital, Finland; Faculty of Veterinary Medicine, Department of Veterinary Biosciences, University of Helsinki, Finland.

In last five years, the Africa has faced two outbreaks of Zaire ebolavirus. These outbreaks have been the largest so far, and latest outbreak is still ongoing and affecting the Democratic Republic of the Congo. We tested in parallel three different Zaire ebolavirus (EBOV) realtime RT-PCRs targeting the nucleoprotein gene (EBOV NP-RT-qPCRs) described by Trombley et al. (2010); Huang et al. (2012) and Weidmann et al. (2004). These assays are used regularly in diagnostic laboratories. The limit of detection (LOD), intra-assay repeatability using different matrixes, sensitivity and specificity were determined. In addition, the primers and probes were aligned with the sequences available in ongoing and past outbreaks in order to check the mismatches. The specificity of all three EBOV NP-RT-qPCRs were excellent (100 %), and LODs were under or 10 copies per PCR reaction. Intra-assay repeatability was good in all assays, however the Ct-values were bit higher using the EDTA-blood based matrix. All of the primers and probes in EBOV NP-RT-qPCR assays have one or more mismatches in the probes and primers when the 2267 Zaire EBOV NP sequences, including strains Ituri from DRC outbreak (year 2018), was aligned. The EBOV strain of Bikoro (year 2018) circulating in DRC was 100 % match in Trombley and Weidmann assay, but had one mismatch in Huang assay.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jviromet.2020.113941DOI Listing
October 2020

Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2.

Euro Surveill 2020 07;25(28)

Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), Singapore.

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2807/1560-7917.ES.2020.25.28.2000291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376845PMC
July 2020

Dysregulation in Akt/mTOR/HIF-1 signaling identified by proteo-transcriptomics of SARS-CoV-2 infected cells.

Emerg Microbes Infect 2020 Dec;9(1):1748-1760

Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden.

How severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections engage cellular host pathways and innate immunity in infected cells remains largely elusive. We performed an integrative proteo-transcriptomics analysis in SARS-CoV-2 infected Huh7 cells to map the cellular response to the invading virus over time. We identified four pathways, ErbB, HIF-1, mTOR and TNF signaling, among others that were markedly modulated during the course of the SARS-CoV-2 infection . Western blot validation of the downstream effector molecules of these pathways revealed a dose-dependent activation of Akt, mTOR, S6K1 and 4E-BP1 at 24 hours post infection (hpi). However, we found a significant inhibition of HIF-1α through 24hpi and 48hpi of the infection, suggesting a crosstalk between the SARS-CoV-2 and the Akt/mTOR/HIF-1 signaling pathways. Inhibition of the mTOR signaling pathway using Akt inhibitor MK-2206 showed a significant reduction in virus production. Further investigations are required to better understand the molecular sequelae in order to guide potential therapy in the management of severe coronavirus disease 2019 (COVID-19) patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/22221751.2020.1799723DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473213PMC
December 2020

Development and Potential Usefulness of the COVID-19 Ag Respi-Strip Diagnostic Assay in a Pandemic Context.

Front Med (Lausanne) 2020 8;7:225. Epub 2020 May 8.

Innovation and Business Development Unit, LHUB-ULB, Université Libre de Bruxelles, Brussels, Belgium.

COVID-19 Ag Respi-Strip, an immunochromatographic (ICT) assay for the rapid detection of SARS-CoV-2 antigen on nasopharyngeal specimen, has been developed to identify positive COVID-19 patients allowing prompt clinical and quarantine decisions. In this original research article, we describe the conception, the analytical and clinical performances as well as the risk management of implementing the COVID-19 Ag Respi-Strip in a diagnostic decision algorithm. Development of the COVID-19 Ag Respi-Strip resulted in a ready-to-use ICT assay based on a membrane technology with colloidal gold nanoparticles using monoclonal antibodies directed against the SARS-CoV and SARS-CoV-2 highly conserved nucleoprotein antigen. Four hundred observations were recorded for the analytical performance study and thirty tests were analyzed for the cross-reactivity study. The clinical performance study was performed in a retrospective multi-centric evaluation on aliquots of 328 nasopharyngeal samples. COVID-19 Ag Respi-Strip results were compared with qRT-PCR as golden standard for COVID-19 diagnostics. In the analytical performance study, the reproducibility showed a between-observer disagreement of 1.7%, a robustness of 98%, an overall satisfying user friendliness and no cross-reactivity with other virus-infected nasopharyngeal samples. In the clinical performance study performed in three different clinical laboratories during the ascendant phase of the epidemiological curve, we found an overall sensitivity and specificity of 57.6 and 99.5%, respectively with an accuracy of 82.6%. The cut-off of the ICT was found at CT <22. User-friendliness analysis and risk management assessment through Ishikawa diagram demonstrate that COVID-19 Ag Respi-Strip may be implemented in clinical laboratories according to biosafety recommendations. The COVID-19 Ag Respi-Strip represents a promising rapid SARS-CoV-2 antigen assay for the first-line diagnosis of COVID-19 in 15 min at the peak of the pandemic. Its role in the proposed diagnostic algorithm is complementary to the currently-used molecular techniques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmed.2020.00225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7227790PMC
May 2020

Mechanism of baricitinib supports artificial intelligence-predicted testing in COVID-19 patients.

EMBO Mol Med 2020 08 24;12(8):e12697. Epub 2020 Jun 24.

Division of Infectious Diseases, ASST Fatebenefratelli Sacco, Milan, Italy.

Baricitinib is an oral Janus kinase (JAK)1/JAK2 inhibitor approved for the treatment of rheumatoid arthritis (RA) that was independently predicted, using artificial intelligence (AI) algorithms, to be useful for COVID-19 infection via proposed anti-cytokine effects and as an inhibitor of host cell viral propagation. We evaluated the in vitro pharmacology of baricitinib across relevant leukocyte subpopulations coupled to its in vivo pharmacokinetics and showed it inhibited signaling of cytokines implicated in COVID-19 infection. We validated the AI-predicted biochemical inhibitory effects of baricitinib on human numb-associated kinase (hNAK) members measuring nanomolar affinities for AAK1, BIKE, and GAK. Inhibition of NAKs led to reduced viral infectivity with baricitinib using human primary liver spheroids. These effects occurred at exposure levels seen clinically. In a case series of patients with bilateral COVID-19 pneumonia, baricitinib treatment was associated with clinical and radiologic recovery, a rapid decline in SARS-CoV-2 viral load, inflammatory markers, and IL-6 levels. Collectively, these data support further evaluation of the anti-cytokine and anti-viral activity of baricitinib and support its assessment in randomized trials in hospitalized COVID-19 patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.202012697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300657PMC
August 2020

Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2.

Cell 2020 05 24;181(4):905-913.e7. Epub 2020 Apr 24.

Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Dr. Bohr-Gasse 3, 1030 Vienna, Austria; Department of Medical Genetics, Life Science Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Electronic address:

We have previously provided the first genetic evidence that angiotensin converting enzyme 2 (ACE2) is the critical receptor for severe acute respiratory syndrome coronavirus (SARS-CoV), and ACE2 protects the lung from injury, providing a molecular explanation for the severe lung failure and death due to SARS-CoV infections. ACE2 has now also been identified as a key receptor for SARS-CoV-2 infections, and it has been proposed that inhibiting this interaction might be used in treating patients with COVID-19. However, it is not known whether human recombinant soluble ACE2 (hrsACE2) blocks growth of SARS-CoV-2. Here, we show that clinical grade hrsACE2 reduced SARS-CoV-2 recovery from Vero cells by a factor of 1,000-5,000. An equivalent mouse rsACE2 had no effect. We also show that SARS-CoV-2 can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by hrsACE2. These data demonstrate that hrsACE2 can significantly block early stages of SARS-CoV-2 infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181998PMC
May 2020

Identification and validation of internal reference genes for real-time quantitative polymerase chain reaction-based studies in Hyalomma anatolicum ticks.

Ticks Tick Borne Dis 2020 07 19;11(4):101417. Epub 2020 Mar 19.

Department of Microbiology, The Public Health Agency of Sweden, Nobels Väg 18, SE-171 82 Solna, Sweden; Department for Laboratory Medicine, Karolinska University Hospital and KI, SE-14186 Huddinge, Sweden; National Veterinary Institute, SE-756 51 Uppsala, Sweden. Electronic address:

Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne viral disease caused by the orthonairovirus CCHF virus (CCHFV). Ticks of the genus Hyalomma are the viral reservoir and they represent the main vector transmitting the virus to their hosts during blood feeding. However, how CCHFV replicates in its natural arthropod host cells and the nature of virus/host interactions are still largely unknown. With the aim of developing tools for use in this field, we identified and validated expression of four candidate endogenous control tick genes in a Hyalomma anatolicum-derived cell line. These genes will be useful for normalization of viral/cellular transcripts in infection/expression studies or as internal controls in molecular epidemiology surveys of pathogens transmitted by Hyalomma ticks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ttbdis.2020.101417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284302PMC
July 2020

Digital Rolling Circle Amplification-Based Detection of Ebola and Other Tropical Viruses.

J Mol Diagn 2020 02 16;22(2):272-283. Epub 2019 Dec 16.

Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, Solna, Sweden. Electronic address:

Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent in 2018 to 2019 in Congo. Thus, immediate diagnostic attention is demanded at the point of care in resource-limited settings, because the performance and the operational parameters of conventional EBOV testing are limited. Especially, their sensitivity, specificity, and coverage of other tropical disease viruses make them unsuitable for diagnostic at the point of care. Here, a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV is presented. For this, a set of PLPs, separately targeting the viral RNA and complementary RNA of all seven EBOV genes, was used in the RCA assay and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous detection of both EBOV RNA types was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. Because PLPs and RCA are inherently multiplexable, we demonstrate the extension of the probe panel for the simultaneous detection of the tropical viruses Ebola, Zika, and Dengue. The demonstrated high specificity, sensitivity, and multiplexing capability in combination with the digital quantification rendered the assay a promising diagnostic tool toward tropical virus detection at the point of care.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmoldx.2019.10.014DOI Listing
February 2020

Viral Hemorrhagic Fevers Other than Ebola and Lassa.

Infect Dis Clin North Am 2019 12;33(4):977-1002

National Institute for Infectious Diseases, Lazzaro Spallanzani, IRCCS, Via Portuense 292, Rome 00149, Italy. Electronic address:

Viral hemorrhagic fevers represent a group of diseases caused by enveloped RNA viruses. The epidemiology is broadly variable, ranging from geographically localized to more diffuse infections. Viral hemorrhagic fevers are classified as category A bioweapon agents by the Centers for Disease Control and Prevention. Viral hemorrhagic fevers are severe febrile illnesses with hemorrhagic phenomena. Laboratory diagnosis takes place in highly specialized reference laboratories. Treatment is essentially supportive. In this article, we focus the attention on yellow fever and viral hemorrhagic fevers other than Ebola and Lassa virus diseases that have been described elsewhere in this issue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.idc.2019.08.003DOI Listing
December 2019

Geographical Variability Affects CCHFV Detection by RT-PCR: A Tool for In-Silico Evaluation of Molecular Assays.

Viruses 2019 10 16;11(10). Epub 2019 Oct 16.

National Institute for Infectious Diseases (INMI) "L. Spallanzani" IRCCS, WHO Collaborating Center for clinical care, diagnosis, response and training on Highly Infectious Diseases, 00149 Rome, Italy.

The Crimean-Congo hemorrhagic fever virus (CCHFV) is considered to be a major emerging infectious threat, according to the WHO R&D blueprint. A wide range of CCHFV molecular assays have been developed, employing varied primer/probe combinations. The high genetic variability of CCHFV often hampers the efficacy of available molecular tests and can affect their diagnostic potential. Recently, increasing numbers of complete CCHFV genomic sequences have become available, allowing a better appreciation of the genomic evolution of this virus. We summarized the current knowledge on molecular methods and developed a new bioinformatics tool to evaluate the existing assays for CCHFV detection, with a special focus on strains circulating in different geographical areas. Twenty-two molecular methods and 181 sequences of CCHFV were collected, respectively, from PubMed and GenBank databases. Up to 28 mismatches between primers and probes of each assay and CCHFV strains were detected through in-silico PCR analysis. Combinations of up to three molecular methods markedly decreased the number of mismatches within most geographic areas. These results supported the good practice of CCHFV detection of performing more than one assay, aimed for different sequence targets. The choice of the most appropriate tests must take into account patient's travel history and geographic distribution of the different CCHFV strains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/v11100953DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833031PMC
October 2019

Taxonomy of the order Bunyavirales: update 2019.

Arch Virol 2019 Jul 7;164(7):1949-1965. Epub 2019 May 7.

United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD, USA.

In February 2019, following the annual taxon ratification vote, the order Bunyavirales was amended by creation of two new families, four new subfamilies, 11 new genera and 77 new species, merging of two species, and deletion of one species. This article presents the updated taxonomy of the order Bunyavirales now accepted by the International Committee on Taxonomy of Viruses (ICTV).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-019-04253-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6641860PMC
July 2019