Recovery of complete genome sequences of Crimean-Congo haemorrhagic fever virus (CCHFV) directly from clinical samples: A comparative study between targeted enrichment and metagenomic approaches.

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, endemic to the Balkans, Africa, Middle East and Asia. There are currently no licensed vaccines or effective antivirals against CCHF. CCHF virus (CCHFV) has a negative sense segmented tripartite RNA genome consisting of the small (S), medium (M) and large (L) segments. Depending on the segment utilised for genetic affiliation, there are up to 7 circulating lineages of CCHFV. The current lack of geographical representation of CCHFV sequences in various repositories highlights a requirement for increased CCHFV sequencing capabilities in endemic regions. We have optimised and established a multiplex PCR tiling methodology for the targeted enrichment of complete genomes of Europe 1 CCHFV lineage directly from clinical samples and compared its performance to a non-targeted enrichment approach on both short-read and long-read sequencing platforms. We have found a statistically significant increase in mapped viral sequencing reads produced with our targeted enrichment approach. This has allowed us to recover near complete S segment sequences and above 90% of the M and L segment sequences for samples with Ct values as high as 31.3. This study demonstrates the superiority of a targeted enrichment approach for recovery of CCHFV genomic sequences from samples with low virus titre. CCHFV is an important vector-borne human pathogen with wide geographical distribution. The validated methodology reported here adds value to front-line public health laboratories employing genomic sequencing for CCHFV Europe 1 lineage surveillance, particularly in the Balkan and Middle Eastern territories currently monitoring the spread of the pathogen. Tracking the genomic evolution of the virus across regions improves risk assessment and directly informs the development of diagnostics, therapeutics, and vaccines.

Development of a Novel Positive Pressure Protective Suit for a Biosafety Level 4 Laboratory in Japan.

Biosafety level 4 (BSL-4) laboratories are necessary to study microorganisms that are highly pathogenic to humans and have no prevention or therapeutic measures. Currently, most BSL-4 facilities have suit-type laboratories to conduct experiments on highly pathogenic microorganisms. In 2021, the first Japanese suit-type BSL-4 laboratory was constructed at Nagasaki University. Positive pressure protection suit (PPPS) is a primary barrier that protects and isolates laboratory workers from pathogens and the laboratory environment. Here, we developed a novel PPPS originally designed to be used in the Nagasaki BSL-4 laboratory. We modified several parts of a domestic chemical protective suit, including its front face shield, cuff, and air supply hose, for safe handling of microbiological agents. The improved suit, PS-790BSL4-AL, showed resistance to several chemicals, including quaternary ammonium disinfectant, and did not show any permeation against blood and phages. To validate the suit's integrity, we also established an airtight test that eliminated individual differences for quantitative testing. In conclusion, our developed suit performs sufficiently as a primary barrier and allows for the safe handling of pathogens in our new BSL-4 laboratory.

Identification of novel orthonairoviruses from rodents and shrews in Gabon, Central Africa.

In Africa, several emerging zoonotic viruses have been transmitted from small mammals such as rodents and shrews to humans. Although no clinical cases of small mammal-borne viral diseases have been reported in Central Africa, potential zoonotic viruses have been identified in rodents in the region. Therefore, we hypothesized that there may be unrecognized zoonotic viruses circulating in small mammals in Central Africa. Here, we investigated viruses that have been maintained among wild small mammals in Gabon to understand their potential risks to humans. We identified novel orthonairoviruses in 24.6 % of captured rodents and shrews from their kidney total RNA samples. Phylogenetic analysis revealed that the novel viruses, Lamusara virus (LMSV) and Lamgora virus, were closely related to Erve virus, which was previously identified in shrews of the genus Crocidura and has been suspected to cause neuropathogenic diseases in humans. Moreover, we show that the LMSV ovarian tumour domain protease, one of the virulence determination factors of orthonairoviruses, suppressed interferon signalling in human cells, suggesting the possible human pathogenicity of this virus. Taken together, our study demonstrates the presence of novel orthonairoviruses that may pose unrecognized risks of viral disease transmission in Gabon.

5-Aminolevulinic acid antiviral efficacy against SARS-CoV-2 omicron variant in vitro.

The coronavirus disease 2019 (COVID 19) pandemic continues to pose a threat to global health. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529) has spread rapidly worldwide and became dominant in many countries. A natural 5-aminolevulinic acid (5-ALA) with sodium ferrous citrate (SFC) has demonstrated antiviral activity in Wuhan, Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 infections in vitro. In this study, we report antiviral activity of 5-ALA, 5-ALA with SFC led to IC50 of 329 and 765/191, respectively after infection with Omicron variant of SARS-CoV-2 in vitro. Our finding suggests that 5-ALA could be used as antiviral drug candidate to treat Omicron variant infected patients.

A screen of FDA-approved drugs with minigenome identified tigecycline as an antiviral targeting nucleoprotein of Crimean-Congo hemorrhagic fever virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Orthonairovirus and is the causative agent of a viral hemorrhagic disease with a case fatality rate of 30%. However, limited studies have been conducted to explore antiviral compounds specific to CCHFV. In this study, we developed a minigenome system of orthonairoviruses, CCHFV and Hazara virus to analyze viral replication and screened an FDA-approved compound library. The transfection of the minigenome components induced marked increase in luciferase expression, indicating the sufficient replication and translation of reporter RNA. Compound library screening identified 14 candidate compounds that significantly decreased luciferase activity. Some of the compounds also inhibited the replication of the infectious Hazara virus. The mechanism of inhibition by tigecycline was further analyzed, and a decrease in the interaction between the viral N protein and RNA by tigecycline was observed. This work provides a basis for validation using animal models and the design of chemical derivatives with stronger activity in future studies on the development of an antiviral against CCHFV.

Unique Evolution of SARS-CoV-2 in the Second Large Cruise Ship Cluster in Japan.

In the initial phase of the novel coronavirus disease (COVID-19) pandemic, a large-scale cluster on the cruise ship Diamond Princess (DP) emerged in Japan. Genetic analysis of the DP strains has provided important information for elucidating the possible transmission process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on a cruise ship. However, genome-based analyses of SARS-CoV-2 detected in large-scale cruise ship clusters other than the DP cluster have rarely been reported. In the present study, whole-genome sequences of 94 SARS-CoV-2 strains detected in the second large cruise ship cluster, which emerged on the Costa Atlantica (CA) in Japan, were characterized to understand the evolution of the virus in a crowded and confined place. Phylogenetic and haplotype network analysis indicated that the CA strains were derived from a common ancestral strain introduced on the CA cruise ship and spread in a superspreading event-like manner, resulting in several mutations that might have affected viral characteristics, including the P681H substitution in the spike protein. Moreover, there were significant genetic distances between CA strains and other strains isolated in different environments, such as cities under lockdown. These results provide new insights into the unique evolution patterns of SARS-CoV-2 in the CA cruise ship cluster.

Antiviral activity of 5-aminolevulinic acid against variants of severe acute respiratory syndrome coronavirus 2.

BACKGROUND: Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began to emerge in 2020 and have been spreading globally during the coronavirus disease 2019 (COVID-19) pandemic. Despite the presence of different COVID-19 vaccines, the discovery of effective antiviral therapeutics for the treatment of patients infected with SARS-CoV-2 are still urgently needed. A natural amino acid, 5-aminolevulinic acid (5-ALA), has exhibited both antiviral and anti-inflammatory activities. In a previous study, we demonstrated an in vitro antiviral effect of 5-ALA against SARS-CoV-2 infection without significant cytotoxicity. In the present study, we sought to investigate whether 5-ALA with or without sodium ferrous citrate (SFC) can inhibit in vitro both the original SARS-CoV-2 Wuhan strain and its variants, including the Alpha, Beta, Gamma and Delta strains. METHODS: The antiviral activity of ALA with or without SFC was determined in Vero-E6 cell. The virus inhibition was quantified by real time RT-PCR. RESULTS: Co-administration of 5-ALA and SFC inhibited the Wuhan, Alpha and Delta variants of SARS-CoV-2 with IC50 values of 235, 173 and 397 µM, respectively, and the Beta and Gamma variants with IC50 values of 1311 and 1516 µM. CONCLUSION: Our study suggests that 5-ALA with SFC warrants accelerated clinical evaluation as an antiviral drug candidate for treating patients infected with SARS-CoV-2 variants.

5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro.

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19, in cell culture. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is worth to be further investigated as an antiviral drug candidate for COVID-19.

Re-emergence of dengue virus serotype 3 infections in Gabon in 2016-2017, and evidence for the risk of repeated dengue virus infections.

OBJECTIVES: Dengue outbreaks, mainly caused by dengue virus serotype 2 (DENV-2), occurred in 2007 and in 2010 in Gabon, Central Africa. However, information on DENV infections has been insufficient since 2010. The aim of this study was to investigate the current DENV infection scenario and the risk of repeated infections in Gabon. METHODS: During 2015-2017, serum samples were collected from enrolled febrile participants and were tested for DENV infection using RT-qPCR. DENV-positive samples were analyzed for a history of previous DENV infection(s) using ELISA. The complete DENV genome was sequenced to analyze the phylogeny of Gabonese DENV strains. RESULTS: DENV-3 was exclusively detected, with a high rate of anti-DENV IgG seropositivity among DENV-3-positive participants. DENV-3 showed higher infection rates in adults and the infection was seasonal with peaks in the rainy seasons. Phylogenetic analysis revealed that Gabonese DENV-3 originated from West African strains and has been circulating continuously in Gabon since at least 2010, when the first DENV-3 case was reported. CONCLUSIONS: These findings indicate stable DENV-3 circulation and the risk of repeated DENV infections in Gabon, highlighting the need for continuous monitoring to control DENV infections.

Development of an RT-LAMP assay for the detection of Lassa viruses in southeast and south-central Nigeria.

Lassa virus (LASV) causes Lassa fever (LF), a viral hemorrhagic fever endemic in West Africa. LASV strains are clustered into six lineages according to their geographic location. To confirm a diagnosis of LF, a laboratory test is required. Here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of LASV in southeast and south-central Nigeria using three primer sets specific for strains clustered in lineage II was developed. The assay detected in vitro transcribed LASV RNAs within 23 min and was further evaluated for detection in 73 plasma collected from suspected LF patients admitted into two health settings in southern Nigeria. The clinical evaluation using the conventional RT-PCR as the reference test revealed a sensitivity of 50% in general with 100% for samples with a viral titer of 9500 genome equivalent copies (geq)/mL and higher. The detection limit was estimated to be 4214 geq/mL. The assay showed 98% specificity with no cross-reactivity to other viruses which cause similar symptoms. These results suggest that this RT-LAMP assay is a useful molecular diagnostic test for LF during the acute phase, contributing to early patient management, while using a convenient device for field deployment and in resource-poor settings.

Genetic characterization of Lassa virus strains isolated from 2012 to 2016 in southeastern Nigeria.

Lassa virus (LASV) is endemic in parts of West Africa where it causes Lassa fever (LF), a viral hemorrhagic fever with frequent fatal outcomes. The diverse LASV strains are grouped into six major lineages based on the geographical location of the isolated strains. In this study, we have focused on the lineage II strains from southern Nigeria. We determined the viral sequences from positive cases of LF reported at tertiary hospitals in Ebonyi and Enugu between 2012 and 2016. Reverse transcription-polymerase chain reaction (RT-PCR) showed that 29 out of 123 suspected cases were positive for the virus among which 11 viral gene sequences were determined. Phylogenetic analysis of the complete coding sequences of the four viral proteins revealed that lineage II strains are broadly divided into two genetic clades that diverged from a common ancestor 195 years ago. One clade, consisting of strains from Ebonyi and Enugu, was more conserved than the other from Irrua, although the four viral proteins were evolving at similar rates in both clades. These results suggested that the viruses of these clades have been distinctively evolving in geographically separate parts of southern Nigeria. Furthermore, the epidemiological data of the 2014 outbreak highlighted the role of human-to-human transmission in this outbreak, which was supported by phylogenetic analysis showing that 13 of the 16 sequences clustered together. These results provide new insights into the evolution of LASV in southern Nigeria and have important implications for vaccine development, diagnostic assay design, and LF outbreak management.

BST-2 controls T cell proliferation and exhaustion by shaping the early distribution of a persistent viral infection.

The interferon inducible protein, BST-2 (or, tetherin), plays an important role in the innate antiviral defense system by inhibiting the release of many enveloped viruses. Consequently, viruses have evolved strategies to counteract the anti-viral activity of this protein. While the mechanisms by which BST-2 prevents viral dissemination have been defined, less is known about how this protein shapes the early viral distribution and immunological defense against pathogens during the establishment of persistence. Using the lymphocytic choriomeningitis virus (LCMV) model of infection, we sought insights into how the in vitro antiviral activity of this protein compared to the immunological defense mounted in vivo. We observed that BST-2 modestly reduced production of virion particles from cultured cells, which was associated with the ability of BST-2 to interfere with the virus budding process mediated by the LCMV Z protein. Moreover, LCMV does not encode a BST-2 antagonist, and viral propagation was not significantly restricted in cells that constitutively expressed BST-2. In contrast to this very modest effect in cultured cells, BST-2 played a crucial role in controlling LCMV in vivo. In BST-2 deficient mice, a persistent strain of LCMV was no longer confined to the splenic marginal zone at early times post-infection, which resulted in an altered distribution of LCMV-specific T cells, reduced T cell proliferation / function, delayed viral control in the serum, and persistence in the brain. These data demonstrate that BST-2 is important in shaping the anatomical distribution and adaptive immune response against a persistent viral infection in vivo.

The cholesterol, fatty acid and triglyceride synthesis pathways regulated by site 1 protease (S1P) are required for efficient replication of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a high mortality rate. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV infection. Here, we report that the cholesterol, fatty acid, and triglyceride synthesis pathways regulated by S1P is involved in SFTSV replication, using CHO-K1 cell line (SRD-12B) that is deficient in site 1 protease (S1P) enzymatic activity, PF-429242, a small compound targeting S1P enzymatic activity, and Fenofibrate and Lovastatin, which inhibit triglyceride and cholesterol synthesis, respectively. These results enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection.

Different effects of two mutations on the infectivity of Ebola virus glycoprotein in nine mammalian species.

Ebola virus (EBOV), which belongs to the genus Ebolavirus, causes a severe and often fatal infection in primates, including humans, whereas Reston virus (RESTV) only causes lethal disease in non-human primates. Two amino acids (aa) at positions 82 and 544 of the EBOV glycoprotein (GP) are involved in determining viral infectivity. However, it remains unclear how these two aa residues affect the infectivity of Ebolavirus species in various hosts. Here we performed viral pseudotyping experiments with EBOV and RESTV GP derivatives in 10 cell lines from 9 mammalian species. We demonstrated that isoleucine at position 544/545 increases viral infectivity in all host species, whereas valine at position 82/83 modulates viral infectivity, depending on the viral and host species. Structural modelling suggested that the former residue affects viral fusion, whereas the latter residue influences the interaction with the viral entry receptor, Niemann-Pick C1.

Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification.

The recent outbreak of Zika virus (ZIKV) disease caused an enormous number of infections in Central and South America, and the unusual increase in the number of infants born with microcephaly associated with ZIKV infection aroused global concern. Here, we developed a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of ZIKV. The assay specifically detected ZIKV strains of both Asian and African genotypes without cross-reactivity with other arboviruses, including Dengue and Chikungunya viruses. The assay detected viral RNA at 14.5 TCID50/mL in virus-spiked serum or urine samples within 15 min, although it was slightly less sensitive than reference real time RT-PCR assay. We then evaluated the utility of this assay as a molecular diagnostic test using 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraíba and Pernambuco, Brazil in 2016. The results of this assay were consistent with those of the reference RT-PCR test. This portable RT-LAMP assay was highly specific for ZIKV, and enable rapid diagnosis of the virus infection. Our results provide new insights into ZIKV molecular diagnostics and may improve preparedness for future outbreaks.

Defining the relative performance of isothermal assays that can be used for rapid and sensitive detection of foot-and-mouth disease virus.

This study describes the first multiway comparison of portable isothermal assays for the detection of foot-and-mouth disease virus (FMDV), benchmarked against real-time reverse transcription RT-PCR (rRT-PCR). The selected isothermal chemistries included reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA). The analytical sensitivity of RT-LAMP was comparable to rRT-PCR (101 RNA copies), while RT-RPA was one log10 less sensitive (102 RNA copies). Diagnostic performance was evaluated using a panel of 35 samples from FMDV-positive cattle and eight samples from cattle infected with other vesicular viruses. Assay concordance for RT-LAMP and RT-RPA was 86-98% and 67-77%, respectively, when compared to rRT-PCR, with discordant samples consistently having high rRT-PCR cycle threshold values (no false-positives were detected for any assay). In addition, a hierarchy of sample preparation methods, from robotic extraction to simple dilution of samples, for epithelial suspensions, serum and oesophageal-pharyngeal (OP) fluid were evaluated. Results obtained for RT-LAMP confirmed that FMDV RNA can be detected in the absence of RNA extraction. However, simple sample preparation methods were less encouraging for RT-RPA, with accurate results only obtained when using RNA extraction. Although the evaluation of assay performance is specific to the conditions tested in this study, the compatibility of RT-LAMP chemistry with multiple sample types, both in the presence and absence of nucleic acid extraction, provides advantages over alternative isothermal chemistries and alternative pen-side diagnostics such as antigen-detection lateral-flow devices. These characteristics of RT-LAMP enable the assay to be performed over a large diagnostic detection window, providing a realistic means to rapidly confirm positive FMD cases close to the point of sampling.

Rapid detection of all known ebolavirus species by reverse transcription-loop-mediated isothermal amplification (RT-LAMP).

Ebola virus disease (EVD), a highly virulent infectious disease caused by ebolaviruses, has a fatality rate of 25-90%. Without a licensed chemotherapeutic agent or vaccine for the treatment and prevention of EVD, control of outbreaks requires accurate and rapid diagnosis of cases. In this study, five sets of six oligonucleotide primers targeting the nucleoprotein gene were designed for specific identification of each of the five ebolavirus species using reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. The detection limits of the ebolavirus species-specific primer sets were evaluated using in vitro transcribed RNAs. The detection limit of species-specific RT-LAMP assays for Zaire ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Bundibugyo ebolavirus was 256 copies/reaction, while the detection limit for Reston ebolavirus was 64 copies/reaction, and the detection time for each of the RT-LAMP assays was 13.3±3.0, 19.8±4.6, 14.3±0.6, 16.1±4.7, and 19.8±2.4min (mean±SD), respectively. The sensitivity of the species-specific RT-LAMP assays were similar to that of the established RT-PCR and quantitative RT-PCR assays for diagnosis of EVD and are suitable for field or point-of-care diagnosis. The RT-LAMP assays were specific for the detection of the respective species of ebolavirus with no cross reaction with other species of ebolavirus and other viral hemorrhagic fever viruses such as Marburg virus, Lassa fever virus, and Dengue virus. The species-specific RT-LAMP assays developed in this study are rapid, sensitive, and specific and could be useful in case of an EVD outbreak.

Functional mutations in spike glycoprotein of Zaire ebolavirus associated with an increase in infection efficiency.

Ebola virus (EBOV) is extremely virulent, and its glycoprotein is necessary for viral entry. EBOV may adapt to its new host humans during outbreaks by acquiring mutations especially in glycoprotein, which allows EBOV to spread more efficiently. To identify these evolutionary selected mutations and examine their effects on viral infectivity, we used experimental-phylogenetic-structural interdisciplinary approaches. In evolutionary analysis of all available Zaire ebolavirus glycoprotein sequences, we detected two codon sites under positive selection, which are located near/within the region critical for the host-viral membrane fusion, namely alanine-to-valine and threonine-to-isoleucine mutations at 82 (A82V) and 544 (T544I), respectively. The fine-scale transmission dynamics of EBOV Makona variants that caused the 2014-2015 outbreak showed that A82V mutant was fixed in the population, whereas T544I was not. Furthermore, pseudotype assays for the Makona glycoprotein showed that the A82V mutation caused a small increase in viral infectivity compared with the T544I mutation. These findings suggest that mutation fixation in EBOV glycoprotein may be associated with their increased infectivity levels; the mutant with a moderate increase in infectivity will fix. Our findings showed that a driving force for Ebola virus evolution via glycoprotein may be a balance between costs and benefits of its virulence.

A loop-mediated isothermal amplification assay for rapid and sensitive detection of bovine papular stomatitis virus.

Bovine papular stomatitis virus (BPSV) causes pustular cutaneous disease in cattle worldwide. This paper describes the development of a specific loop-mediated isothermal amplification (LAMP) assay to detect BPSV which did not cross-react with other parapoxviruses. To assess analytical sensitivity of this LAMP assay, DNA was extracted from serially diluted BPSV from which the infectious titer was determined by a novel assay based on calf kidney epithelial cells. The LAMP assay had equivalent analytical sensitivity to quantitative PCR, and could detect as few as 86 copies of viral DNA per reaction. These results suggest that the assay is a specific and sensitive technique to rapidly diagnose bovine papular stomatitis in domestic animals.

Deployment of a Reverse Transcription Loop-Mediated Isothermal Amplification Test for Ebola Virus Surveillance in Remote Areas in Guinea.

To strengthen the laboratory diagnostic capacity for Ebola virus disease (EVD) in the remote areas of Guinea, we deployed a mobile field laboratory and implemented reverse transcription loop-mediated isothermal amplification (RT-LAMP) for postmortem testing. We tested 896 oral swab specimens and 21 serum samples, using both RT-LAMP and reverse transcription-polymerase chain reaction (RT-PCR). Neither test yielded a positive result, and the results from RT-LAMP and RT-PCR were consistent. More than 95% of the samples were tested within 2 days of sample collection. These results highlight the usefulness of the RT-LAMP assay as an EVD diagnostic testing method in the field or remote areas.