The proprotein convertase SKI-1/S1P is a critical host factor for Nairobi sheep disease virus infectivity

Nairobi sheep disease virus (NSDV) is a tick-borne viral disease of sheep and goats belongs to Orthonairovirus from Bunyavirales order and characterized by fever, haemorrhagic gastroenteritis, abortion, and high mortality. This virus is genetically related to human-pathogenic Crimean-Congo haemorrhagic fever virus (CCHFV) made it proposed as a model organism for researching CCHFV pathogenesis. However, little is known about NSDV pathogenesis and virus-host interactions. The life cycle of human-pathogenic CCHFV is depends on SKI-1/S1P, an important enzyme in the cell’s cholesterol control mechanism. Previous studies have demonstrated that the CCHFV GPC is cleaved by SKI-1/S1P at the consensus motif RRLL519 (amino acid position refers to CCHFV-IbAr 10,200), and this proteolytic cleavage has a strong impact on virus infectivity. Therefore, the aim of this study was to observe the role of SKI-1/S1P expression in NSDV replication and infectivity. The result presented in this study highlights the critical role played by the host cell protease SKI-1/S1P in NSDV replication and infectivity. The findings imply that SKI-1/S1P may be an essential orthonairovirus host factor, which is consistent with the early observation on the function of SKI-1/S1P in the CCHFV life cycle. This study also offers the first proof of the inhibitory small molecule, PF-429242’s antiviral action against NSDV. These results could be the base for further research to assess PF-429242 effectiveness against different orthonairovirus, such as the human pathogenic CCHFV.

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Comparative Structural and Functional Analysis of Bunyavirus and Arenavirus Cap-Snatching Endonucleases

The cap-snatching mechanism exists in Segmented negative strand RNA viruses (sNSVs) of the arena-, bunya- and orthomyxovirus for mRNA synthesis. Authors focused on that the location of cap-binding domain was not confirmed in bunya- and arenavirus. To confirm the locations and profile of cap-snatching nuclease, they analyzed three dimensional structures, shift of melting temperature of endonuclease crystal in with/without divalent metal ions, Hantaan (one of Bunyavirus) endonuclease activity rates in comparison with other sNSVs, mutational analysis of endonuclease activity, iron induced stability of Hantaan endonuclease, structural characterization of divalent metal ions binding to Lassa (one of Arenavirales) endonuclease and mutational analysis of endonuclease activity, iron induced stability of Lassa endonuclease. These results showed that there are 2 types of endonucleases in sNSVs. One is for orthomyxovirus and bunyavirus and contains characteristic catalytic histidine. The other is for arenavirus without histidine and shows poorer activity.

(NN)

Optimal Expression of the envelope Glycoprotein of Orthobrornaviruses Determines the Production of Mature Virus Particles

Borna disease virus 1, a member of the genus Orthobornavirus, is characterized by its long term gene expression. For improving reproduction RNA virus-based episomal vector system, authors focused on that expression of G protein which is type I surface glycoprotein and contains signal peptide is important for Bornavirus infection to cell. They confirmed the importance of G expression for reproduction of infectious viral particles. As the results, excessive G expression of most Bornavirus except canary bornavirus didn’t affect reproduction but reduces infectious reproduced particles with mature G and genomic RNA. These results suggest that limitation of G expression is necessary for normal Borna viral particle production, because increased pre-G inhibits cleavage of G and introduction of mature-G and viral genomic RNA into particles.

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Single-cell profiling of lncRNA expression during Ebola virus infection in rhesus macaquas

Not bulk tissue analysis but single-cell RNA sequences are necessary for uncovering current gene expression value and tissue specificity. Especially, Ebola virus (EBOV) causes one of most lethal infectious disease and strong inflammatory to humans. Single-cell analysis of circulating monocytes could uncover that correct gene expression value, regulation, and tissue or cell specificity. As the results, they identified 2 times lncRNA by annotating them with single-cell RNA sequences. Expression of lncRNA was in fewer cells than mRNA. Some lncRNA were regulated on EBOV infection. These results show that single-cell RNA sequence is effective for measuring correct lncRNA expression. Some of lncRNA expression can be in only specific cells. LncRNA whose expression is condition or cell-type specific are candidates of disease biomarkers.

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“Rule of Six”: How Does the Sendai Virus RNA Polymerase Keep Count?

The “Rule of six” stipulates that the Paramyxovirus polymerase efficiently replicate 6n+0 nucleotides genomes especially. Sendai virus (SeV), a member of paramyxovirus, was used through this series of experiments in this study. As the results, SeV RNA polymerase showed selectivity for RNA following to rule of six without C protein. Swap editing site of SeV RNA was effective for length correction. Experiment of constructs with extra 3’-OH sequences showed that the 3’-OH congruence was not an important signal for SeV RNA polymerase. Furthermore, 2 promoters, working independently, were confirmed. Initiation for one promoter was antigenomic promoter, for another was genomic promoter and both obeyed to rule of six. These results showed that the observance of the rule of six can be necessary for polymerase recognition of certain nucleotides properly positioned regarding the nucleoprotein interaction points for appropriate replication.

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Mechanistic basis for potent neutralization of Sin Nombre hantavirus by a human monoclonal antibody

Sin Nombre virus (SNV) belonging to Hantaviridae family, is responsible for causing hantavirus cardiopulmonary syndrome, a severe illness associated with high fatality. The transmission of SNV to humans can occur via inhalation of aerosolized excreta from rodent. Gn an Gc, the spike proteins on hantavirus, play an essential role as recognized compartment for antibodies. Prior research suggested that Gn-targeting mAbs potentially enhance neutralization ability against hantavirus. As an effective neutralizing human monoclonal antibody, SNV-42 was isolated from a memory B cell originating from a convalescent SNV donor.  This study aims to evaluate the neutralizing mechanism of SNV-42 through from genetic analysis and protein structure analysis. SNV-42 maintains gene segment which serves a vital part in promoting affinity maturation of antibodies, thereby enhancing antibody specificity. The results of crystallographic analysis on SNV-42 protein binding to SNV Gn protein suggests SNV-42 is capable of interfering with various stages of the viral entry pathway. Subsequently, overlaying SNV-42 and SNV (Gn−Gc) ultrastructure arrangement indicates SNV-42 combine to the distal region of SNV membrane. Furthermore, the researchers conduct a simulation to investigate the interaction between SNV-42 protein and SNV Gn spike protein, suggesting SNV-42 might contribute to receptor blocking and fusion inhibition.

(HW)

Preclinical efficacy of oncolytic VSV-IFNβ in treating cancer: A systematic review

VSV-IFNβ　is made of vesicular stomatitis virus (VSV) with inserting IFNβ gene. It is an oncolytic virus which infects and kills tumor cells selectively. Inserting IFNβ is for mitigate leading synthesis of IFN and other pro-inflammatory proteins to evade innate antiviral immunity. There are 4 routes of administration: intertumoral, intraperitoneal, locoregional and intervenors for both sarcoma and carcinoma. In these treatments, there are some significant effectivities, however, it can lead neurotoxicity especially in systemically established myeloma in nude mouse model. Authors conclude that further research are necessary to fully understand the side effect and to optimize as an oncolytic virotherapeutic agent.

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The organization of double-strand RNA in the chinkugunya virus replication organelle

The chikungunya virus (CHIKV) is the Alphavirus genus in the family Togaviridae. CHIKV cause Chikungunya fever which is a zoonosis. When CHIKV infects a cell, they make spherules on cellular-plasma membrane. The spherule contains duplex viral RNA (negative strand and synthesized positive sense) and two-megadalton protein complex that works as enzyme for RNA replication. Here, they uncovered that the double strand RNA in spherule of CHIKV is shorter than unconstrained double-strand RNA and CHIKV RNA has five ~25-32nm conformations. Finally, the RNA occupies the spherule homogenous density, however perpendicular form from the point of cell-virus connection (membrane neck) to the center of spherule is a preferred orientation. These results can show that RNA synthesis is taken at a single state like neck complex where protein complex is in.
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Immunogenicity of poxvirus-based vaccines against Nipah virus

Nipah virus (NiV) is a causative agent with broad host ranges including livestock and humans. The Pteropus species of fruit bats as reservoir of NiV show no symptoms, while human infection has severe outcome and high mortality. Thus, new vaccination covering wide range of species with easy administration is of utmost importance. Poxvirus, such as humans-modified vaccinia virus Ankara (MVA) for humans, recombinant raccoon poxvirus (RCN) for animals have been previously shown potential vectors for infection through mucosal and dermal administration. In this study, two vaccine candidates, namely MVA-FG and RCN-FG, were constructed by combining fusion (F) and glycoprotein (G) of NiV with MVA or RCN, following to characterization in vitro, and in vivo administration to mice subcutaneously (SC) an intranasally (IN). Along with post-immunization monitoring, level of immunity was evaluated in mice serum. Conclusively, neither of the immunized mice showed any weight loss or clinical signs. Immunized mice serum was detected with significant levels of anti-F and G circulating antibodies through SC or IN. Also, immunized mice exhibited cytokine expression and different phenotype of CD8a+ T cell in splenocytes and lung cells. Based on these data, authors suggested MVA-FG and RCN-FG as potential vaccine candidates against NiV.

(HW)

Conserved allosteric inhibitory site on the respiratory syncytial virus and human metapneumovirus RNA-dependent RNA polymerases

Respiratory syncytial virus (RSV) and Human metapneumovirus (HMPV) are two of the most common causes of respiratory infections in children, elderly, and immunocompromised people worldwide. Inhibitors for viral replication using small molecule inhibitors are being developed, but the binding location and molecular process of this inhibitors are still largely understood. This study analyzed the interaction between the RSV and HMPV polymerases and MRK-1, a non-nucleoside pneumovirus inhibitor. According to a functional analysis of the RSV polymerase in the presence of MRK-1, the molecule inhibits both initiation and early elongation, indicating that it works as an allosteric inhibitor to stop the conformational changes that the polymerase experiences in the early stages of RNA synthesis. According to the Northern blot analysis of minigenome-generated RNAs, the increase in MRK-1 concentration decreased both mRNA and antigenome accumulation, indicating that the ligand suppresses the function of both processes. The results presented in this study provide beneficial information on the structure-function characteristics of the polymerases of RSV and HMPV and the insight into the interaction of a small molecule inhibitors that targets them. 

(MA)

Cross-protective antibodies against common endemic respiratory viruses

 Respiratory syncytial virus (RSV), human metapneumovirus (HMPV), and human parainfluenza virus types one (HPIV1) and three (HPIV3) pose major problems as it causes pneumonia and bronchiolitis in at-risk individuals including infants and immunocompromised adults. Additionally, pathogenic viruses in adults are diversity, so cross-protective mAbs neutralising these viruses could serve as an alternative way for attaining a wider population against respiratory viral infections.  
  In this study, two cross-protective neutralizing mAb, namely 3X1 (targeting HPIV3 and HPIV1) and MXR (targeting RSV and HMPV) were discovered by labelled viral fusion protein. 3X1 mAb was discovered by a bait-and-switch strategy involved the isolation of HPIV3 preF tetramer-marked human splenocytes capable of neutralising with HPIV1, selecting the neutralizing mAb binding to specific region of both HPIV1 and HPIV3. MXR mAb were obtained by isolating from single isotype-switched B cells that contained specific alleles for heavy and light chain and bind to F proteins of RSV and HMPV. It proved that both 3X1 and MXR mAbs effectively neutralized the targeted viruses in vitro and in vivo. Analysis with cryo-electron microscopy allowed to identify specific binding neutralizing region in antigen. Furthermore, a cocktail combing 3X1 and MXR mAbs exhibited cross-protective neutralizing ability against these viruses in mice. 

(HW)

Natural history of nonhuman primates after conjunctival exposure to Ebola virus

The Ebola viruses (EBOV) are pathogenic agents causing severely viral haemorrhagic fever.  The development of treatment and vaccination necessitates non-human primate models. In the nature, the transmission of the disease can occur through infected blood or secretion.  However, EBOV in medical facilities also transmit through accidental injection. Thus, monkey handlers in laboratory may face risks of exposing to EBOV during intramuscular injection of EBOV to monkey. This study aims to establish an animal experiment on monkey through conjunctival infection with the intention of reducing the risk of needle accidence.  In this study, Cynomolgus macaques in three challenging cohorts were anesthetized before being infected via the eyelid mucosa in low and high dosage.  Animals were monitored and blood specimens were collected to evaluate including hematologic and serum biochemical analysis, viral RNA and neutralizing antibody. In conclusion, conjunctive infection has the potential to induce lethality in infected monkeys in dose-dependent manner. A low dosage of viruses results in a minimal level of lethality among infected monkeys, whereas a high dosage uniformly induces lethality. Interestingly, viral RNA was detected in all infected monkeys, regardless of the clinical signs, but viral titre was only detected from infected animals with clinical signs of EBOV infection.
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Mechanistic insight into the efficient packaging of antigenomic S RNA into Rift Valley fever virus particles

 The infectious Rift Valley fever virus (RVFV) particle is packaged with L, M and S RNAs, in addition to their antigenomic RNA counterparts, and two envelope glycoproteins, Gn and Gc. The Gn and Gc glycoproteins are encoded in M segment and Gn is hypothesized to function as the matrix protein. The L segment encodes L protein. The S RNA uses ambisense coding strategy where the nucleocapsid and nonstructural mRNAs are transcribed from genomic S RNA and antigenomic S RNA respectively. Packaging of RVFV particle is associated with interaction between Gn and viral ribonucleoprotein complexes. However, Gn is reported to bind antigenomic S RNA efficiently compared to antigenomic L and M RNAs. This study characterized regions of viral RNA that directly interact with Gn using UV-crosslinking and immunoprecipitation followed by high-throughput sequencing. Multiple Gn-binding sites in genomic and antigenomic RVFV RNAs were identified. A prominent Gn-binding site was identified within 3’ noncoding region of the antigenomic S RNA. It was established that the prominent Gn-binding site has a significant role in effective packaging of antigenomic S RNA into virions. Therefore, immediately after viral infection, nonstructural proteins which are antagonist of interferon are synthesized and expressed, subsequently suppressing expression of interferon-β mRNA.

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Multiplication of defective Ebola virus in a complementary permissive cell line

Ebola virus (EBOV) causes haemorrhagic fever in humans and nonhuman primates with high morbidity and fatality. The small molecule drugs are not available until this time, even though two antibody-based drugs are available. By using altered DNA to create counterfeit EBOV, reverse genetics technology enables the study of the viral structure and operation and enable the development of EBOV antiviral agent identification. In this study, a recombinant Ebola virus that has the glycoprotein (GP) gene replaced with Cre recombinase (Cre) gene was generated using reverse genetic system. The recombinant virus, upon infection in a complementary permissive cell line, can multiply itself and produce reporter protein and GP in the presence of exogenous Cre. The design of this recombinant virus has several significant benefits. The Cre gene embedded in the viral genome is crucial for the recombinant virus life cycle, immunity responses are not affected by the recombinant virus infection, it could be utilized to infect the Cre-reporting animals, and it has the identical component to wild type virus. Consequently, this recombinant virus is an ideal resource to develop a diagnostic tools and procedures, vaccine, and the manufacturing of antiviral agents. It could also be used to observe the virus-host interactome and further, the design could be adapted for other viruses.

(MA)