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SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with high mutation-resilience under escape mutation screening. A computationally designed antibody based on one of the Y489-binding antibodies, NIV-10/FD03, was able to bind XBB with any 486 mutation and neutralized XBB.1.5. The structural basis for the mutation-resilience of this Y489-binding antibody group may provide important insights into the design of therapeutics resistant to viral escape. In this study, the authors isolated SARS-CoV-2 receptor binding site monoclonal antibodies resistant to Omicron mutations. An amino acid in the receptor binding domain, tyrosine-489, is a virus-vulnerable site and a common footprint of broadly neutralizing antibodies. [ABSTRACT FROM AUTHOR]

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern1–4. Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral entry, raising concerns that the replication capacity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here we have evaluated the replicative ability and pathogenicity of BA.4 and BA.5 isolates in wild-type Syrian hamsters, human ACE2 (hACE2) transgenic hamsters and hACE2 transgenic mice. We have observed no obvious differences among BA.2, BA.4 and BA.5 isolates in growth ability or pathogenicity in rodent models, and less pathogenicity compared to a previously circulating Delta (B.1.617.2 lineage) isolate. In addition, in vivo competition experiments revealed that BA.5 outcompeted BA.2 in hamsters, whereas BA.4 and BA.2 exhibited similar fitness. These findings suggest that BA.4 and BA.5 clinical isolates have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2.Results indicate that the sublineages BA.4 and BA.5 of SARS-CoV-2 Omicron variants have similar pathogenicity to that of the BA.2 sublineage in rodents, highlighting the importance of evaluating viral replication and pathogenesis using clinical isolates. [ABSTRACT FROM AUTHOR]

In cultured cells, SARS-CoV-2 infects cells via multiple pathways using different host proteases. Recent studies have shown that the furin and TMPRSS2 (furin/TMPRSS2)-dependent pathway plays a minor role in infection of the Omicron variant. Here, we confirm that Omicron uses the furin/TMPRSS2-dependent pathway inefficiently and enters cells mainly using the cathepsin-dependent endocytosis pathway in TMPRSS2-expressing VeroE6/TMPRSS2 and Calu-3 cells. This is the case despite efficient cleavage of the spike protein of Omicron. However, in the airways of TMPRSS2-knockout mice, Omicron infection is significantly reduced. We furthermore show that propagation of the mouse-adapted SARS-CoV-2 QHmusX strain and human clinical isolates of Beta and Gamma is reduced in TMPRSS2-knockout mice. Therefore, the Omicron variant isn't an exception in using TMPRSS2 in vivo, and analysis with TMPRSS2-knockout mice is important when evaluating SARS-CoV-2 variants. In conclusion, this study shows that TMPRSS2 is critically important for SARS-CoV-2 infection of murine airways, including the Omicron variant. The SARS-CoV-2 variant of concern Omicron, has been reported to mainly use the endocytosis pathway for viral entry, bypassing the furin/TMPRSS2 pathway utilised by other variants. Here, the authors test a panel of SARS-CoV-2 variants in TMPRSS2 knock out mice to show that all tested variants, including Omicron, utilise this pathway in vivo. [ABSTRACT FROM AUTHOR]

Crimean–Congo hemorrhagic fever virus (CCHFV) causes a zoonotic disease, Crimean–Congo hemorrhagic fever (CCHF) endemic in Africa, Asia, the Middle East, and Southeastern Europe. However, the prevalence of CCHF is not monitored in most of the endemic countries due to limited availability of diagnostic assays and biosafety regulations required for handling infectious CCHFV. In this study, we established a protocol to purify the recombinant CCHFV nucleoprotein (NP), which is antigenically highly conserved among multiple lineages/clades of CCHFVs and investigated its utility in an enzyme-linked immunosorbent assay (ELISA) to detect CCHFV-specific antibodies. The NP gene was cloned into the pCAGGS mammalian expression plasmid and human embryonic kidney 293 T cells were transfected with the plasmid. The expressed NP molecule was purified from the cell lysate using cesium-chloride gradient centrifugation. Purified NP was used as the antigen for the ELISA to detect anti-CCHFV IgG. Using the CCHFV NP-based ELISA, we efficiently detected CCHFV-specific IgG in anti-NP rabbit antiserum and CCHFV-infected monkey serum. When compared to the commercially available Blackbox CCHFV IgG ELISA kit, our assay showed equivalent performance in detecting CCHFV-specific IgG in human sera. These results demonstrate the usefulness of our CCHFV NP-based ELISA for seroepidemiological studies. [ABSTRACT FROM AUTHOR]

Pteropine orthoreovirus (PRV) causes respiratory tract illness (RTI) in humans. PRVs were isolated from throat swabs collected from 9 of 91 wild bats captured on the Mindanao Islands, The Philippines, in 2013. The nucleic acid sequence of the whole genome of each of these isolates was determined. Phylogenetic analysis based on predicted amino acid sequences indicated that the isolated PRVs were novel strains in which re-assortment events had occurred in the viral genome. Serum specimens collected from 76 of 84 bats were positive for PRV-neutralizing antibodies suggesting a high prevalence of PRV in wild bats in the Philippines. The bat-borne PRVs isolated in the Philippines were characterized in comparison to an Indonesian PRV isolate, Miyazaki-Bali/2007 strain, recovered from a human patient, revealing that the Philippine bat-borne PRVs had similar characteristics in terms of antigenicity to those of the Miyazaki-Bali/2007 strain, but with a slight difference (e.g., growth capacity in vitro). The impact of the Philippine bat-borne PRVs should be studied in human RTI cases in the Philippines. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease causing severe hemorrhagic symptoms with a nearly 30 % case-fatality rate in humans. The experimental use of CCHF virus (CCHFV), which causes CCHF, requires high-biosafety-level (BSL) containment. In contrast, pseudotyping of various viral glycoproteins (GPs) onto vesicular stomatitis virus (VSV) can be used in facilities with lower BSL containment, and this has facilitated studies on the viral entry mechanism and the measurement of neutralizing activity, especially for highly pathogenic viruses. In the present study, we generated high titers of pseudotyped VSV bearing the CCHFV envelope GP and analyzed the mechanisms involved in CCHFV infection. A partial deletion of the CCHFV GP cytoplasmic domain increased the titer of the pseudotyped VSV, the entry mechanism of which was dependent on the CCHFV envelope GP. Using the pseudotype virus, DC-SIGN (a calcium-dependent [C-type] lectin cell-surface molecule) was revealed to enhance viral infection and act as an entry factor for CCHFV. [ABSTRACT FROM AUTHOR]

Middle East respiratory syndrome (MERS) coronavirus (Co-V) contains a single spike (S) protein, which binds to a receptor molecule, dipeptidyl peptidase 4 (DPP4; also known as CD26), and serves as a neutralizing antigen. Pseudotyped viruses are useful for measuring neutralization titers against highly infectious viruses as well as for studying their mechanism of entry. In this study, we constructed a series of cytoplasmic deletion mutants of MERS-CoV S and compared the efficiency with which they formed pseudotypes with vesicular stomatitis virus. A pseudotype bearing an S protein with the C-terminal 16 amino acids deleted (MERSpv-St16) reached a maximum titer that was approximately tenfold higher than that of a pseudotype bearing a non-truncated full-length S protein. Using MERSpv-St16, we demonstrated the inability of rat DPP4 to serve as a functional receptor for MERS-CoV, suggesting that rats are not susceptible to MERS-CoV infection. This study provides novel information that enhances our understanding of the host range of MERS-CoV. [ABSTRACT FROM AUTHOR]

In this study, we improved a method for rapid determination of viral RNA sequences (RDV) to overcome the limitations of previous versions. The RDV ver4.0 method can detect RNA sequences with at least 1,000 copies as starting material. A novel virus, which was isolated from field-collected Aedes aegypti larvae in the Phasi Charoen district of Thailand using C6/36 cells, was identified using the RDV ver4.0 protocol. The virus was named Phasi Charoen virus (PhaV). We used a high-throughput pyrosequencing approach to obtain more information about the genome sequence of PhaV. Analysis of a phylogenic tree based on amino acid sequences strongly suggested that PhaV belongs to the family Bunyaviridae. [ABSTRACT FROM AUTHOR]

With the aim of elucidating evolutionary features of GB virus C/hepatitis G virus (GBV-C/HGV), molecular evolutionary analyses were conducted using the entire coding region of this virus. In particular, the rate of nucleotide substitution for this virus was estimated to be less than 9.0 × 10−6 per site per year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as outgroup, indicated that there were three major clusters (the HG, GB, and Asian types) in GBV-C/HGV, and the divergence between the ancestor of GB- and Asian-type strains and that of HG-type strains first took place more than 7000–10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response of the host by means of producing escape mutants, implying that it may have evolved other systems for persistent infection. [ABSTRACT FROM AUTHOR]

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5′ noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5′ NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662–1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5′- or 3′- deleted mutants of the HCV 5′ NCR as competitor RNAs for binding of p25 to wild-type HCV 5′ NCR. Competitor RNAs lacking nucleotides (nt) 47–74 or nt 279–331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome. [ABSTRACT FROM AUTHOR]

Hog cholera virus (HoCV) 5′ terminus of the ALD and GPE(−) strains were analyzed by using rapid amplification of cDNA end method (5′RACE). An additional nine nucleotides were found at the 5′ termini of genomic RNA in the ALD and GPE(−) strains of HoCV. These nine nucleotides were also conserved in BVDV and were suggested to form a hairpin structure at the 5′ terminus by computer-assisted analysis. It seems possible that the secondary structure and/or the 5′ terminus sequence has a significant role in the HoCV virus genome. [ABSTRACT FROM AUTHOR]

The emergence of orthoreoviruses as the causative agent of human respiratory illness over the past few years has led to a demand to determine their viral genome sequences. The whole genome sequencing of such RNA viruses using traditional methods, such as Sanger dideoxy sequencing following rapid amplification of cDNA ends presents a laborious challenge due to the numerous preparatory steps required before sequencing can commence. We developed a practical, time-efficient novel combination method capable of reducing the total time required from months to less than a week in the determination of whole genome sequence of Pteropine orthoreoviruses (PRV); through a combination of viral RNA purification and enrichment, adaptor ligation, reverse transcription, cDNA circularization and amplification, and next generation sequencing. We propose to call the method 'modified rolling circular amplification with adaptor ligation - next generation sequencing (mRCA-NGS)'. Here, we describe the technological focus and advantage of mRCA-NGS and its expansive application, exemplified through the phylogenetic understanding of the Miyazaki-Bali/2007 PRV. [ABSTRACT FROM AUTHOR]