Search

International audience; The actin cytoskeleton and actin-dependent molecular and cellular events are responsible for the organization of eukaryotic cells and their functions. Viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), depend on host cell organelles and molecular components for cell entry and propagation. Thus, it is not surprising that they also interact at many levels with the actin cytoskeleton of the host. There have been many studies on how different viruses reconfigure and manipulate the actin cytoskeleton of the host during successive steps of their life cycle. However, we know relatively little about the interactions of SARS-CoV-2 with the actin cytoskeleton. Here, we describe how the actin cytoskeleton is involved in the strategies used by different viruses for entry, assembly, and egress from the host cell. We emphasize what is known and unknown about SARS-CoV-2 in this regard. This review should encourage further investigation of the interactions of SARS-CoV-2 with cellular components, which will eventually be helpful for developing novel antiviral therapies for mitigating the severity of COVID-19.

Reverse genetics systems for viruses, the technology used to generate gene-engineered recombinant viruses from artificial genes, enable the study of the roles of the individual nucleotides and amino acids of viral genes and proteins in infectivity, replication, and pathogenicity. The successful development of a reverse genetics system for poliovirus in 1981 accelerated the establishment of protocols for other RNA viruses important for human health. Despite multiple efforts, rotavirus (RV), which causes severe gastroenteritis in infants, was refractory to reverse genetics analysis, and the first complete reverse genetics system for RV was established in 2017. This novel technique involves use of the fusogenic protein FAST (fusion-associated small transmembrane) derived from the bat-borne Nelson Bay orthoreovirus, which induces massive syncytium formation. Co-transfection of a FAST-expressing plasmid with complementary DNAs encoding RV genes enables rescue of recombinant RV. This review focuses on methodological insights into the reverse genetics system for RV and discusses applications and potential improvements to this system.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is the cause of the ongoing coronavirus disease 2019 (COVID‐19) pandemic. The S protein is the key viral protein for associating with ACE2, the receptor for SARS‐CoV‐2. There are many kinds of posttranslational modifications in S protein. However, the detailed mechanism of palmitoylation of SARS‐CoV‐2 S remains to be elucidated. In our current study, we characterized the palmitoylation of SARS‐CoV‐2 S. Both the C15 and cytoplasmic tail of SARS‐CoV‐2 S were palmitoylated. Fatty acid synthase inhibitor C75 and zinc finger DHHC domain‐containing palmitoyltransferase (ZDHHC) inhibitor 2‐BP reduced the palmitoylation of S. Interestingly, palmitoylation of SARS‐CoV‐2 S was not required for plasma membrane targeting of S but was critical for S‐mediated syncytia formation and SARS‐CoV‐2 pseudovirus particle entry. Overexpression of ZDHHC2, ZDHHC3, ZDHHC4, ZDHHC5, ZDHHC8, ZDHHC9, ZDHHC11, ZDHHC14, ZDHHC16, ZDHHC19, and ZDHHC20 promoted the palmitoylation of S. Furthermore, those ZDHHCs were identified to associate with SARS‐CoV‐2 S. Our study not only reveals the mechanism of S palmitoylation but also will shed important light into the role of S palmitoylation in syncytia formation and virus entry., Highlights SARS‐CoV‐2 S protein is palmitoylatedin both N terminal and C terminal cysteines.Palmitoylation of S is critical for S mediated syncytia formation.Palmitoylation of S promotes SARS‐CoV‐2pp entry.Multiple ZDHHCs promoted palmitoylation of SARS‐CoV‐2 S.

The S2 subunit serves a crucial role in infectious bronchitis virus (IBV) infection, particularly in facilitating membrane fusion. Using reverse genetic techniques, mutant strains of the S2 locus exhibited substantially different syncytium-forming abilities in chick embryonic kidney cells. To determine the precise formation mechanism of syncytium, we demonstrated the co-ordinated role of Abl2 and its mediated cytoskeletal regulatory pathway within the S2 subunit. Using a combination of fluorescence quantification, RNA silencing, and protein profiling techniques, the functional role of S2 subunits in IBV-infected cells was exhaustively determined. Our findings imply that Abl2 is not the primary cytoskeletal regulator, the viral S2 component is involved in indirect regulation, and the three different viral strains activate various cytoskeletal regulatory pathways through Abl2. CRK, CRKL, ABI1, NCKAP1, and ENAH also play a role in cytoskeleton regulation. Our research provides a point of reference for the development of an intracellular regulatory network for the S2 subunit and a foundation for the rational design of antiviral drug targets against Abl2.

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era., A study of SARS-CoV-2 variants examining their transmission, infectivity, and potential resistance to therapies provides insights into the biology of the Delta variant and its role in the global pandemic.

Background Respiratory syncytial virus (RSV) is a primary cause of morbidity and mortality worldwide, affecting infants, young children, and immune-compromised patients; however, currently no vaccine is available for prevention of RSV infections. The overwhelming majority of our knowledge of how RSV causes infection is based upon studies that have been carried out using traditional 2D methods, with cells cultured on flat plastic dishes. Although these simplified culture systems are essential to gain an insight into the fundamentals of host-pathogen interactions, cells in 2D are not exposed to the same conditions as cells in 3D tissues in the body and are therefore a poor representation of thein vivo microenvironment. In this study, we aim to develop the first 3D culture model for RSV infection using A549 cells to test its utility for RSV pathogenesis. Methods To generate spheroids, A549 cells were cultured using ultra-low attachment plates to generate 25 × 103 cell spheroids. The viability of the spheroids was assessed by trypan blue exclusion assay and flow cytometry showing prominent live cells throughout the spheroids confirming high viability over seven days of incubation. Results Immunostaining of A549 spheroids inoculated with RSV, showed time-dependent dissemination of the viral antigen RSV-F within the spheroid, resulting in syncytia formation and a 3-fold increase in mucin secretion compared to the uninfected cells. Additionally, RSV successfully replicated in the spheroids producing infectious virus as early as day one post-inoculation and was sustained for up to 7 days post-inoculation. Conclusions Results show that A549 spheroids are susceptible and permissive for RSV since they exhibit the characteristics of RSV infection including syncytia formation and mucin overexpression, suggesting that A549 spheroids can be used a promising model for studying RSV in vitro.

The direct oncolytic effect of Newcastle disease virus (NDV) depends on the following two aspects: the susceptibility of cancer cells to virus infection and the ability of virus itself to lyse cancer cells. First, we investigate the susceptibility of cancer cells to NDV infection, HepG2, MDA-MB-231, and SH-SY5Y cells were susceptible, A549, MCF7, and LoVo cells were less susceptible. To investigate the molecular mechanism responsible for cancer cell susceptibility, transcriptome sequencing was carried out. We found that the levels of alpha-sialic acid acyltransferase were upregulated in MDA-MB-231 cells compared with MCF7 cells, and the interferon was downregulated. Second, to optimize the oncolytic capacity of the wild-type rClone30, a series of chimeric viruses rClone30-Anh(HN), rClone30-Anh(F), and rClone30-Anh(HN-F) were constructed by exchanging the HN gene, F gene or both of non-lytic rClone30 strain with lytic strain Anhinga. rClone30-Anh(F) and rClone30-Anh(HN-F) enhanced the oncolytic effect of the rClone30, and this enhancement is more obvious in the susceptible cells. The oncolytic mechanism of rClone30-Anh(F) was analyzed by transcriptome analyses, in comparison with rClone30, rClone30-Anh(F) upregulated the expression of ATG5, Beclin 1, and MAP1LC3B, thus activating autophagy and promoting the production of syncytia. In conclusion, our study provides a strategy to enhance the oncolytic effect of rClone30.

Oncolytic virotherapy uses replication-competent virus as a means of treating cancer. Whereas this field has shown great promise as a viable treatment method, the limited spread of these viruses throughout the tumor microenvironment remains a major challenge. To overcome this issue, researchers have begun looking at syncytia formation as a novel method of increasing viral spread. Several naturally occurring fusogenic viruses have been shown to possess strong oncolytic potential and have since been studied to gain insight into how this process benefits oncolytic virotherapy. Whereas these naturally fusogenic viruses have been beneficial, there are still challenges associated with their regular use. Because of this, engineered/recombinant fusogenic viruses have also been created that enhance nonfusogenic oncolytic viruses with the beneficial property of syncytia formation. The purpose of this review is to examine the existing body of literature on syncytia formation in oncolytics and offer direction for potential future studies. Keywords: syncytia, oncolytic virotherapy, fusogenic virus, fusion protein

Introduction Cancer has become one of the most critical health issues of modern times. To overcome the ineffectiveness of current treatment options, research is being done to explore new therapeutic modalities. One such novel treatment is oncolytic virotherapy (OV) which uses tumor tropic viruses to specifically target and kill malignant cells. While OV has shown significant promise in recent clinical trials, the therapeutic use of viruses poses a number of unique challenges. In particular, obtaining effective viral spread throughout the tumor microenvironment remains problematic. Previous work has suggested this can be overcome by forcing oncolytic viruses to induce syncytia formation. Methods In the current work, we generated a series of recombinant myxoma viruses expressing exogenous fusion proteins from other viral genomes and examined their therapeutic potential in vitro and in vivo. Results Similar to previous studies, we observed that the expression of these fusion proteins during myxoma infection induced the formation of multinucleated syncytia which increased viral spread and lytic potential compared to non-fusogenic controls. Contrary to expectations, however, the treatment of established tumors with these viruses resulted in decreased therapeutic efficacy which corresponded with reduced viral persistence. Discussion These findings indicate that enhanced viral spread caused by syncytia formation can actually reduce the efficacy of OV and supports a number of previous works suggesting that the in vitro properties of viruses frequently fail to predict their in vivo efficacy.

International audience; The most severe forms of coronavirus disease 2019 (COVID-19) are often associated with the presence of syncytia in the lungs resulting from cell-cell fusion mediated by the SARS-CoV-2 spike protein. In this issue, Rajah and colleagues show that the SARS-CoV-2 alpha, beta, and delta variants promote enhanced syncytia formation as compared to the original strain.

Only a handful of cell types, including fibroblasts, epithelial, and endothelial cells, can support human cytomegalovirus (CMV) replication in vitro, in striking contrast to the situation in vivo. While the susceptibility of epithelial and endothelial cells to CMV infection is strongly modulated by their anatomical site of origin, multiple CMV strains have been successfully isolated and propagated on fibroblasts derived from different organs. As oral mucosal cells are likely involved in CMV acquisition, we sought to evaluate the ability of infant labial fibroblasts to support CMV replication, compared to that of commonly used foreskin and fetal lung fibroblasts. No differences were found in the proportion of cells initiating infection, or in the amounts of viral progeny produced after exposure to the fibroblast-adapted CMV strain AD169 or to the endothelial cell-adapted strain TB40/E. Syncytia formation was, however, significantly enhanced in infected labial and lung fibroblasts compared to foreskin-derived cells, and did not occur after infection with AD169. Together, these data indicate that fibroblast populations derived from different tissues are uniformly permissive to CMV infection but retain phenotypic differences of potential importance for infection-induced cell–cell fusion, and ensuing viral spread and pathogenesis in different organs.

Tetraspanins are transmembrane glycoproteins that have been shown increasing interest as host factors in infectious diseases. In particular, they were implicated in the pathogenesis of both non-enveloped (human papillomavirus (HPV)) and enveloped (human immunodeficiency virus (HIV), Zika, influenza A virus, (IAV), and coronavirus) viruses through multiple stages of infection, from the initial cell membrane attachment to the syncytium formation and viral particle release. However, the mechanisms by which different tetraspanins mediate their effects vary. This review aimed to compare and contrast the role of tetraspanins in the life cycles of HPV, HIV, Zika, IAV, and coronavirus viruses, which cause the most significant health and economic burdens to society. In doing so, a better understanding of the relative contribution of tetraspanins in virus infection will allow for a more targeted approach in the treatment of these diseases.

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

A distinctive feature of the SARS-CoV-2 spike protein is its ability to efficiently fuse cells, thus producing syncytia found in COVID-19 patients. This commentary proposes how this ability enables spike to cause COVID-19 complications as well as side effects of COVID-19 vaccines, and suggests how these effects can be prevented..

Cell–cell fusion is a fundamental and complex process that occurs during reproduction, organ and tissue growth, cancer metastasis, immune response, and infection. All enveloped viruses express one or more proteins that drive the fusion of the viral envelope with cellular membranes. The same proteins can mediate the fusion of the plasma membranes of adjacent cells, leading to the formation of multinucleated syncytia. While cell–cell fusion triggered by alpha- and gammaherpesviruses is well-studied, much less is known about the fusogenic potential of betaherpesviruses such as human cytomegalovirus (HCMV) and human herpesviruses 6 and 7 (HHV-6 and HHV-7). These are slow-growing viruses that are highly prevalent in the human population and associated with several diseases, particularly in individuals with an immature or impaired immune system such as fetuses and transplant recipients. While HHV-6 and HHV-7 are strictly lymphotropic, HCMV infects a very broad range of cell types including epithelial, endothelial, mesenchymal, and myeloid cells. Syncytia have been observed occasionally for all three betaherpesviruses, both during in vitro and in vivo infection. Since cell–cell fusion may allow efficient spread to neighboring cells without exposure to neutralizing antibodies and other host immune factors, viral-induced syncytia may be important for viral dissemination, long-term persistence, and pathogenicity. In this review, we provide an overview of the viral and cellular factors and mechanisms identified so far in the process of cell–cell fusion induced by betaherpesviruses and discuss the possible consequences for cellular dysfunction and pathogenesis.

Since the D614G substitution in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged, the variant strain has undergone a rapid expansion to become the most abundant strain worldwide. Therefore, this substitution may provide an advantage for viral spreading. To explore the mechanism, we analyzed 18 viral isolates containing S proteins with either G614 or D614 (S-G614 and S-D614, respectively). The plaque assay showed a significantly higher virus titer in S-G614 than in S-D614 isolates. We further found increased cleavage of the S protein at the furin substrate site, a key event that promotes syncytium formation, in S-G614 isolates. The enhancement of the D614G substitution in the cleavage of the S protein and in syncytium formation has been validated in cells expressing S protein. The effect on the syncytium was abolished by furin inhibitor treatment and mutation of the furin cleavage site, suggesting its dependence on cleavage by furin. Our study pointed to the impact of the D614G substitution on syncytium formation through enhanced furin-mediated S cleavage, which might increase the transmissibility and infectivity of SARS-CoV-2 strains containing S-G614. IMPORTANCE Analysis of viral genomes and monitoring of the evolutionary trajectory of SARS-CoV-2 over time has identified the D614G substitution in spike (S) as the most prevalent expanding variant worldwide, which might confer a selective advantage in transmission. Several studies showed that the D614G variant replicates and transmits more efficiently than the wild-type virus, but the mechanism is unclear. By comparing 18 virus isolates containing S with either D614 or G614, we found significantly higher virus titers in association with higher furin protease-mediated cleavage of S, an event that promotes syncytium formation and virus infectivity, in the S-G614 viruses. The effect of the D614G substitution on furin-mediated S cleavage and the resulting enhancement of the syncytium phenotype has been validated in S-expressing cells. This study suggests a possible effect of the D614G substitution on S of SARS-CoV-2; the antiviral effect through targeting furin protease is worthy of being investigated in proper animal models.

The respiratory syncytial virus (RSV) causes respiratory infection and bronchiolitis, requiring hospitalization mainly in infants. The interaction between RSV, envelope glycoproteins G and F, and cell surface heparan sulfate proteoglycans (HSPG) is required for binding and entry into the host cells. A G2-S16 polyanionic carbosilane dendrimer was identified as a possible RSV inhibitor. We speculated that the G2-S16 dendrimer adheres to the host cell-surface HSPG, acts through binding to HS receptors, and prevents further RSV infection. The G2-S16 dendrimer was non-toxic when applied intranasally to Balb/c mice, and interestingly enough, this G2-S16 dendrimer inhibits 85% RSV. Therefore, our G2-S16 dendrimer could be a candidate for developing a new possible therapy against RSV infection. This work has been (partially) funded by the RD16/0025/0019, projects as part of Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (2013–2016) and co-financed by Instituto de Salud Carlos III (Subdirección General de Evaluación) and Fondo Europeo de Desarrollo Regional (FEDER), RETIC PT17/0015/0042, Fondo de Investigacion Sanitaria (FIS) (grant number PI19/01638) and EPIICAL project. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, the Consolider Program, and CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work has been supported partially by the EUROPARTNER: Strengthening and spreading international partnership activities of the Faculty of Biology and Environmental Protection for interdisciplinary research and innovation of the University of Lodz Programme: NAWA International Academic Partnership Programme. This article/publication is based upon work from COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology). Sí

Measles virus (MeV), an enveloped RNA virus in the family Paramyxoviridae, is still an important cause of childhood morbidity and mortality worldwide. MeV usually causes acute febrile illness with skin rash, but in rare cases persists in the brain, causing a progressive neurological disorder, subacute sclerosing panencephalitis (SSPE). The disease is fatal, and no effective therapy is currently available. Although transsynaptic cell-to-cell transmission is thought to account for MeV propagation in the brain, neurons do not express the known receptors for MeV. Recent studies have shown that hyperfusogenic changes in the MeV fusion (F) protein play a key role in MeV propagation in the brain. However, how such mutant viruses spread in neurons remains unexplained. Here, we show that cell adhesion molecule 1 (CADM1; also known as IGSF4A, Necl-2, and SynCAM1) and CADM2 (also known as IGSF4D, Necl-3, SynCAM2) are host factors that enable MeV to cause membrane fusion in cells lacking the known receptors and to spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the envelope. However, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same cell membrane, causing the fusion protein triggering and membrane fusion. Knockdown of CADM1 and CADM2 inhibits syncytium formation and virus transmission between neurons that are both mediated by hyperfusogenic F proteins. Thus, our results unravel the molecular mechanism (receptor-mimicking cis-acting fusion triggering) by which MeV spreads transsynaptically between neurons, thereby causing SSPE. IMPORTANCE Measles virus (MeV), an enveloped RNA virus, is the causative agent of measles, which is still an important cause of childhood morbidity and mortality worldwide. Persistent MeV infection in the brain causes a fatal progressive neurological disorder, subacute sclerosing panencephalitis (SSPE), several years after acute infection. However, how MeV spreads in neurons, which are mainly affected in SSPE, remains largely unknown. In this study, we demonstrate that cell adhesion molecule 1 (CADM1) and CADM2 are host factors enabling MeV spread between neurons. During enveloped virus entry, a cellular receptor generally interacts in trans with the attachment protein on the viral membrane (envelope). Remarkably, CADM1 and CADM2 interact in cis with the MeV attachment protein on the same membrane, triggering the fusion protein and causing membrane fusion, as viral receptors usually do in trans. Careful screening may lead to more examples of such “receptor-mimicking cis-acting fusion triggering” in other viruses.

This commentary proposes how the coronavirus SARS- CoV-2 can contribute to clinical manifestations of COVID-19 by producing syncytia.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high fatality rate, caused by SFTS virus (SFTSV). Because little is known about the nature of SFTSV, basic studies are required for the developments of vaccines and effective therapies. In the present study, we identified the amino acid residue important for membrane fusion induced by the SFTSV glycoprotein (GP). Syncytium formations were observed in cells expressing the GPs of SFTSV Japanese strain (YG-1 and SPL030). In contrast, no or only weak syncytium formations were induced in cells expressing GP of SFTSV Chinese strain (HB29). The replacement of arginine at amino acid residue 962 with serine in HB29 GP (R962S) induced membrane fusion, while the replacement of serine at residue 962 with arginine in YG1 GP (S962R) did not. These data indicate that serine at residue 962 in the SFTSV-GP is critical for inducing membrane fusion and viral infection.

Respiratory syncytial virus (RSV) remains a leading cause of infant mortality worldwide and exhaustive international efforts are underway to develop a vaccine. However, vaccine development has been hindered by a legacy of vaccine-enhanced disease, poor viral immunogenicity in infants, and genetic and physical instabilities. Natural infection with RSV does not prime for enhanced disease encouraging development of live-attenuated RSV vaccines for infants; however, physical instabilities of RSV may limit vaccine development. The role of RSV strain-specific differences on viral physical stability remains unclear. We have previously demonstrated that the RSV fusion (F) surface glycoprotein is responsible for mediating significant differences in thermostability between strains A2 and A2-line19F. In this study, we performed a more comprehensive analysis to characterize the replication and physical stability of recombinant RSV A and B strains that differed only in viral attachment (G) and/or F surface glycoprotein expression. We observed significant differences in thermal stability, syncytia size, pre-fusion F incorporation and viral growth kinetics in vitro, but limited variations to pH and freeze-thaw inactivation among several tested strains. Consistent with earlier studies, A2-line19F showed significantly enhanced thermal stability over A2, but also restricted growth kinetics in both HEp2 and Vero cells. As expected, no significant differences in susceptibility to UV inactivation were observed. These studies provide the first analysis of the physical stability of multiple strains of RSV, establish a key virus strain associated with enhanced thermal stability compared to conventional lab strain A2, and further support the pivotal role RSV F plays in virus stability.

The human metapneumovirus (HMPV) fusion protein (F) mediates fusion of the viral envelope and cellular membranes to establish infection. HMPV F from some, but not all, viral strains promotes fusion only after exposure to low pH. Previous studies have identified several key residues involved in low pH triggering, including H435 and a proposed requirement for glycine at position 294. We analyzed the different levels of fusion activity, protein expression and cleavage of three HMPV F proteins not previously examined. Interestingly, low pH-triggered fusion in the absence of G294 was identified in one F protein, while a novel histidine residue (H434) was identified that enhanced low pH promoted fusion in another. The third F protein failed to promote cell-to-cell fusion, suggesting other requirements for F protein triggering. Our results demonstrate HMPV F triggering is more complex than previously described and suggest a more intricate mechanism for fusion protein function and activation.

Background Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR). Methods We changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3GREM-EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells. Results We successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3GREM-EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows. Conclusion The new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.

Pneumoviruses represent a major public health burden across the world. Respiratory syncytial virus (RSV) and human metapneumovirus (HMPV), two of the most recognizable pediatric infectious agents, belong to this family. These viruses are enveloped with a non-segmented negative-sense RNA genome, and their replication occurs in specialized cytosolic organelles named inclusion bodies (IB). The critical role of IBs in replication of pneumoviruses has begun to be elucidated, and our current understanding suggests they are highly dynamic structures. From IBs, newly synthesized nucleocapsids are transported to assembly sites, potentially via the actin cytoskeleton, to be incorporated into nascent virions. Released virions, which generally contain one genome, can then diffuse in the extracellular environment to target new cells and reinitiate the process of infection. This is a challenging business for virions, which must face several risks including the extracellular immune responses. In addition, several recent studies suggest that successful infection may be achieved more rapidly by multiple, rather than single, genomic copies being deposited into a target cell. Interestingly, recent data indicate that pneumoviruses have several mechanisms that permit their transmission en bloc, i.e. transmission of multiple genomes at the same time. These mechanisms include the well-studied syncytia formation as well as the newly described formation of long actin-based intercellular extensions. These not only permit en bloc viral transmission, but also bypass assembly of complete virions. In this review we describe several aspects of en bloc viral transmission and how these mechanisms are reshaping our understanding of pneumovirus replication, assembly and spread.

Glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) plays a key role in multiple events during infection including virus entry, cell-to-cell spread, and virus-induced syncytia formation. Here, we provide evidence that an arginine/lysine cluster located at the transmembrane-cytoplasm interface of gD critically contributes to viral spread and cell-cell fusion. Our studies began with the discovery that packaging of gD into virions is almost completely blocked in the absence of tegument protein UL16. We subsequently identified a novel, direct, and regulated interaction between UL16 and gD, but this was not important for syncytia formation. However, a mutational analysis of the membrane-proximal basic residues of gD revealed that they are needed for the gBsyn phenotype, salubrinal-induced fusion of HSV-infected cells, and cell-to-cell spread. Finally, we found that these same gD tail basic residues are not required for cell fusion induced by a gKsyn variant.

HSV-2 spread is predominantly dependent on cell-to-cell contact. However, the underlying mechanisms remain to be determined. Here we demonstrate that HSV-2 gJ, which was previously assigned no specific function, promotes HSV-2 cell-to-cell spread and syncytia formation. In the context of viral infection, knockout or knockdown of gJ impairs HSV-2 cell-to-cell spread among epithelial cells or from epithelial cells to neuronal cells, which leads to decreased virus production, whereas ectopic expression of gJ enhances virus production. Mechanistically, gJ increases the expression levels of HSV-2 proteins, and also enhances viral protein expression and replication of heterologous viruses like HIV-1 and JEV, suggesting that HSV-2 gJ likely functions as a regulator of viral protein expression and virus production. Findings in this study provide a basis for further understanding the role of gJ in HSV-2 replication.

Recombinant measles viruses (MVs) have oncolytic activity against a variety of human cancers. However, their kinetics of spread within tumors has been unexplored. We established an intravital imaging system using the dorsal skin fold chamber, which allows for serial, non-invasive imaging of tumor cells and replication of a fusogenic and a hypofusogenic MV. Hypofusogenic virus-infected cells were detected at the earliest 3 days post-infection (dpi), with peak infection around 6 dpi. In contrast, the fusogenic virus replicated faster: infected cells were detectable 1 dpi and cells were killed quickly. Infection foci were significantly larger with the fusogenic virus. Both viruses formed syncytia. The spatial relationships between cells have a major influence on the outcome of therapy with oncolytic viruses. Keywords: virotherapy, oncolytic, intravital imaging, virus dynamics, measles, population dynamics

Nipah virus (NiV) is a highly pathogenic zoonotic virus with a broad species tropism, originating in pteropid bats. Human outbreaks of NiV disease occur almost annually, often with high case-fatality rates. The specific events that lead to pathogenesis are not well defined, but the disease has both respiratory and encephalitic components, with relapsing encephalitis occurring in some cases more than a year after initial infection. Several cell types are targets of NiV, dictated by the expression of the ephrin-B2/3 ligand on the cell’s outer membrane, which interact with the NiV surface proteins. Vascular endothelial cells (ECs) are major targets of infection. Cytopathic effects (CPE), characterized by syncytia formation and cell death, and an ensuing vasculitis, are a major feature of the disease. Smooth muscle cells (SMCs) of the tunica media that line small blood vessels are infected in humans and animal models of NiV disease, although pathology or histologic changes associated with antigen-positive SMCs have not been reported. To gain an understanding of the possible contributions that SMCs might have in the development of NiV disease, we investigated the susceptibility and potential cytopathogenic changes of human SMCs to NiV infection in vitro. SMCs were permissive for NiV infection and resulted in high titers and prolonged NiV production, despite a lack of cytopathogenicity, and in the absence of detectable ephrin-B2/3. These results indicate that SMC might be important contributors to disease by producing progeny NiV during an infection, without suffering cytopathogenic consequences.

New affordable therapeutic protocols for COVID-19 are urgently needed despite the increasing number of effective vaccines and monoclonal antibodies. To this end, there is increasing attention towards computational methods for drug repositioning andde novodrug design.Here, we systematically integrated multiple data-driven computational approaches to perform virtual screening and prioritize candidate drugs for the treatment of COVID-19. From the set of prioritized drugs, we selected a subset of representative candidates to test in human cells. Two compounds, 7-hydroxystaurosporine and bafetinib, showed synergistic antiviral effects in ourin vitroexperiments, and strongly inhibited viral-induced syncytia formation. Moreover, since existing drug repositioning methods provide limited usable information forde novodrug design, we extracted and prioritized the chemical substructures of the identified drugs, providing a chemical vocabulary that may help to design new effective drugs.

The emergence of the highly-pathogenic severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of COVID-19 (coronavirus disease-2019), has caused an unprecedented global health crisis, as well as societal and economic disruption. The SARS-CoV-2 spike (S), a surface-anchored trimeric class-I fusion glycoprotein essential for entry into host cells, represents a key target for developing vaccines and therapeutics capable of blocking virus invasion. The emergence of several SARS-CoV-2 spike variants that facilitate virus spread and may affect the efficacy of recently developed vaccines, creates great concern and highlights the importance of identifying antiviral drugs to reduce SARS-CoV-2-related morbidity and mortality. Nitazoxanide, a thiazolide originally developed as an antiprotozoal agent with recognized broad-spectrum antiviral activity in-vitro and in clinical studies, was recently shown to be effective against several coronaviruses, including SARS-CoV-2. Using biochemical and pseudovirus entry assays, we now demonstrate that nitazoxanide interferes with the SARS-CoV-2 spike biogenesis, hampering its maturation at an endoglycosidase H-sensitive stage, and hindering its fusion activity in human cells. Besides membrane fusion during virus entry, SARS-CoV-2 S-proteins in infected cells can also trigger receptor-dependent formation of syncytia, observed in-vitro and in COVID-19 patients tissues, facilitating viral dissemination between cells and possibly promoting immune evasion. Utilizing two different quantitative cell-cell fusion assays, we show that nitazoxanide is effective in inhibiting syncytia formation mediated by different SARS-CoV-2 spike variants in human lung, liver and intestinal cells. The results suggest that nitazoxanide may represent a useful tool in the fight against COVID-19 infections, inhibiting SARS-CoV-2 replication and preventing spike-mediated syncytia formation.

A new grass carp reovirus (GCRV), healthy grass carp reovirus (HGCRV), was isolated from grass carp in 2019. Its complete genome sequence was determined and contained 11 dsRNAs with a total size of 23,688 bp and 57.2 mol% G+C content, encoding 12 proteins. All segments had conserved 5’ and 3’ termini. Sequence comparisons showed that HGCRV was closely related to GCRV-873 (GCRV-I, 69.57–96.71% protein sequence identity) but shared only 22.65–45.85% and 23.37–43.39% identities with GCRV-HZ08 and Hubei grass carp disease reovirus (HGDRV), respectively. RNA-dependent RNA-polymerase (RdRp) protein-based phylogenetic analysis showed that HGCRV clustered with Aquareovirus-C (AqRV-C) prior to joining a branch common with other aquareoviruses. Further analysis using VP6 amino acid sequences from Chinese GCRV strains showed that HGCRV was in the same evolutionary cluster as GCRV-I. Thus, HGCRV could be a new GCRV isolate of GCRV-I but is distantly related to other known GCRVs. Grass carp infected with HGCRV did not exhibit signs of hemorrhage. Interestingly, the isolate induced a typical cytopathic effect in fish cell lines, such as infected cell shrank, apoptosis, and plague-like syncytia. Further analysis showed that HGCRV could proliferate in grass carp liver (L28824), gibel carp brain (GiCB), and other fish cell lines, reaching a titer of up to 7.5 × 104copies/μL.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a ribonucleic acid–based (RNA-based) lineage B β-coronavirus characterized by 10-20 times higher infectivity and transmissibility even across species than previous coronaviruses. The significant infectivity rate of SARS-CoV-2 is due to its different host cell entry mechanisms that are mainly via angiotensin-converting enzyme 2 (ACE2) receptors contrasting earlier coronaviruses that used mainly the endosomal route. Due to the widespread distribution of ACE2 receptors throughout our body, various routes of infectivity are possible, highlighting the necessity of employing multiple forms of protection besides face masks to limit inter-human transmissibility. SARS-CoV-2 exhibits other remarkable features such as the ability to escape the immune system repeated genomic mutations that make it difficult to design a vaccine to address all viral strains and form huge host cell syncytia leading to massive tissue destruction. If we accept SARS-CoV-2 primary reservoir from bats, we should investigate the routes of viral inter-species propagation. In this article, a new theory is proposed- that the dissemination of the virus from the bats to other species and humans could have been possible via an insect vector, as insects possess significant amounts of both ACE2 receptors and a disintegrin and metalloprotease 17 (ADAM-17) enzymes that are essential for virus infectivity. .

Sargassum fusiforme, a plant used as a medicine and food, is regarded as a marine vegetable and health supplement to improve life expectancy. Here, we demonstrate that S. fusiforme extract (SFE) has antiviral effects against respiratory syncytial virus (RSV) in vitro and in vivo mouse model. Treatment of HEp2 cells with a non-cytotoxic concentration of SFE significantly reduced RSV replication, RSV-induced cell death, RSV gene transcription, RSV protein synthesis, and syncytium formation. Moreover, oral inoculation of SFE significantly improved RSV clearance from the lungs of BALB/c mice. Interestingly, the phenolic compounds eicosane, docosane, and tetracosane were identified as active components of SFE. Treatment with a non-cytotoxic concentration of these three components elicited similar antiviral effects against RSV infection as SFE in vitro. Together, these results suggest that SFE and its potential components are a promising natural antiviral agent candidate against RSV infection.

The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination., SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects (syncytium formation). We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity but greatly enhanced syncytium-forming ability. Notably, the D614G change found in globally predominant SARS-CoV-2 strains increased infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, these two features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. Since proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both host proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCE The development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function, and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here, we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity, and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, a process that contributes to the destruction of the virus-infected cell.

In this study, the lumpy skin disease virus was isolated on the lamb testis cell culture for the first time inIraq. Forty skin nodules were collected from cows suspected with lumpy skin disease (LSD) in Baghdadgovernorate. Real-time PCR detected LSD virus in 100% of these skin nodules. After that, the lamb testiscell culture was prepared for virus isolation. The prepared samples (0.5 ml) were inoculated into preparedlamb testis cell culture. Cytopathic effects (CPE) of the virus have appeared after 24hr post-inoculation andcompleted within 72hr. The noticed CPE were cell rounding, aggregation of cells, syncytia formation, anddetached of the cells from the cell sheet. Fluids of lamb testis cell culture were examined by the real-timePCR to confirm that the observed CPE was due to the LSD virus. Cell culture fluids were positive by thereal-time PCR (100%). Real-time PCR was sensitive for the detection of the LSD virus DNA in skin nodules,and it was a suitable confirmatory diagnosis tool of the virus in the tissue culture fluid. Finally, the locallyisolated LSD virus in this study is valuable in future studies for making a vaccine to control the disease.

Cytopathic Effect (CPE) refers to the structural changes of host cells caused by virus invasion. Infection with the virus causes the host cell to lyse, or when the cell dies because it cannot replicate. Both of these effects occur due to CPE. If the virus causes these morphological changes in the host cell, it is said to be cytopathic. Common examples of CPE include rounding of infected cells, fusion with neighboring cells to form syncytia, and the appearance of nuclear or cytoplasmic inclusions. EPC and other changes in cell morphology are just a few of the many effects of cytocidal viruses.

Despite occasional reports of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy, the question of placental infection and its consequences for the newborn remain unanswered. Herein, we analyzed the placentas of 31 coronavirus disease 2019-positive mothers by reverse transcriptase PCR, immunohistochemistry, and in situ hybridization. Only one case of placental infection was detected, which was associated with intrauterine demise of the fetus. Differentiated primary trophoblasts were then isolated from nonpathologic human placentas at term, differentiated, and exposed to SARS-CoV-2 virions. Unlike for positive control cells Vero E6, the virus inside cytotrophoblasts and syncytiotrophoblasts or in the supernatant 4 days after infection was undetectable. As a mechanism of defense, we hypothesized that trophoblasts at term do not express angiotensin-converting enzyme 2 and transmembrane protease serine 2 (TMPRSS2), the two main host membrane receptors for SARS-CoV-2 entry. The quantification of these proteins in the placenta during pregnancy confirmed the absence of TMPRSS2 at the surface of the syncytium. Surprisingly, a transiently induced experimental expression of TMPRSS2 did not allow the entry or replication of the virus in differentiated trophoblasts. Altogether, these results underline that trophoblasts are not likely to be infected by SARS-CoV-2 at term, but raise concern about preterm infection.

To the Editor An elegant study reported dysmorphic cells and syncytia in the deceased's lungs for COVID-19. The authors reasonably considered that most of these syncytia-forming cells were pneumocytes, as identified by specific biomarkers. However, cellular dysmorphism and syncytia are pathological features common in other respiratory infections caused by different viruses, including the human respiratory syncytial virus (HRSV) and Epstein-Barr virus (EBV), as correctly documented...

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects. We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced the ability to form lethal syncytia. Notably, the D614G change found in globally predominant SARS-CoV-2 strains restored infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, two unique features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent.IMPORTANCEThe development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, which contributes to the destruction of the virus-infected cell.

Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion, but also fusion-from-without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during Covid-19 can proceed at low levels of S protein and may not be effectively prevented by antibodies., Graphical Abstract

Respiratory syncytial virus (RSV) is a common, contagious infection of the lungs and the respiratory tract. RSV is characterized by syncytia, which are multinuclear cells created by cells that have fused together. We use a mathematical model to study how different assumptions about the viral production and lifespan of syncytia change the resulting infection time course. We find that the effect of syncytia on viral titer is only apparent when the basic reproduction number for infection via syncytia formation is similar to the reproduction number for cell free viral transmission. When syncytia fusion rate is high, we find the presence of syncytia can lead to slowly growing infections if viral production is suppressed in syncytia. Our model provides insight into how the presence of syncytia can affect the time course of a viral infection.

Development of specific antivirals is an urgent unmet need for SARS-coronavirus 2 (SARS-CoV-2) infections. This study focuses on host proteases that proteolytically activate the SARS-CoV-2 spike protein, critical for its fusion after binding to angiotensin-converting enzyme 2 (ACE2), as antiviral targets. We first validated cleavage at a putative furin substrate motif at SARS-CoV-2 spike by expressing it in VeroE6 cells and found prominent syncytium formation. Both cleavage and syncytium were abolished by treatment with furin inhibitors decanoyl-RVKR-chloromethylketone (CMK) and naphthofluorescein but not by transmembrane protease serine 2 (TMPRSS2) inhibitor camostat. CMK and naphthofluorescein showed antiviral effects in SARS-CoV-2-infected cells by decreasing viral production and cytopathic effects. Further analysis revealed that, similar to camostat, CMK blocks virus entry, but it further suppresses the cleavage of spike and syncytium. Naphthofluorescein instead acts primarily by suppressing viral RNA transcription. Therefore, furin inhibitors may become promising antivirals for prevention and treatment of SARS-CoV-2 infections., Graphical Abstract, Highlights ● The furin cleavage site in the SARS-CoV-2 spike protein mediates syncytium formation. ● The SARS-CoV-2 spike-mediated syncytium is suppressed by specific furin inhibitors. ● Furin inhibitors block SARS-CoV-2 viral entry and viral replication. ● Furin inhibitors are potential antivirals for SARS-CoV-2 infection and pathogenesis., Development of effective antivirals is an urgent unmet need for SARS-CoV-2 infections. Cheng et al. find that the cleavage of the furin substrate site in the viral spike protein is critical for viral production and cytopathic effects. Two inhibitors targeting furin are potential antivirals to control SARS-CoV-2 infection and pathogenesis.

Nipah virus (NiV) is a zoonotic bat henipavirus in the family Paramyxoviridae. NiV is deadly to humans, infecting host cells by direct fusion of the viral and host cell plasma membranes. This membrane fusion process is coordinated by the receptor-binding attachment (G) and fusion (F) glycoproteins. Upon G-receptor binding, F fuses membranes via a cascade that sequentially involves F-triggering, fusion pore formation, and viral or genome entry into cells. Using NiV as an important paramyxoviral model, we identified two novel regions in F that modulate the membrane fusion cascade. For paramyxoviruses and other viral families with class I fusion proteins, the heptad repeat 1 (HR1) and HR2 regions in the fusion protein prefusion conformation bind to form a six-helix bundle in the postfusion conformation. Here, structural comparisons between the F prefusion and postfusion conformations revealed that a short loop region (N1) undergoes dramatic spatial reorganization and a short alpha helix (N4) undergoes secondary structural changes. The roles of the N1 and N4 regions during the membrane fusion cascade, however, remain unknown for henipaviruses and paramyxoviruses. By performing alanine scanning mutagenesis and various functional analyses, we report that specific residues within these regions alter various steps in the membrane fusion cascade. While the N1 region affects early F-triggering, the N4 region affects F-triggering, F thermostability, and extensive fusion pore expansion during syncytium formation, also uncovering a link between F-G interactions and F-triggering. These novel mechanistic roles expand our understanding of henipaviral and paramyxoviral F-triggering, viral entry, and cell-cell fusion (syncytia), a pathognomonic feature of paramyxoviral infections. IMPORTANCE Henipaviruses infect bats, agriculturally important animals, and humans, with high mortality rates approaching ∼75% in humans. Known human outbreaks have been concentrated in Southeast Asia and Australia. Furthermore, about 20 new henipaviral species have been recently discovered in bats, with geographical spans in Asia, Africa, and South America. The development of antiviral therapeutics requires a thorough understanding of the mechanism of viral entry into host cells. In this study, we discovered novel roles of two regions within the fusion protein of the deadly henipavirus NiV. Such roles were in allowing viral entry into host cells and cell-cell fusion, a pathological hallmark of this and other paramyxoviruses. These novel roles were in the previously undescribed N1 and N4 regions within the fusion protein, modulating early and late steps of these important processes of viral infection and henipaviral disease. Notably, this knowledge may apply to other henipaviruses and more broadly to other paramyxoviruses.

Introduction Most of the typical chemokine receptors (CKRs) have been identified as coreceptors for a variety of human and simian immunodeficiency viruses (HIVs and SIVs). This study evaluated CCRL2 to examine if it was an HIV/SIV coreceptor. Methods The Human glioma cell line, NP-2, is normally resistant to infection by HIV and SIV. The cell was transduced with amplified cluster of differentiation 4 (CD4) as a receptor and CCR5, CXCR4 and CCRL2 as coreceptor candidates to produce NP-2/CD4/coreceptor cells (). The cells were infected with multiplicity of infection (MOI) 1.0. Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa staining. Proviral DNA was detected by polymerase chain reaction (PCR), and reverse transcriptase (RT) activity was measured. Results IFA detected viral antigens of the primary isolates, HIV-1HAN2 and HIV-2MIR in infected NP-2/CD4/CCRL2 cells, indicated CCRL2 as a functional coreceptor. IFA results were confirmed by the detection of proviral DNA and measurement of RT-activity in the spent cell supernatants. Additionally, MGC was detected in HIV-2MIR-infected NP-2/CD4/CCCRL2 cells. HIV-2MIR were found more potent users of CCRL2 than HIV-1HAN2. Moreover, GWAS studies, gene ontology and cell signaling pathways of the HIV-associated genes show interaction of CCRL2 with HIV/SIV envelope protein. Conclusions In vitro experiments showed CCRL2 to function as a newly identified coreceptor for primary HIV-2 isolates conveniently. The findings contribute additional insights into HIV/SIV transmission and pathogenesis. However, its in vivo relevance still needs to be evaluated. Confirming in vivo relevance, ligands of CCRL2 can be investigated as potential targets for HIV entry-inhibitor drugs.

This study compared different detection methods of human/simian immunodeficiency virus (HIV/SIV) infections in the cell line systems; notably, i) Indirect immunofluorescence assay (IFA), ii) integrated proviral DNA detection, iii) detection of syncytia, iv) measurement of reverse transcriptase (RT) activity. RTs of various retroviruses require cations, including Mg2+, Mn2+, Ni2+, and Cu2+, for their enzyme-activities. The study further compared the roles of Mg2+ and Mn2+ as cofactors for RT activities of freshly harvested HIV-1, HIV-2, and SIV. The NP-2/CD4/coreceptor cells were seeded for overnight and infected with viral inoculums at a multiplicity of infection (MOI) 1.0. The cells were passaged regularly in a 2-3 days interval and maintained up to 2 weeks. Infected cells were detected by indirect immunofluorescence assay (IFA). Multinucleated giant cells (MGC) in syncytia were quantified by Giemsa-staining. Proviral DNA was detected by PCR, and reverse transcriptase (RT) activity was measured. Two different cations, Mg2+ and Mn2+ were used as cofactors for RT assay. We found all the strains of HIV-1, HV-2 and SIV to infection in the cell line conveniently. IFA had identified all the viral infections in the infected cells. Proviral DNA detection, syncytia formation was observed in the infected cells. We found a better performance of Mg2+ as cofactor over Mn2+ in RT assay for HIV-1, HIV-2, SIV. Different four detection techniques of HIV/SIV infections show high level of agreement in the NP-2-based cell line system. Mg2+ remains a better cofactor for RT.

Simple Summary Bovine respiratory syncytial virus (BRSV) is an important pathogen of both dairy and beef cattle, and causes huge economic losses annually across the world. This study reports the identification, isolation, and molecular characterization of a new BRSV (subgroup III) strain collected from respiratory distressed cattle in Turkey. The three field isolates obtained showed 100% similarity to each other at the nucleotide (nt) level and were found to be 99.49% and 99.22% identical to another Turkish strain—KY499619—at both (nt) and amino acid (aa) levels, respectively. They were also 97.43% (nt) and 98.44% (aa) similar to the American reference strain KU159366. This important information will inform Turkish BRSV diagnostic and control strategies, as well as highlight the urgent need to better understand the burden that BRSV is placing on the Turkish agricultural sector. Abstract Bovine respiratory disease (BRD) is a huge economic burden on the livestock industries of countries worldwide. Bovine respiratory syncytial virus (BRSV) is one of the most important pathogens that contributes to BRD. In this study, we report the identification and first isolation, with molecular characterization, of a new BRSV strain from lung specimens of three beef cows in Turkey that died from respiratory distress. After the screening of lung tissues for BRD-associated viruses using a multiscreen antigen-ELISA, a BRSV antigen was detected. This was then confirmed by real-time RT-PCR specific for BRSV. Following confirmation, virus isolation was conducted in MDBK cell cultures and clear CPE, including syncytia compatible with BRSV, were detected. RT-nested PCR, using F gene-specific primers, was performed on the cultured isolates, and the products were sequenced and deposited to Genbank with accession numbers MT179304, MT024766, and MT0244767. Phylogenetic analysis of these sequences indicated that the cattle were infected with BRSV from subgroup III and were closely related to previously identified American and Turkish strains, but contained some amino acid and nucleotide differences. This research paves the way for further studies on the molecular characteristics of natural BRSV isolates, including full genome analysis and disease pathogenesis, and also contributes to the development of robust national strategies against this virus.

A metal nanoparticle composite, namely TPNT1, which contains Au-NP (1 ppm), Ag-NP (5 ppm), ZnO-NP (60 ppm) and ClO2 (42.5 ppm) in aqueous solution was prepared and characterized by spectroscopy, transmission electron microscopy, dynamic light scattering analysis and potentiometric titration. Based on the in vitro cell-based assay, TPNT1 inhibited six major clades of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with effective concentration within the range to be used as food additives. TPNT1 was shown to block viral entry by inhibiting the binding of SARS-CoV-2 spike proteins to the angiotensin-converting enzyme 2 (ACE2) receptor and to interfere with the syncytium formation. In addition, TPNT1 also effectively reduced the cytopathic effects induced by human (H1N1) and avian (H5N1) influenza viruses, including the wild-type and oseltamivir-resistant virus isolates. Together with previously demonstrated efficacy as antimicrobials, TPNT1 can block viral entry and inhibit or prevent viral infection to provide prophylactic effects against both SARS-CoV-2 and opportunistic infections.