Native Virus Particles Research Articles

Viral Protein VP1 Virus-like Particles (VLP) of CVB4 Induces Protective Immunity against Lethal Challenges with Diabetogenic E2 and Wild Type JBV Strains in Mice Model.

Several epidemiological studies demonstrated that coxsackievirus B4 (CVB4) causes viral pancreatitis and can ultimately result in type 1 diabetes mellitus (T1D). Prevention of CVB4 infection is therefore highly desirable. There is currently no vaccine or antiviral therapeutic reagent in clinical use. VLP are structurally similar to native virus particles and therefore are far better immunogens than any other subunit vaccines. Many studies have shown the potential of capsid protein VP1 on providing protective effects from different viral strains. In this study, we contributed towards the development of a CVB4 VLP-based vaccine from the total protein VP1 of the diabetogenic CVB4E2 strain and assessed whether it could induce a protective immunity against both the wild-type CVB4JBV and the diabetogenic CVB4E2 strains in mice model. Serum samples, taken from mice immunized with VLP, were assayed in vitro for their anti-CVB4 neutralizing activity and in vivo for protective activity. We show that VLP vaccine generates robust immune responses that protect mice from lethal challenges. Results demonstrate that CVB4 VP1 capsid proteins expressed in insect cells have the intrinsic capacity to assemble into non-infectious VLP, which afforded protection from CVB4 infection to mice when used as a vaccine.

Perturbation-HDX reveals glycosylation sites as weakest links in dengue capsid quaternary structure.

Dengue virus (DENV) is a mosquito-borne, icosahedral flavivirus that represents a significant threat to human health worldwide. Vaccine development for dengue has long eluded scientists due to serotype and strain diversity associated with altered viral capsid dynamics. Despite being largely conserved, dengue strains show distinctive reversible fluctuations referred to as ‘breathing’. DENV capsids have T=3 icosahedral symmetry, consisting of 180 copies of out envelope (E) protein. E-protein dimer contacts confer stability of the native virus particle. To localize and rank the strengths of E-protein interfaces on the surface, we carried out urea and temperature perturbation of DENV particles analyzed using amide hydrogen-deuterium exchange mass spectrometry. Our results highlight the weakest links on the virion surface. These sites are spanning the glycosylation sites of dengue. We have isolated and characterized the viable deglycosylated mutant which shows a different temperature stability profile. These sensitive sites are prime targets for drug development and predict sites that may change due to the selective pressure that impact viral evolution.

Reactivity of Viral Nucleic Acids with Chlorine and the Impact of Virus Encapsidation.

Free chlorine disinfection is widely applied to inactivate viruses by reacting with their biomolecules, which include nucleic acids, proteins, and lipids. Knowing the reactivities of viral genomes with free chlorine and the protection that encapsidation provides would ultimately help predict virus susceptibility to the disinfectant. The relative reactivities of different viral genome types and the impact of viral higher order structure with free chlorine are poorly characterized. Here, we studied the reactivity of viral genomes representing four genome types from virus particles with diverse structures, namely, (+)ssRNA (MS2), dsRNA (φ6), ssDNA (φX174), and dsDNA (T3) with free chlorine. We compared the reactivities of these viral nucleic acids when they were suspended in phosphate buffer solutions (naked forms) and when they were in the native virus particles (encapsidated forms). The reactivities of nucleic acids were tracked by polymerase chain reaction (PCR)-based assays. The naked dsDNA of T3 was the least reactive with free chlorine, with an average second order rate constant normalized by the number of bases in the measured regions (in M-1 s-1 b-1) that was 34×, 65×, and 189× lower than those of the dsRNA of φ6, ssRNA of MS2, and ssDNA of φX174, respectively. Moreover, different regions in the ssRNA genome of MS2 and the dsRNA genome of φ6 exhibited statistically different reaction kinetics. The genomes within virus particles reacted slower than the naked genomes overall, but the extent of these differences varied among the four viruses. The results on viral nucleic acid reactivity help explain different susceptibilities of viruses to inactivation by free chlorine and also provide a valuable comparison of the susceptibilities of different nucleic acids to oxidants.

Nucleic Acid Photolysis by UV254 and the Impact of Virus Encapsidation.

Determining the influence of higher order structure on UVC photolysis will help inform predictions of nucleic acid fate and microorganism inactivation. We measured the direct UV254 photolysis kinetics of four model viral genomes composed of single-stranded and double-stranded RNA (ssRNA and dsRNA, respectively), as well as single-stranded and double-stranded DNA (ssDNA and dsDNA, respectively), in ultrapure water, in phosphate buffered saline (PBS), and encapsidated in their native virus particles. The photolysis rate constants of naked nucleic acids measured by qPCR (RT-qPCR for RNA) and normalized by the number of bases measured in a particular sequence exhibited the following trend: ssDNA > ssRNA ≈ dsDNA > dsRNA. In PBS, naked ssRNA bases reacted, on average, 24× faster than the dsRNA bases, whereas naked ssDNA bases reacted 4.3× faster than dsDNA bases. Endogenous indirect photolysis involving 1O2 and ·OH was ruled out as a major contributing factor in the reactions. A comparison of our measured rate constants with rate constants reported in the literature shows a general agreement among the nucleic acid UV254 direct photolysis kinetics. Our results underscore the high resistance of dsRNA to UVC photolysis and demonstrate the role that nucleic acid structure and solution chemistry play in photoreactivity.

Marburg virus-like particles produced in insect cells induce neutralizing antibodies in rhesus macaques.

Marburg virus (MARV), which is one of the most virulent agents in the world, causes lethal haemorrhagic fever in humans and nonhuman primates (NHPs) with a mortality rate of up to 90%. Currently, there is no effective treatment or approved vaccine for MARV for human use to control disease outbreak and spread. Virus-like particles (VLPs), which are morphologically identical to the native infectious virus particle, are efficacious as vaccines against many viruses, including human papilloma virus (HPV), porcine circovirus (PCV) type 2 and hepatitis B virus (HBV). In this study, we generated MARV virus-like particles (VLPs) by co-expressing a glycoprotein (GP) and matrix protein (VP40) using the baculovirus expression system. Rhesus macaques vaccinated with MARV VLPs mixed with adjuvant Poria cocos polysaccharides (PCP-II) produced a GP-specific IgG titer of up to 1:1280 and virus-neutralizing antibody titers that reached 1:320. MARV VLPs also elicited interferon-γ (IFN-γ) and interleukin-4 (IL-4) secretion associated with T-helper 1 cell (Th1)- and T-helper 2 cell (Th2)-mediated immunity, as detected using enzyme-linked immunospot (ELISpot) assays. These data indicate that MARV VLPs mixed with adjuvant PCP-II have excellent immunogenicity in rhesus macaques and may be a promising candidate vaccine against MARV.

Generation of recombinant protein shells of Johnson grass chlorotic stripe mosaic virus in tobacco plants and their use as drug carrier

The development and use of virus-like particles (VLPs) is a growing field with a powerful potential in generation of nanoparticles. In the present study we have attempted to generate and use empty shells of Johnson grass chlorotic stripe mosaic virus (JgCSMV, a member of the genus Aureusvirus, family Tombusviridae) as VLP nanoparticles for drug loading. In order to successfully produce recombinant JgCSMV-derived VLPs, we followed an approach based on cloning of the JgCSMV CP gene into pBI121 vector and introduction of the latter into Agrobacterium rhizogenes and transformation of tobacco cells for coat protein expression. Expression in tobacco tissue was demonstrated in transformed hairy roots as a model system. Recombinant VLPs were purified, analyzed by immune assay and visulalized by electron microscopy. Next, we explored the possibility of using JgCSMV-derived VLPs as a nanocontainer for loading the anticancer drug doxorubicin (DOX), taking advantage of the reversible swelling of VLPs in vitro. The results showed that transformed hairy roots produced high levels of the recombinant protein that readily assembled to form empty shells with overall structure similar to native virus particles. In addition, we demonstrated that JgCSMV-VLPs could function as vehicles able to load the chemotherapeutic drug doxorubicin. To our knowledge, this is the first research addressing the question of how this icosahedral virus (JgCSMV) can be used for the production of nanocontainers for biomedical applications.

Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents.

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1038-1045, 2016.

Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential “differentiating infected from vaccinated animals” (DIVA) vaccine candidates for the surveillance and eradication of PPR.

Assembly of Sesbania mosaic virus: Role of the coat protein N-terminal R domain

Coat proteins of several isometric viruses consist of two domains, a disordered N-terminal R-domain consisting of several positively charged residues and a shell (S) domain characterized by a jelly roll β-barrel structure. The three-dimensional structure of Sesbania mosaic virus (SeMV), a T=3 plant virus, has been determined at 3 Å resolution. The full length coat protein, when expressed in E. coli, assembles into T=3 icosahedral shells (VLPs) resembling native virus particles. In the present investigations, the role of N-terminal R domain in the assembly of VLPs was explored by replacing the R domain with a presumably globular domain (SeMV-P10) and other intrinsically disordered (SeMV-P8, and SeMV-VPg) SeMV encoded domains. The R domain was also replaced with the non-viral globular B-domain of Staphyloccocus aureus protein A. These domains were of nearly the same size as that of the R-domain. Most of the chimeric coat proteins, when expressed in E.coli, formed VLPs, which could be purified by ultra-centrifugation. The purified VLPs were examined by transmission electron microscopy (TEM), which suggested that a fraction of the expressed proteins could assemble into T=3 VLPs, although often, the particles were heterogeneous. Interestingly, the SeMV NΔ65B CP could also be purified by Ni-NTA chromatography as a dimer which assembled into T=1 VLPs under crystallization conditions. The structure of NΔ65B-CP VLPs revealed that the assembled particles were devoid of divalent metal ions at the canonical site and there was no density corresponding to the B domain. However, the S domain could be superimposed on that of SeMV NΔ65VLPs determined earlier. The other VLPs- SeMVNΔ65P10 CP, SeMVNΔ65P8 CP and SeMVNΔ65VPg could not be crystallized because of their heterogeneity. These studies suggest a subtle interplay between the length, sequence and structure of the R-domain polypeptide and the assembly of particles.

Graphical analysis of flow cytometer data for characterizing controlled fluorescent protein display on λ phage

As native virus particles typically cannot be resolved using a flow cytometer, the general practice is to use fluorescent dyes to label the particles. In this work, an attempt was made to use a common commercial flow cytometer to characterize a phage display strategy that allows for controlled levels of protein display, in this case, eGFP. To achieve this characterization, a number of data processing steps were needed to ensure that the observed phenomena were indeed capturing differences in the phages produced. Phage display of eGFP resulted in altered side scatter and fluorescence profile, and sub-populations could be identified within what would otherwise be considered uniform populations. Surprisingly, this study has found that side scatter may be used in the future to characterize the display of nonfluorescent proteins.

A Human PrM Antibody That Recognizes a Novel Cryptic Epitope on Dengue E Glycoprotein

Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.

Vaccination with coxsackievirus B3 virus-like particles elicits humoral immune response and protects mice against myocarditis

Coxsackievirus B3 (CVB3), along with other enteroviruses, is involved in about 50% of myocarditis cases and in the pathogenesis of dilated cardiomyopathy. Prevention of CVB3 infection is therefore highly desirable. Virus-like particles (VLPs) are structurally similar to native virus particles and therefore are far better immunogens than any other subunit vaccines. Recombinant baculoviruses carrying either the intact, entire coding region of CVB3 or the four individual coding regions for virus proteins 1-4 (VP1-4) were constructed. Expression of CVB3 capsid proteins in insect cells infected with recombinant baculovirus was detected by immunofluorescence and Western blot analysis. Sucrose gradient ultracentrifugation fractions of the infected cell lysates contained peaks of CVB3 antigen with an approximate density of 1.14g/ml. Electron microscopy demonstrated the presence of VLP in these sucrose fractions. The CVB3 VLP was non-infectious in tissue culture. SWR (H-2(q)) mice vaccinated with CVB3 VLP developed antibodies to CVB3 capsid proteins after the first boost. Antibody titre was comparable to the level induced by an attenuated CVB3 vaccine. Vaccinated animals were protected from myocarditis when subsequently challenged with cardiovirulent CVB3 (chimera-2). Vaccination with VLP produced from the complete CVB3 coding region gave a greater immune response and afforded better protection than with VLP from the quadruple expression vector. These results demonstrate that CVB3 capsid proteins expressed in insect cells have the intrinsic capacity to assemble into non-infectious VLP, which afforded protection from CVB3 infection to mice when used as a vaccine.

Mutant Plant Viruses with Cell Binding Motifs Provide Differential Adhesion Strengths and Morphologies

The ability of Tobacco mosaic virus (TMV) to tolerate various amino acid insertions near its carboxy terminus is well-known. Typically these inserts are based on antigenic sequences for vaccine development with plant viruses as carriers. However, we determined that the structural symmetries and the size range of the viruses could also be modeled to mimic the extracellular matrix proteins by inserting cell-binding sequences to the virus coat protein. The extracellular matrix proteins play important roles in guiding cell adhesion, migration, proliferation, and stem cell differentiation. Previous studies with TMV demonstrated that the native and phosphate-modified virus particles enhanced stem cell differentiation toward bone-like tissues. Based on these studies, we sought to design and screen multiple genetically modified TMV mutants with reported cell adhesion sequences to expand the virus-based tools for cell studies. Here, we report the design of these mutants with cell binding amino acid motifs derived from several proteins, the stabilities of the mutants against proteases during purification and storage, and a simple and rapid functional assay to quantitatively determine adhesion strengths by centrifugal adhesion assay. Among the mutants, we found that cells on TMV expressing RGD motifs formed filopodial extensions with weaker attachment profiles, whereas the cells on TMV expressing collagen I mimetic sequence displayed little spreading but higher attachment strengths.

Virus Capsid Coating of Gold Nanoparticles via Cysteine–Au Interactions and Their Effective Cellular Uptakes

Abstract This manuscript describes the synthesis of virus capsid protein-coated Au nanoparticle (VP–AuNP) without the use of the inherent self-assembly of virus proteins into virus particles. Covalent binding between Au and cysteines in the virus proteins keep the cell-surface binding sites on the external surface. Based on this method, various sizes of VP–AuNP can be created in a similar manner to native virus particles. We clarified the optimum size of the VP–AuNP for internalization into cells.

Epitope mapping and cross-reactivity analysis of the monoclonal antibodies against hexon protein of human adenovirus type 3

Human adenovirus 3 (HAdV-3) occurs epidemically in China in recent years. It can cause life-threatening infection in young children and immunocompromised patients. Development of reliable diagnostic reagents for adenovirus infection is important in surveillance and control of the infection. In this study, three monoclonal antibodies (MAbs) 5D4, 7B2 and 3D10 were generated against the recombinant HAdV-3 hexon protein. Western-blots analysis showed that all three MAbs recognized the native HAdV-3 hexon protein, but only 5D4 reacted with the native HAdV-3 virus particles by indirect enzyme-linked immunosorbent assay (ELISA). The epitopes recognized by these MAbs were further investigated by epitope analysis with a series of peptides of the hexon protein. The epitope for 3D10 was located to the sequence 339GVLAGQASQLNA 350 and present in all the currently recognized serotypes of human adenovirus. The MAb 7B2 showed broad reactivity with the peptides from group B, group E, and most serotypes of group D, but not with those from group A, C and F adenoviruses. The epitope of 5D4, located in the hypervariable region 3 (HVR3), was only shared by HAdV-3 and HAdV-7. Further ELISA demonstrated that 5D4 only recognized HAdV-3 and HAdV-7 virus particles. The MAbs and the identified epitopes may play roles in clinical serotype-specific diagnosis, viral structure analysis, as well as in the identification of new serotypes of human adenovirus.

Purification and analysis of polyhistidine-tagged human parvovirus B19 VP1 and VP2 expressed in insect cells

Human parvovirus B19 is an autonomously replicating human pathogen with a specific tropism for human erythroid progenitor cells. There is an interest in producing empty nucleocapsids of B19 as they can be used as tools in molecular biology and diagnostics. Native B19 virus particles are formed from two structural viral proteins, VP1 and VP2. The VP2 protein alone is able to self assemble and consequently form virus-like particles (VLPs) in heterologous expression systems. Purification of recombinant VLPs has been conducted using various traditional methods. These include laborious and time-consuming, e.g. cesium chloride or sucrose gradient ultracentrifugation steps, allowing limited working volumes to be processed. Therefore, an alternative purification method enabling process scale-up was developed and evaluated. Polyhistidine-tagged versions of B19 VP1 and VP2 capsid proteins were engineered and produced using the baculovirus expression system. The recombinant protein products were purified by immobilized metal-ion affinity chromatography (IMAC) and analyzed by SDS-PAGE, immunoblotting, electron microscopy, and enzyme-linked immunosorbent assays. Further, the immunological properties of the recombinant proteins were evaluated. The results showed that the VP2 fusion protein assembled into capsid-like structures and that both VP1 and VP2 following purification by IMAC have potential as antigens for diagnosis of a B19 infection.

Structure of recombinant capsids formed by the β-annulus deletion mutant — rCP (Δ48–59) of Sesbania mosaic virus

A unique feature of several T = 3 icosahedral viruses is the presence of a structure called the β-annulus formed by extensive hydrogen bonding between protein subunits related by icosahedral three-fold axis of symmetry. This unique structure has been suggested as a molecular switch that determines the T = 3 capsid assembly. In order to examine the importance of the β-annulus, a deletion mutant of Sesbania mosaic virus coat protein in which residues 48–59 involved in the formation of the β-annulus were deleted retaining the rest of the residues in the amino terminal segment (rCP (Δ48–59)) was constructed. When expressed in Escherichia coli, the mutant protein assembled into virus like particles of sizes close to that of the wild type virus particles. The purified capsids were crystallized and their three dimensional structure was determined at 3.6 Å resolution by X-ray crystallography. The mutant capsid structure closely resembled that of the native virus particles. However, surprisingly, the structure revealed that the assembly of the particles has proceeded without the formation of the β-annulus. Therefore, the β-annulus is not essential for T = 3 capsid assembly as speculated earlier and may be formed as a consequence of the particle assembly. This is the first structural demonstration that the virus particle morphology with and without the β-annulus could be closely similar.

Location and Role of Free Cysteinyl Residues in the Sindbis Virus E1 and E2 Glycoproteins

Sindbis virus is a single-stranded positive-sense RNA virus. It is composed of 240 copies of three structural proteins: E1, E2, and capsid. These proteins form a mature virus particle composed of two nested T=4 icosahedral shells. A complex network of disulfide bonds in the E1 and E2 glycoproteins is developed through a series of structural intermediates as virus maturation occurs (M. Mulvey and D. T. Brown, J. Virol. 68:805-812, 1994; M. Carleton et al., J. Virol. 71:1558-1566, 1997). To better understand the nature of this disulfide network, E1 and E2 cysteinyl residues were labeled with iodoacetamide in the native virus particle and analyzed by liquid chromatography-tandem mass spectrometry. This analysis identified cysteinyl residues of E1 and E2, which were found to be label accessible in the native virus particle, as well as those that were either label inaccessible or blocked by their involvement in disulfide bonds. Native virus particles alkylated with iodoacetamide demonstrated a 4-log decrease in viral infectivity. This suggests that the modification of free cysteinyl residues results in the loss of infectivity by destabilizing the virus particle or that a rearrangement of disulfide bonds, which is required for infectivity, is blocked by the modification. Although modification of these residues prevented infectivity, it did not alter the ability of virus to fuse cells after exposure to acidic pH; thus, modification of free cysteinyl residues biochemically separated the process of infection from the process of membrane fusion.

Structured particles for antigen presentation

Traditional antiviral vaccines have relied mostly on attenuated or chemically inactivated live viruses. However, the potential problems related either to an incomplete inactivation procedure or to the reversion of an attenuated vaccine strain result in growing concerns for their use in human vaccination approaches. Virus-like particles (VLPs) are based on the expression of virus capsid proteins, which spontaneously assemble in particles structurally similar to native virus particles, without containing viral genetic material. Thus, VLPs represent a non-replicating, non-infectious particulate antigen delivery system able to present conformationally structured viral proteins to the immune system. We have developed a candidate HIV-1 vaccine model based on HIV-1 Pr55gag VLPs (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a HIV-1 isolate of the clade A (HIV-VLPAs). HIV-VLPAs induce in Balb/c mice systemic and mucosal neutralizing antibodies as well as cytotoxic T lymphocytes, by intraperitoneal as well as intranasal administration. HIV-VLPAs efficiently induce maturation and activation of MDDCs which show an enhanced Th1- and Th2-specific cytokine production. In addition, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4+ T cells, in an ex vivo immunization assay. The versatility of the presentation system, together with the strong immunogenicity, make the VLP approach a highly valuable tool for vaccination strategies.

Early endocytosis pathways in SSN-1 cells infected by dragon grouper nervous necrosis virus

Many fish undergo betanodavirus infection. To study the infection process of dragon grouper nervous necrosis virus (DGNNV), native virus and virus-like particles (VLPs) were used to analyse the binding and internalization in SSN-1 cells. The binding of DGNNV and VLPs to SSN-1 cells was demonstrated using Western blotting and immunofluorescence microscopy. As estimated by indirect ELISA, the DGNNV particles bound SSN-1 cells in a dose-dependent manner up to 8 x 10(4) particles per cell. The binding of VLPs was sensitive to neuraminidase and tunicamycin, suggesting that cell-surface sialic acid is involved in binding. The penetration of DGNNV into cells, which was monitored by electron microscopy, appeared to occur mainly via the spherical pit and membrane ruffling pathways. Occasionally, a spherical pit was engulfed by membrane ruffling so as to form a large figure-of-eight-shaped vesicle with an open connection. Our observations suggest that DGNNV utilizes both micro- and macropinocytosis pathways to enter SSN-1 cells.