Intact Virus Research Articles

Quantitative Assessment of the Physical Virus Titer and Purity by Ultrasensitive Flow Virometry

AbstractRapid quantification of viruses is vital for basic research on viral diseases as well as biomedical application of virus‐based products. Here, we report the development of a high‐throughput single‐particle method to enumerate intact viral particles by ultrasensitive flow virometry, which detects single viruses as small as 27 nm in diameter. The nucleic acid dye SYTO 82 was used to stain the viral (or vector) genome, and a laboratory‐built nano‐flow cytometer (nFCM) was employed to simultaneously detect the side‐scatter and fluorescence signals of individual viral particles. Using the bacteriophage T7 as a model system, intact virions were completely discriminated from empty capsids and naked viral genomes. Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses, which could be used for gene delivery, therapeutic products derived from phage cocktails, and infected cell supernatants for veterinary vaccine production.

Quantitative Assessment of the Physical Virus Titer and Purity by Ultrasensitive Flow Virometry.

Rapid quantification of viruses is vital for basic research on viral diseases as well as biomedical application of virus‐based products. Here, we report the development of a high‐throughput single‐particle method to enumerate intact viral particles by ultrasensitive flow virometry, which detects single viruses as small as 27 nm in diameter. The nucleic acid dye SYTO 82 was used to stain the viral (or vector) genome, and a laboratory‐built nano‐flow cytometer (nFCM) was employed to simultaneously detect the side‐scatter and fluorescence signals of individual viral particles. Using the bacteriophage T7 as a model system, intact virions were completely discriminated from empty capsids and naked viral genomes. Successful measurement of the physical virus titer and purity was demonstrated for recombinant adenoviruses, which could be used for gene delivery, therapeutic products derived from phage cocktails, and infected cell supernatants for veterinary vaccine production.

FRET-Based Detection and Quantification of HIV-1 Virion Maturation.

HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.

Virus Mechanics under Molecular Crowding.

Viruses avoid exposure of the viral genome to harmful agents with the help of a protective protein shell known as the capsid. A secondary effect of this protective barrier is that macromolecules that may be in high concentration on the outside cannot freely diffuse across it. Therefore, inside the cell and possibly even outside, the intact virus is generally under a state of osmotic stress. Viruses deal with this type of stress in various ways. In some cases, they might harness it for infection. However, the magnitude and influence of osmotic stress on virus physical properties remains virtually unexplored for single-stranded RNA viruses-the most abundant class of viruses. Here, we report on how a model system for the positive-sense RNA icosahedral viruses, brome mosaic virus (BMV), responds to osmotic pressure. Specifically, we study the mechanical properties and structural stability of BMV under controlled molecular crowding conditions. We show that BMV is mechanically reinforced under a small external osmotic pressure but starts to yield after a threshold pressure is reached. We explain this mechanochemical behavior as an effect of the molecular crowding on the entropy of the "breathing" fluctuation modes of the virus shell. The experimental results are consistent with the viral RNA imposing a small negative internal osmotic pressure that prestresses the capsid. Our findings add a new line of inquiry to be considered when addressing the mechanisms of viral disassembly inside the crowded environment of the cell.

Entry and Disposition of Zika Virus Immune Complexes in a Tissue Culture Model of the Maternal-Fetal Interface.

Zika virus (ZIKV) infections have been associated with an increased incidence of severe microcephaly and other neurodevelopmental disorders in newborn babies. Passive immunization with anti-ZIKV neutralizing antibodies has the potential to become a feasible treatment or prophylaxis option during pregnancy. Prior to clinical use, such antibodies should be assessed for their ability to block ZIKV passage to the fetus. We used human placental and mammalian cell monolayers that express FcRn and laboratory preparations of anti-ZIKV antibodies as a model system to investigate the disposition of ZIKV/antibody immune complexes (ICs) at the maternal-fetal interface. We further characterized solution properties of the ICs to evaluate whether these are related to in vitro effects. We found that both ZIKV and ZIKV envelope glycoprotein can enter and passage through epithelial cells, especially those that overexpress FcRn. In the presence of ZIKV antibodies, Zika virus entry was bimodal, with reduced entry at the lowest (0.3–3 ng/mL) and highest (µg/mL) antibody concentrations. Intermediate concentrations attenuated inhibition or enhanced viral entry. With respect to anti-ZIKV antibodies, we found that their degradation was accelerated when presented as ICs containing increased amounts of ZIKV immunogen. Of the two monoclonal antibodies tested, the preparation with higher aggregation also exhibited higher degradation. Our studies confirm that intact Zika virus and its envelope immunogen have the potential to enter and be transferred across placental and other epithelial cells that express FcRn. Presence of anti-ZIKV IgG antibodies can either block or enhance cellular entry, with the antibody concentration playing a complex role in this process. Physicochemical properties of IgG antibodies can influence their degradation in vitro.

The Structure of the Potato Virus A Particles Elucidated by Small Angle X-Ray Scattering and Complementary Techniques

Potato virus A (PVA) protein coat contains on its surface partially unstructured N-terminal domain of the viral coat protein (CP), whose structural and functional characteristics are important for understanding the mechanism of plant infection with this virus. In this work, we investigated the properties and the structure of intact PVA and partially trypsinized PVAΔ32 virions using small-angle X-ray scattering (SAXS) and complimentary methods. It was shown that after the removal of 32 N-terminal amino acids of the CP, the virion did not disintegrate and remained compact, but the helical pitch of the CP packing changed. To determine the nature of these changes, we performed ab initio modeling, including the multiphase procedure, with the geometric bodies (helices) and restoration of the PVA structure in solution using available high-resolution structures of the homologous CP from the PVY potyvirus, based on the SAXS data. As a result, for the first time, a low-resolution structure of the filamentous PVA virus, both intact and partially degraded, was elucidated under conditions close to natural. The far-UV circular dichroism spectra of the PVA and PVAΔ32 samples differed significantly in the amplitude and position of the main negative maximum. The extent of thermal denaturation of these samples in the temperature range of 20-55°C was also different. The data of transmission electron microscopy showed that the PVAΔ32 virions were mostly rod-shaped, in contrast to the flexible filamentous particles typical of the intact virus, which correlated well with the SAXS results. In general, structural analysis indicates an importance of the CP N-terminal domain for the vital functions of PVA, which can be used to develop a strategy for combating this plant pathogen.

Amplification-Free Detection of Viruses in Minutes using Single-Particle Imaging and Machine Learning

The increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the current COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive viral diagnostic methods. To address this need, we developed a novel method to rapidly detect and identify intact virus particles using wide-field fluorescence imaging, advanced image analysis and deep learning. Our method utilises cation-mediated labelling of enveloped viruses, a labelling method not specific to any virus.

Biological and Clinical Consequences of Integrin Binding via a Rogue RGD Motif in the SARS CoV-2 Spike Protein.

Although ACE2 (angiotensin converting enzyme 2) is considered the primary receptor for CoV-2 cell entry, recent reports suggest that alternative pathways may contribute. This paper considers the hypothesis that viral binding to cell-surface integrins may contribute to the high infectivity and widespread extra-pulmonary impacts of the SARS-CoV-2 virus. This potential is suggested on the basis of the emergence of an RGD (arginine-glycine-aspartate) sequence in the receptor-binding domain of the spike protein. RGD is a motif commonly used by viruses to bind cell-surface integrins. Numerous signaling pathways are mediated by integrins and virion binding could lead to dysregulation of these pathways, with consequent tissue damage. Integrins on the surfaces of pneumocytes, endothelial cells and platelets may be vulnerable to CoV-2 virion binding. For instance, binding of intact virions to integrins on alveolar cells could enhance viral entry. Binding of virions to integrins on endothelial cells could activate angiogenic cell signaling pathways; dysregulate integrin-mediated signaling pathways controlling developmental processes; and precipitate endothelial activation to initiate blood clotting. Such a procoagulant state, perhaps together with enhancement of platelet aggregation through virions binding to integrins on platelets, could amplify the production of microthrombi that pose the threat of pulmonary thrombosis and embolism, strokes and other thrombotic consequences. The susceptibility of different tissues to virion–integrin interactions may be modulated by a host of factors, including the conformation of relevant integrins and the impact of the tissue microenvironment on spike protein conformation. Patient-specific differences in these factors may contribute to the high variability of clinical presentation. There is danger that the emergence of receptor-binding domain mutations that increase infectivity may also enhance access of the RGD motif for integrin binding, resulting in viral strains with ACE2 independent routes of cell entry and novel integrin-mediated biological and clinical impacts. The highly infectious variant, B.1.1.7 (or VUI 202012/01), includes a receptor-binding domain amino acid replacement, N501Y, that could potentially provide the RGD motif with enhanced access to cell-surface integrins, with consequent clinical impacts.

N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles.

Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on the sequence of the major capsid protein VP1, further enhance this public threat. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells were characterized in detail using a set of biophysical and structural tools. We used native mass spectrometry, gas-phase electrophoretic mobility molecular analysis, and proteomics to get clear insights into particle size, structure, and composition, as well as stability. Generally, noroviruses have been known to form mainly T = 3 particles. Importantly, we identified a major truncation in the capsid proteins as a likely cause for the formation of T = 1 particles. For vaccine development, particle production needs to be a reproducible, reliable process. Understanding the underlying processes in capsid size variation will help to produce particles of a defined capsid size presenting antigens consistent with intact virions. Next to vaccine production itself, this would be immensely beneficial for bio-/nano-technological approaches using viral particles as carriers or triggers for immunological reactions.

Application of Heparin Affinity Chromatography to Produce a Differential Vaccine without Eliciting Antibodies against the Nonstructural Proteins of the Serotype O Foot-and-Mouth Disease Viruses.

Although polyethylene glycol (PEG) application is the most widely used method in removing nonstructural proteins (NSPs) for foot-and-mouth disease (FMD) vaccine production, some NSPs remaining in the antigen could elicit antibodies against these proteins after repeated vaccinations in livestock. Therefore, the purpose of this study was to purify the FMD virus (FMDV) via affinity chromatography using a heparin ligand to remove most proteins, including NSPs. Chromatography showed an intact virus (146S) particle recovery of 70% or more for three different strains of serotype O FMDV (two locally isolated strains and one genetically modified strain). The experimental vaccine made with antigens eluted via heparin affinity chromatography elicited virus-neutralizing antibodies against homologous viruses but did not induce antibodies against NSPs even after five immunizations in goats; this indicated that the NSPs were effectively removed from the vaccine antigen. This method can then be used to produce a higher-quality vaccine compared with PEG application in terms of the purity of the FMD vaccine. Therefore, this result would be an important groundwork for advanced FMD vaccine manufacturing in the near future.

Convalescent Memory T Cell Immunity in Individuals with Mild or Asymptomatic SARS-CoV-2 Infection May Result from an Evolutionarily Adapted Immune Response to Coronavirus and the 'Common Cold'.

Recent studies have shown a significant level of T cell immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in convalescent coronavirus disease 2019 (COVID-19) patients and unexposed healthy individuals. Also, SARS-CoV-2-reactive T memory cells occur in unexposed healthy individuals from endemic coronaviruses that cause the ‘common cold.’ The finding of the expression of adaptive SARS-CoV-2-reactive T memory cells in unexposed healthy individuals may be due to multiple cross-reactive viral protein targets following previous exposure to endemic human coronavirus infections. The opinion of the authors is that determination of protein sequence homologies across seemingly disparate viral protein libraries may provide epitope-matching data that link SARS-CoV-2-reactive T memory cell signatures to prior administration of cross-reacting vaccines to common viral pathogens. Exposure to SARS-CoV-2 initiates diverse cellular immune responses, including the associated ‘cytokine storm’. Therefore, it is possible that the intact virus possesses a required degree of conformational matching, or stereoselectivity, to effectively target its receptor on multiple cell types. Therefore, conformational matching may be viewed as an evolving mechanism of viral infection and viral replication by an evolutionary modification of the angiotensin-converting enzyme 2 (ACE2) receptor required for SARS-CoV-2 binding and host cell entry. The authors propose that convalescent memory T cell immunity in individuals with mild or asymptomatic SARS-CoV-2 infection may result from an evolutionarily adapted immune response to coronavirus and the ‘common cold’.

Application of Capsid Integrity (RT-)qPCR to Assessing Occurrence of Intact Viruses in Surface Water and Tap Water in Japan

Capsid integrity (RT-)qPCR has recently been developed to discriminate between intact forms from inactivated forms of viruses, but its applicability to identifying integrity of viruses in drinking water has remained limited. In this study, we investigated the application of capsid integrity (RT-)qPCR using cis-dichlorodiammineplatinum (CDDP) with sodium deoxycholate (SD) pretreatment (SD-CDDP-(RT-)qPCR) to detect intact viruses in surface water and tap water. A total of 63 water samples (surface water, n = 20; tap water, n = 43) were collected in the Kanto region in Japan and quantified by conventional (RT)-qPCR and SD-CDDP-(RT-)qPCR for pepper mild mottle virus (PMMoV) and seven other viruses pathogenic to humans (Aichivirus (AiV), noroviruses of genotypes I and II, enterovirus, adenovirus type 40 and 41, and JC and BK polyomaviruses). In surface water, PMMoV (100%) was more frequently detected than other human pathogenic viruses (30%–60%), as determined by conventional (RT-)qPCR. SD-CDDP-(RT-)qPCR also revealed that intact PMMoV (95%) was more common than intact human pathogenic viruses (20%–45%). In the tap water samples, most of the target viruses were not detected by conventional (RT-)qPCR, except for PMMoV (9%) and AiV (5%). PMMoV remained positive (5%), whereas no AiV was detected when tested by SD-CDDP-(RT-)qPCR, indicating that some PMMoV had an intact capsid, whereas AiV had damaged capsids. The presence of AiV in the absence of PMMoV in tap water produced from groundwater may demonstrate the limitation of PMMoV as a viral indicator in groundwater. In addition to being abundant in surface water, PMMoV was detected in tap water, including PMMoV with intact capsids. Thus, the absence of intact PMMoV may be used to guarantee the viral safety of tap water produced from surface water.

Mobile Health (mHealth) Viral Diagnostics Enabled with Adaptive Adversarial Learning

Deep-learning (DL)-based image processing has potential to revolutionize the use of smartphones in mobile health (mHealth) diagnostics of infectious diseases. However, the high variability in cellphone image data acquisition and the common need for large amounts of specialist-annotated images for traditional DL model training may preclude generalizability of smartphone-based diagnostics. Here, we employed adversarial neural networks with conditioning to develop an easily reconfigurable virus diagnostic platform that leverages a dataset of smartphone-taken microfluidic chip photos to rapidly generate image classifiers for different target pathogens on-demand. Adversarial learning was also used to augment this real image dataset by generating 16,000 realistic synthetic microchip images, through style generative adversarial networks (StyleGAN). We used this platform, termed smartphone-based pathogen detection resource multiplier using adversarial networks (SPyDERMAN), to accurately detect different intact viruses in clinical samples and to detect viral nucleic acids through integration with CRISPR diagnostics. We evaluated the performance of the system in detecting five different virus targets using 179 patient samples. The generalizability of the system was confirmed by rapid reconfiguration to detect SARS-CoV-2 antigens in nasal swab samples (n = 62) with 100% accuracy. Overall, the SPyDERMAN system may contribute to epidemic preparedness strategies by providing a platform for smartphone-based diagnostics that can be adapted to a given emerging viral agent within days of work.

Distribution and Evolution of the Bacteriophage WO and Its Antagonism With Wolbachia.

The symbiosis system comprising eukaryotic hosts, intracellular bacterium Wolbachia, and temperate bacteriophages WO is widely spread through nearly half the number of arthropod species. The relationships between the three components of the system are extremely intricate. Even though the bacteriophage WO can have diverse influences on the ecology and evolution of Wolbachia, little is known about the distribution and evolution of the phages. To the best of our knowledge, this study is the first to report that in infected fig wasps (Ceratosolen solmsi, Kradibia gibbosae, and Wiebesia pumilae), the genomes of all the Wolbachia strains had only one cryptic WO prophage, which contained defects in the genomic structural modules. This phenomenon was contrary to the widely accepted understanding that Wolbachia with cryptic prophages usually possesses at least one intact WO prophage consisting of gene sequences of the head, baseplate, and tail modules, through which the prophage could form intact virions. In addition to the genetic structure features, the phylogenetic relationships of WO and Wolbachia also revealed that bacteriophage WO can horizontally spread among a certain genus or a group of insect hosts, nearly free from the restriction of the affiliation of Wolbachia. Combined with the vertical transmission along with Wolbachia, the wide spread of WO phages can be explained. Furthermore, the gender preference and functional module preference for transcriptional activity of the genes in cryptic WOs implied the antagonized coevolutionary pattern between WO prophages and their Wolbachia hosts.

Virus Irradiation and COVID-19 Disease

Virus irradiation has been performed for many decades for basic research studies, sterilization, and vaccine development. The COVID-19 outbreak is currently causing an enormous effort worldwide for finding a vaccine against coronavirus. High doses of -rays can be used for the development of vaccines that exploit inactivated virus. This technique has been gradually replaced by more practical methods, in particular the use of chemicals, but irradiation remains a simple and effective method used in some cases. The technique employed for inactivating a virus has an impact on its ability to induce an adaptive immune response able to confer effective protection. We propose here that accelerated heavy ions can be used to inactivate SARS-CoV-2 viruses with small damage to the spike proteins of the envelope, and can then provide an intact virion for vaccine development.

Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers.

Filoviruses such as Ebola and Marburg virus bud from the host membrane as enveloped virions. This process is achieved by the matrix protein VP40. When expressed alone, VP40 induces budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and generation of membrane curvature are intrinsic properties of VP40. There has been no direct information on the structure of VP40 matrix layers within viruses or virus-like particles. We present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, and within intact Marburg viruses. VP40 dimers assemble extended chains via C-terminal domain interactions. These chains stack to form 2D matrix lattices below the membrane surface. These lattices form a patchwork assembly across the membrane and suggesting that assembly may begin at multiple points. Our observations define the structure and arrangement of the matrix protein layer that mediates formation of filovirus particles.

Full-length three-dimensional structure of the influenza A virus M1 protein and its organization into a matrix layer.

Matrix proteins are encoded by many enveloped viruses, including influenza viruses, herpes viruses, and coronaviruses. Underneath the viral envelope of influenza virus, matrix protein 1 (M1) forms an oligomeric layer critical for particle stability and pH-dependent RNA genome release. However, high-resolution structures of full-length monomeric M1 and the matrix layer have not been available, impeding antiviral targeting and understanding of the pH-dependent transitions involved in cell entry. Here, purification and extensive mutagenesis revealed protein-protein interfaces required for the formation of multilayered helical M1 oligomers similar to those observed in virions exposed to the low pH of cell entry. However, single-layered helical oligomers with biochemical and ultrastructural similarity to those found in infectious virions before cell entry were observed upon mutation of a single amino acid. The highly ordered structure of the single-layered oligomers and their likeness to the matrix layer of intact virions prompted structural analysis by cryo-electron microscopy (cryo-EM). The resulting 3.4-Å-resolution structure revealed the molecular details of M1 folding and its organization within the single-shelled matrix. The solution of the full-length M1 structure, the identification of critical assembly interfaces, and the development of M1 assembly assays with purified proteins are crucial advances for antiviral targeting of influenza viruses.

Antennal gland of shrimp as an entry for WSSV infection

White spot syndrome virus (WSSV) is an extremely destructive virus in shrimp farming which has caused a serious economic loss to global shrimp industry. Learning its infection routes is the basis for WSSV prevention and control. The digestive tract and gill were generally considered to be the target tissues for WSSV entry into shrimp. However, whether there is any other target tissue for WSSV entry into the shrimp is still not clear. In the present study, we firstly found that the transcripts of WSSV could be detected in the antennal gland of the Pacific white shrimp Litopenaeus vannamei cultured in the sea water without any signs of disease. Considering that the antennal gland is an important organ which functions in regulating the osmotic pressure and balance of ions in shrimp, we want to know whether it is also an entry for WSSV infection to shrimp. The spatial expressions of WSSV in shrimp tissues including antennal gland were firstly analyzed in shrimp naturally infected with WSSV, and the data showed that WSSV could be detected in antennal gland of shrimp at the early stages of WSSV infection, and intact virus particles were observed under transmission electronic microscope. Reverse perfusion of WSSV into antennal gland could cause the infection and lead to the death of shrimp. Incubation of WSSV with antennal gland tissues in vitro also led to the replication of WSSV in cultured antennal gland. These data indicated that antennal gland was an entry of WSSV into shrimp to cause the infection. The effect of salinity on WSSV infection was analyzed, and the data showed that salinity stress could induce faster WSSV replication in antennal gland, and accelerate the process of disease. Our study confirmed a new infection route of WSSV, and salinity stress could lead to the disease outbreak caused by WSSV. These data will provide useful information for the development of strategy for WSSV prevention and control.

Occult Colonic Perforation in a Patient With Coronavirus Disease 2019 After Interleukin-6 Receptor Antagonist Therapy.

BackgroundInterleukin-6 blockade (IL-6) has become a focus of therapeutic investigation for the coronavirus disease 2019 (COVID-19).MethodsWe report a case of a 34-year-old with COVID-19 pneumonia receiving an IL-6 receptor antagonist (IL-6Ra) who developed spontaneous colonic perforation. This perforation occurred despite a benign abdominal exam and in the absence of other known risk factors associated with colonic perforation.ResultsExamination of the colon by electron microscopy revealed numerous intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions abutting the microvilli of the colonic mucosa. Multiplex immunofluorescent staining revealed the presence of the SARS-CoV-2 spike protein on the brush borders of colonic enterocytes that expressed angiotensin-converting enzyme 2. However, no viral particles were observed within the enterocytes to suggest direct viral injury as the cause of colonic perforation.ConclusionsThese data and absence of known risk factors for spontaneous colonic perforation implicate IL-6Ra therapy as the potential mediator of colonic injury in this case. Furthermore, this report provides the first in situ visual evidence of the virus in the colon of a patient presenting with colonic perforation adding to growing evidence that intact infectious virus can be present in the stool.

The viral protein fragment theory of COVID-19 pathogenesis

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the current pandemic of coronavirus disease 2019 (COVID-19) that has killed nearly one million people so far. While this is a respiratory virus, surprisingly, it has been recognized that patients with cardiovascular disease are likely to be affected severely and die of COVID-19. This phenomenon cannot be explained by the generally accepted logic that the SARS-CoV-2 infection/replication is the sole determinant of the actions of the virus to define the fate of host cells. I herein propose the viral protein fragment theory of COVID-19 pathogenesis based on my observations in cultured human vascular cells that SARS-CoV-2 spike protein can activate cell signaling events without the rest of the viral components. It is generally thought that SARS-CoV-2 and other single-stranded RNA viruses attach to the host cells through the interactions between surface proteins of the viral capsid and the host cell receptors; the fusion and the entry of the viral components, resulting in the replication of the viruses; and the host cell responses are the consequence of these events. I hypothesize that, as humans are infected with SARS-CoV-2, the virus releases (a) fragment(s) of the spike protein that can target host cells for eliciting cell signaling without the rest of the viral components. Thus, COVID-19 patients are subjected to the intact virus infecting the host cells for the replication and amplification as well as the spike protein fragments that are capable of affecting the host cells. I propose that cell signaling elicited by the spike protein fragments that occur in cardiovascular cells would predispose infected individuals to develop complications that are seen in severe and fatal COVID-19 conditions. If this hypothesis is correct, then the strategies to treat COVID-19 should include, in addition to agents that inhibit the viral replication, therapeutics that inhibit the viral protein fragment-mediated cardiovascular cell signaling.