Virus Infectivity Assays Research Articles

Evaluating the antiviral efficacy and specificity of chlorogenic acid and related herbal extracts against SARS-CoV-2 variants via spike protein binding intervention

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus infect the respiratory tract through surface proteins, causing similar symptoms. Since the onset of the COVID-19 pandemic, both viruses have posed significant ongoing global health threats. Like the influenza virus, SARS-CoV-2 evolves into variants that can reduce vaccine efficacy. Thus, herbal medicines are being explored as supplementary options to enhance protection against these infections. This study aimed to investigate the therapeutic potential of chlorogenic acid (3-CQA) and related extracts from green coffee beans and Echinacea purpurea against SARS-CoV-2 variants and H1N1 infection. The methods employed included an ELISA-based trimeric spike protein binding assay, viral infection assays, plaque assays, and molecular docking studies. Results showed that 3-CQA blocked spike protein/angiotensin converting enzyme 2 (ACE2) binding for most SARS-CoV-2 variants of concern, except the Delta variant. Extracts from Green coffee bean and E. purpurea effectively blocked all variants tested. Additionally, antibodies blocked spike protein binding up to Omicron BA.2. Molecular docking suggested that 3-CQA binding to Omicron BA.1, BA.2, and BA.4, though not to the Delta spike protein, may lead to steric hindrance, preventing receptor-binding domain interactions with ACE2. Finally, both 3-CQA and E. purpurea extract showed preventive effects against H1N1 viral infection, though with lower potency compared to SARS-CoV-2. In conclusion, 3-CQA has potential as a phytoconstituent marker for herbs with bioactive properties against SARS-CoV-2 and H1N1 viral infections.

On-Chip Droplet Splitting with High Volume Ratios Using a 3D Conical Microstructure-Based Microfluidic Device.

This work reports a simple microfluidic method for splitting a mother droplet into two daughter droplets with high and precise volume ratios. To achieve this, a droplet-splitting microfluidic device embedded with a three-dimensional (3D) conical microstructure is fabricated, in which the high splitting ratios of monodisperse mother droplets are achieved. The volume ratio of the split daughter droplets can reach up to 265. In addition, we examined factors that affect the splitting ratio of the daughter droplets and found that the ratio is affected by the flow rates of the two individual outlet channels, the injection length of the conical microstructure, and the diameter of the original mother droplets. Numerical simulations of these parameters were conducted to gain a clearer understanding of the splitting behavior. The proposed droplet splitting device with a conical microstructure enables on-chip sample extraction and droplet volume control, which can be a powerful tool for various droplet-based applications in microfluidic devices such as viral infectivity assays and sequencing heterogeneous populations.

Anti-coronavirus and anti-pulmonary inflammation effects of iridoids, the common component from Chinese herbal medicines for the treatment of COVID-19.

The practice of Chinese herbal medicines for the treatment of COVID-19 in China played an essential role for the control of mortality rate and reduction of recovery time. The iridoids is one of the main constituents of many heat-clearing and detoxifying Chinese medicines that were largely planted and frequently used in clinical practice. Twenty-three representative high content iridoids from several staple Chinese medicines were obtained and tested by a SARS-CoV-2 pseudo-virus entry-inhibition assay on HEK-293T/ACE2 cells, a live HCoV-OC43 virus infection assay on HRT-18 cells, and a SARS-CoV-2 3CL protease inhibitory FRET assay followed by molecular docking simulation. The anti-pulmonary inflammation activities were further evaluated on a TNF-α induced inflammation model in A549 cells and preliminary SARs were concluded. The results showed that specnuezhenide (7), cornuside (12), neonuezhenide (15), and picroside III (21) exhibited promising antiviral activities, and neonuezhenide (15) could inhibit 3CL protease with an IC50 of 14.3μM. Docking computation showed that compound 15 could bind to 3CL protease through a variety of hydrogen bonding and hydrophobic interactions. In the anti-pulmonary inflammation test, cornuside (12), aucubin (16), monotropein (17), and shanzhiside methyl ester (18) could strongly decrease the content of IL-1β and IL-8 at 10μM. Compound 17 could also upregulate the expression of the anti-inflammatory cytokine IL-10 significantly. The iridoids exhibited both anti-coronavirus and anti-pulmonary inflammation activities for their significance of existence in Chinese herbal medicines, which also provided a theoretical basis for their potential utilization in the pharmaceutical and food industries.

Implications of high PsSAD expression and oleic acid content in defining the Sujata phenotype of Papaver somniferum

Papaver somniferum L. produces therapeutically-useful alkaloids like morphine, codeine, papaverine, and thebaine that accumulate in the latex of its capsule. Apart from its alkaloids, the edible seeds of the plant have high nutritional value and culinary use, with the seed oil being rich in the health-promoting unsaturated fatty acids. Sujata is low alkaloid-producing latex-less culinary variety of P. somniferum developed from an alkaloid-rich gum harvest parent (Sampada) for curtailing the narcotic menace of morphine and opium (dried latex). Earlier, it has been shown that the expression of stearoyl-acyl-carrier protein desaturase (PsSAD) in the young capsule and the proportion of unsaturated fatty acids in the seeds are higher in Sujata as compared to Sampada. Here, we studied bacterial endophytes isolated from Sujata tissues for their role in defining the fatty acid (saturated stearic versus unsaturated oleic) profile in its leaves and seeds. Besides, a potyvirus infecting P. somniferum was characterized through its coat protein gene sequencing and transmission electron microscopy and the two genotypes (Sujata and Sampada) contrasting in their phenotypes (oil/fatty acid, alkaloid and latex profiles) were compared for their susceptibility towards it through indicator plant- and qRT-PCR-based virus infectivity assays. In both the assays, Sujata was found to have higher susceptibility to the potyvirus, as compared to Sampada. The most plausible reason for this could be the higher PsSAD expression in the leaves of Sujata, which resulted in higher relative levels of oleic acid and lower relative levels of stearic acid in its leaves as compared to that of Sampada.

RNA-binding protein PTENα blocks RIG-I activation to prevent viral inflammation.

A timely inflammatory response is crucial for early viral defense, but uncontrolled inflammation harms the host. Retinoic acid-inducible gene I (RIG-I) has a pivotal role in detecting RNA viruses, yet the regulatory mechanisms governing its sensitivity remain elusive. Here we identify PTENα, an N-terminally extended form of PTEN, as an RNA-binding protein with a preference for the CAUC(G/U)UCAU motif. Using both in vivo and in vitro viral infection assays, we demonstrated that PTENα restricted the host innate immune response, relying on its RNA-binding capacity and phosphatase activity. Mechanistically, PTENα directly bound to viral RNA and enzymatically converted its 5'-triphosphate to 5'-monophosphate, thereby reducing RIG-I sensitivity. Physiologically, brain-intrinsic PTENα exerted protective effects against viral inflammation, while peripheral PTENα restricted host antiviral immunity and, to some extent, promoted viral replication. Collectively, our findings underscore the significance of PTENα in modulating viral RNA- and RIG-I-mediated immune recognition, offering potential therapeutic implications for infectious diseases.

Pseudotyped virus infection of multiplexed ACE2 libraries reveals SARS-CoV-2 variant shifts in receptor usage.

Pairwise compatibility between virus and host proteins can dictate the outcome of infection. During transmission, both inter- and intraspecies variabilities in receptor protein sequences can impact cell susceptibility. Many viruses possess mutable viral entry proteins and the patterns of host compatibility can shift as the viral protein sequence changes. This combinatorial sequence space between virus and host is poorly understood, as traditional experimental approaches lack the throughput to simultaneously test all possible combinations of protein sequences. Here, we created a pseudotyped virus infection assay where a multiplexed target-cell library of host receptor variants can be assayed simultaneously using a DNA barcode sequencing readout. We applied this assay to test a panel of 30 ACE2 orthologs or human sequence mutants for infectability by the original SARS-CoV-2 spike protein or the Alpha, Beta, Gamma, Delta, and Omicron BA1 variant spikes. We compared these results to an analysis of the structural shifts that occurred for each variant spike's interface with human ACE2. Mutated residues were directly involved in the largest shifts, although there were also widespread indirect effects altering interface structure. The N501Y substitution in spike conferred a large structural shift for interaction with ACE2, which was partially recreated by indirect distal substitutions in Delta, which does not harbor N501Y. The structural shifts from N501Y greatly influenced the set of animal orthologs the variant spike was capable of interacting with. Out of the thirteen non-human orthologs, ten exhibited unique patterns of variant-specific compatibility, demonstrating that spike sequence changes during human transmission can toggle ACE2 compatibility and potential susceptibility of other animal species, and cumulatively increase overall compatibilities as new variants emerge. These experiments provide a blueprint for similar large-scale assessments of protein compatibility during entry by diverse viruses. This dataset demonstrates the complex compatibility relationships that occur between variable interacting host and virus proteins.

A systematic mutation analysis of 13 major SARS-CoV-2 variants

SARS-CoV-2 evolves constantly with various novel mutations. Due to their enhanced infectivity, transmissibility and immune evasion, a comprehensive understanding of the association between these mutations and the respective functional changes is crucial. However, previous mutation studies of major SARS-CoV-2 variants remain limited. Here, we performed systematic analyses of full-length amino acids mutation, phylogenetic features, protein physicochemical properties, molecular dynamics and immune escape as well as pseudotype virus infection assays among thirteen major SARS-CoV-2 variants. We found that Omicron exhibited the most abundant and complex mutation sites, higher indices of hydrophobicity and flexibility than other variants. The results of molecular dynamics simulation suggest that Omicron has the highest number of hydrogen bonds and strongest binding free energy between the S protein and ACE2 receptor. Furthermore, we revealed 10 immune escape sites in 13 major variants, some of them were reported previously, but four of which (i.e. 339/373/477/496) are first reported to be specific to Omicron, whereas 462 is specific to Epslion. The infectivity of these variants was confirmed by the pseudotype virus infection assays. Our findings may help us understand the functional consequences of the mutations within various variants and the underlying mechanisms of the immune escapes conferred by the S proteins.

The glycosylation sites in RBD of spike protein attenuate the immunogenicity of PEDV AH2012/12

Porcine epidemic diarrhea (PED) is a highly contagious swine intestinal disease caused by PED virus (PEDV). Vaccination is a promising strategy to prevent and control PED. Previous studies have confirmed that glycosylation could regulate the immunogenicity of viral antigens. In this study, we constructed three recombinant PEDVs which removed the glycosylation sites in RBD. Viral infection assays revealed that similar replication characteristics between the recombinant viruses and parental PEDV. Although animal challenging study demonstrated that the glycosylation sites in RBD do not affect the pathogenicity of PEDV, we found that removing the glycosylation sites on the RBD regions could promote the IgG and neutralization titer in vivo, suggesting deglycosylation in RBD could enhance the immunogenicity of PEDV. These findings demonstrated that removal of the glycosylation sites in RBD is a promising method to develop PEDV vaccines.

Atranorin inhibits Zika virus infection in human glioblastoma cell line SNB-19 via targeting Zika virus envelope protein

BackgroundZika virus (ZIKV) is a single-stranded RNA flavivirus transmitted by mosquitoes. Its infection is associated with neurological complications such as neonatal microcephaly and adult Guillain–Barré syndrome, posing a serious threat to the health of people worldwide. Therefore, there is an urgent need to develop effective anti-ZIKV drugs. Atranorin is a lichen secondary metabolite with a wide range of biological activities, including anti-inflammatory, antibacterial and antioxidant, etc. However, the antiviral activity of atranorin and underlying mechanism has not been fully elucidated. PurposeWe aimed to determine the anti-ZIKV activity of atranorin in human glioma cell line SNB-19 and investigate the potential mechanism from the perspective of viral life cycle and the host cell functions. MethodsWe first established ZIKV-infected human glioma cells (SNB-19) model and used Western Blot, RT-qPCR, immunofluorescence, fluorescence-activated cell sorting (FACS) and plaque assay to evaluate the anti-ZIKV activity of atranorin. Then we assessed the regulation effect of atranorin on ZIKV induced IFN signal pathway activation by RT-qPCR. Afterward, we introduced time-of-addition assay, viral adsorption assay, viral internalization assay and transferrin uptake assay to define which step of ZIKV lifecycle is influenced by atranorin. Finally, we performed virus infectivity assay, molecular docking and thermal shift assay to uncover the target protein of atranorin on ZIKV. ResultsOur study showed that atranorin could protect SNB-19 cells from ZIKV infection, as evidenced by inhibited viral protein expression and progeny virus yield. Meanwhile, atranorin attenuated the activation of IFN signal pathway and downstream inflammatory response that induced by ZIKV infection. The results of time-of-addition assay indicated that atranorin acted primarily by disturbing the viral entry process. After ruling out the effect of atranorin on AXL receptor tyrosine kinase (AXL) dependent virus adsorption and clathrin-mediated endocytosis, we confirmed that atranorin directly targeted the viral envelope protein and lowered ZIKV infectivity by thermal shift assay and virus infectivity assay respectively. ConclusionWe found atranorin inhibits ZIKV infection in SNB-19 cells via targeting ZIKV envelope protein. Our study provided an experimental basis for the further development of atranorin and a reference for antiviral drug discovery from natural resources.

Virological Characteristics of Five SARS-CoV-2 Variants, Including Beta, Delta and Omicron BA.1, BA.2, BA.5.

SARS-CoV-2 variants of concern (VOCs) show increasing transmissibility and infectivity and induce substantial injuries to human health and the ecology. Therefore, it is vital to understand the related features for controlling infection. In this study, SARS-CoV-2 WIV04 (prototype) and five VOCs (Beta, Delta, Omicron BA.1, BA.2 and BA.5 variants) were inoculated in Vero cells to observe their growth activities. Apart from evaluating the environmental stability at different temperatures, residual virus titers and infectivity at different temperatures (4 °C, room temperature (RT) and 37 °C) were measured over 7 days. The experiment also assessed the infectivity for different incubation durations. The growth capacity assay suggested that the WIV04, Beta and Delta variants replicated efficiently in Vero cells compared with Omicron Variants, and BA.2 replicated more efficiently in Vero cells than BA.1 and BA.5. In addition, all variants exhibited longer survivals at 4 °C and could remain infectious after 7 days, compared to RT' survival after 5 days and at 37 °C after 1 day. The virus infection assay indicated that the Omicron variant had a weaker ability to infect cells compared to the WIV04, Beta and Delta strains, and a longer infection time was required for these strains, except for BA.2.

The RNA-binding domain of DCL3 is required for long-distance RNAi signaling.

The online version contains supplementary material available at 10.1007/s42994-023-00124-6.

A viral protein targets mitochondria and chloroplasts by subverting general import pathways and specific receptors.

Many plant proteins and some proteins from plant pathogens are dually targeted to chloroplasts and mitochondria, and are supposed to be transported along the general pathways for organellar protein import, but this issue has not been explored yet. Moreover, organellar translocon receptors exist as families of several members whose functional specialization in different cargos is supposed but not thoroughly studied. This article provides novel insights into such topics showing for the first time that an exogenous protein, the melon necrotic spot virus coat protein, exploits the common Toc/Tom import systems to enter both mitochondria and chloroplasts while identifying the involved specific receptors.

A Rapid and Novel Multiplex PCR Assay for Simultaneous Detection of Multiple Viruses Associated with Bovine Gastroenteritis.

Bovine viral diarrheal virus (BVDV) and bovine coronavirus (BCoV) are prevalent viral infections in buffalo calves that result in significant economic losses globally. However, Bovine picobirnavirus (BPBV) Group I and II has been an emerging causes of gastrointestinal infection as has been detected with mixed of BVDV as well as BCV. To combat economic losses and viral infection, a rapid and innovative multiplex-PCR assay (M-PCR) was developed to simultaneously identify BVDV, BCV, and BPBV. The assay employed three primer pairs, each specific to a particular virus. Notably, the primers for BCV and BVDV, targeting the transmembrane (M) Mebus gene and 5'UTR genes, respectively, were self-designed. To validate the assay, 300 samples of buffalo calf feces were subjected to the standardized multiplex PCR. The results demonstrated that 54 (18%) samples tested positive for multiple viruses, with 16.67% samples infected by BVDV, 0.9% by BCoV, and 0.13% by BPBV, as detected by the M-PCR assay. In summary, this developed assay is characterized by high specificity, sensitivity, throughput, and speed, enabling the simultaneous detection of the three viruses in a single reaction tube. Consequently, it holds potential for epidemiological investigations. It is worth noting that, to the best of our knowledge, this is the first reported multiplex assay for the worldwide detection of BVDV, BCoV, and BPBV. This novel assay promises to aid in the detection of mixed infections in the gastrointestinal tract.

A rapid, high-throughput, viral infectivity assay using automated brightfield microscopy with machine learning.

Infectivity assays are essential for the development of viral vaccines, antiviral therapies, and the manufacture of biologicals. Traditionally, these assays take 2-7 days and require several manual processing steps after infection. We describe an automated viral infectivity assay (AVIATM), using convolutional neural networks (CNNs) and high-throughput brightfield microscopy on 96-well plates that can quantify infection phenotypes within hours, before they are manually visible, and without sample preparation. CNN models were trained on HIV, influenza A virus, coronavirus 229E, vaccinia viruses, poliovirus, and adenoviruses, which together span the four major categories of virus (DNA, RNA, enveloped, and non-enveloped). A sigmoidal function, fit between virus dilution curves and CNN predictions, results in sensitivity ranges comparable to or better than conventional plaque or TCID50 assays, and a precision of ∼10%, which is considerably better than conventional infectivity assays. Because this technology is based on sensitizing CNNs to specific phenotypes of infection, it has potential as a rapid, broad-spectrum tool for virus characterization, and potentially identification.

Genetic signatures associated with the virulence of porcine epidemic diarrhea virus AH2012/12.

Porcine epidemic diarrhea (PED) caused by PED virus (PEDV) remains a big threat to the swine industry worldwide. Vaccination with live attenuated vaccine is a promising method to prevent and control PED, because it can elicit a more protective immunity than the killed vaccine, subunit vaccine, and so on. In this study, we found two obvious deletions in the genome of a high passage of AH2012/12. We further confirmed the second deletion which contains seven amino acids at the carboxy-terminus of the S2 gene and the start codon of ORF3 can reduce its pathogenicity in vivo. Animal experiments indicated that the recombinant PEDV with deleted carboxy-terminus of S gene showed higher IgG, IgA, neutralization antibodies, and protection effects against virus challenge than the killed vaccine. These data reveal that the engineering of the carboxy-terminus of the S2 gene may be a promising method to develop live attenuated vaccine candidates of PEDV.

P1 of turnip mosaic virus interacts with NOD19 for vigorous infection.

P1 protein, the most divergent protein of virus members in the genus Potyvirus of the family Potyviridae, is required for robust infection and host adaptation. However, how P1 affects viral proliferation is still largely elusive. In this work, a total number of eight potential P1-interacting Arabidopsis proteins were identified by the yeast-two-hybrid screening using the turnip mosaic virus (TuMV)-encoded P1 protein as the bait. Among which, the stress upregulated NODULIN 19 (NOD19) was selected for further characterization. The bimolecular fluorescent complementation assay confirmed the interaction between TuMV P1 and NOD19. Expression profile, structure, and subcellular localization analyses showed that NOD19 is a membrane-associated protein expressed mainly in plant aerial parts. Viral infectivity assay showed that the infection of turnip mosaic virus and soybean mosaic virus was attenuated in the null mutant of Arabidopsis NOD19 and NOD19-knockdown soybean seedlings, respectively. Together, these data indicate that NOD19 is a P1-interacting host factor required for robust infection.

Inactivation of SARS-CoV-2 on Surfaces by Cold-Plasma-Generated Reactive Species.

A Cold Atmospheric Plasma (CAP) apparatus was designed and developed for SARS-CoV-2 killing as evaluated by pseudotyped viral infectivity assays. The reactive species generated by the plasma system was fully characterized by using Optical Emission Spectroscopy (OES) measurement under given conditions such as plasma power, flow rate, and treatment time. A variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) were identified from plasma plume with energies of 15-72 eV in the frequency range between 500-1000 nm. Systematic virus killing experiments were carried out, and the efficacy of CAP treatment in reducing SARS-CoV-2 viral infectivity was significant following treatment for 8 s, with further enhancement of killing upon longer exposures of 15-120 s. We correlated killing efficacy with the reactive species in terms of type, intensity, energy, and frequency. These experimental results demonstrate effective cold plasma virus killing via ROS and RNS under ambient conditions.

A High Viral Load in Urine Correlates With Acute Kidney Injury and Poor Outcomes in Hospitalized Patients With Severe Fever With Thrombocytopenia Syndrome: A Noninvasive and Convenient Prognostic Marker.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with an extensive geographical distribution and high mortality rate. To date, the role of SFTS virus (SFTSV) in urine is still elusive. We aimed to explore the relationship between urinary bunyavirus and acute kidney injury (AKI) and mortality in patients with SFTS. Urine samples were collected from 102 patients to quantify SFTSV load in urine (U-SFTSV). Patient renal function was evaluated on admission. Receiver operating characteristic (ROC) curve and logistic regression analysis were performed to evaluate the predictive value of U-SFTSV. Viral infectivity assays in Vero cells were performed from 10 urine samples. The U-SFTSV level was positively correlated with SFTSV load in plasma (r = 0.624) and indicators of renal damage. The U-SFTSV level was identified as an independent risk factor for SFTS-associated AKI (odds ratio, 3.631; P = .019). The U-SFTSV showed great value in predicting the fatal outcome of SFTS patients with high area under curve (0.881). The Kaplan-Meier survival comparison showed that patients with U-SFTSV levels greater than 6379 copies/mL were at a higher risk of death within 28 days after onset. In addition, 4 urine samples with high U-SFTSV levels were infectious. Our large cohort study identified that the U-SFTSV level is a novel convenient and noninvasive predictive biomarker for incidence of AKI and poor outcome of patients with SFTS. Urine specimens could be a source of SFTSV infection in humans.

Collaborative effects of 2019-nCoV-Spike mutants on viral infectivity

BackgroundThe emerging mutants of the 2019-nCoV coronavirus are posing unprecedented challenges to the pandemic prevention. A thorough, understanding of the mutational characterization responsible for the pathogenic mechanisms of mutations in 2019-nCoV-Spike is indispensable for developing effective drugs and new vaccines. MethodsWe employed computational methods and viral infection assays to examine the interaction pattern and binding affinity between ACE2 and both single- and multi-mutants of the Spike proteins. ResultsUsing data from the CNCB-NGDC databank and analysis of the 2019-nCoV-Spike/ACE2 interface crystal structure, we identified 31 amino acids that may significantly contribute to viral infectivity. Subsequently, we performed molecular dynamics simulations for 589 single-mutants that emerged from the nonsynonymous substitutions of the aforementioned 31 residues. Ultimately, we discovered 8 single-mutants that exhibited significantly higher binding affinities (<−65.00 kcal/mol) to ACE2 compared with the wild-type Spike protein (−55.07 kcal/mol). The random combination of these 8 single-mutants yielded 184 multi-mutants, of which 60 multi-mutants exhibit markedly enhanced binding affinities (<−65.00 kcal/mol). Moreover, the binding free energy analyses of all 773 mutants (including 589 single- and 184 multi-mutants) revealed that Y449R and S494R had a synergistic effect on the binding affinity with other mutants, which were confirmed by virus infection assays of six randomly selected multi-mutants. More importantly, the findings of virus infection assay further validated a strong association between the binding free energy of Spike/ACE2 complex and the viral infectivity. ConclusionsThese findings will greatly contribute to the future surveillance of viruses and rational design of therapeutics.

Characterization of ferret Pit1 as a receptor of feline leukemia virus subgroup B.

Feline leukemia virus (FeLV) is a retrovirus that causes immune suppression and immunodeficiency, leading to opportunistic infections and leukemia/lymphoma in cats. Today, a variety of domestic mammals are kept in houses, and it is important to evaluate the possibility of interspecies transmission of FeLV. In this study, we assessed the infectivity of FeLV-B in ferrets that belong to Mustelidae. By pseudotype virus infection assay, we revealed that a ferret cell line, Mpf cells, is resistant to FeLV-B infection. The mRNA expression level of the FeLV-B receptor, Pit-1, was approximately half that of cat FEA cells in ferret Mpf cells. There was no significant difference in receptor usage between ferret's and cat's Pit1. These data may indicate the presence of the post-transcriptional modification and/or the restriction factor(s) against the FeLV-B infection in ferrets.