Infected Cell Cultures Research Articles

BA.1, BA.2 and BA.2.75 variants show comparable replication kinetics, reduced impact on epithelial barrier and elicit cross-neutralizing antibodies.

The Omicron variant of SARS-CoV-2 is capable of infecting unvaccinated, vaccinated and previously-infected individuals due to its ability to evade neutralization by antibodies. With multiple sub-lineages of Omicron emerging in the last 12 months, there is inadequate information on the quantitative antibody response generated upon natural infection with Omicron variant and whether these antibodies offer cross-protection against other sub-lineages of Omicron variant. In this study, we characterized the growth kinetics of Kappa, Delta and Omicron variants of SARS-CoV-2 in Calu-3 cells. Relatively higher amounts infectious virus titers, cytopathic effect and disruption of epithelial barrier functions was observed with Delta variant whereas infection with Omicron sub-lineages led to a more robust induction of interferon pathway, lower level of virus replication and mild effect on epithelial barrier. The replication kinetics of BA.1, BA.2 and BA.2.75 sub-lineages of the Omicron variant were comparable in cell culture and natural infection in a subset of individuals led to a significant increase in binding and neutralizing antibodies to the Delta variant and all the three sub-lineages of Omicron but the level of neutralizing antibodies were lowest against the BA.2.75 variant. Finally, we show that Cu2+, Zn2+ and Fe2+ salts inhibited in vitro RdRp activity but only Cu2+ and Fe2+ inhibited both the Delta and Omicron variants in cell culture. Thus, our results suggest that high levels of interferons induced upon infection with Omicron variant may counter virus replication and spread. Waning neutralizing antibody titers rendered subjects susceptible to infection by Omicron variants and natural Omicron infection elicits neutralizing antibodies that can cross-react with other sub-lineages of Omicron and other variants of concern.

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Characterization of Megabat-Favored, CA-Dependent Susceptibility to Retrovirus Infection.

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK).

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Marek's disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK-particularly its kinase activity-is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection.

Virulence Protein Pgp3 Is Insufficient To Mediate Plasmid-Dependent Infectivity of Chlamydia trachomatis.

Chlamydia trachomatis is the most common cause of infectious blindness and sexually transmitted bacterial infection globally. C. trachomatis contains a conserved chlamydial plasmid with eight coding sequences. Plasmid-cured Chlamydia strains are attenuated and display reduced infectivity in cell culture and in vivo genital infection of female mice. Mutants that do not express the plasmid-encoded proteins Pgp3, a secreted protein with unknown function, or Pgp4, a putative regulator of pgp3 and other chromosomal loci, display an infectivity defect similar to plasmid-deficient strains. Our objective was to determine the combined and individual contributions of Pgp3 and Pgp4 to this phenotype. Deletion of pgp3 and pgp4 resulted in an infectivity defect detected by competition assay in cell culture and in mice. The pgp3 locus was placed under the control of an anhydrotetracycline-inducible promoter to examine the individual contributions of Pgp3 and Pgp4 to infectivity. Expression of pgp3 was induced 100- to 1,000-fold after anhydrotetracycline administration, regardless of the presence or absence of pgp4. However, secreted Pgp3 was not detected when pgp4 was deleted, confirming a role for Pgp4 in Pgp3 secretion. We discovered that expression of pgp3 or pgp4 alone was insufficient to restore normal infectivity, which required expression of both Pgp3 and Pgp4. These results suggest Pgp3 and Pgp4 are both required for infectivity during C. trachomatis infection. Future studies are required to determine the mechanism by which Pgp3 and Pgp4 influence chlamydial infectivity as well as the potential roles of Pgp4-regulated loci.

Inactivation of the UL37 Deamidase Enhances Virus Replication and Spread of the HSV-1(VC2) Oncolytic Vaccine Strain and Secretion of GM-CSF.

The HSV-1 (VC2) live-attenuated vaccine strain was engineered with specific deletions in the amino termini of glycoprotein K (gK) and membrane protein UL20, rendering the virus unable to enter neurons and establish latency. VC2 replicates efficiently in epithelial cell culture but produces lower viral titers and smaller viral plaques than its parental HSV-1 (F) wild-type virus. VC2 is an effective live-attenuated vaccine against HSV-1 and HSV-2 infections in mice and guinea pigs and an anti-tumor immunotherapeutic and oncolytic virus against melanoma and breast cancer in mouse models. Previously, we reported that the gK/UL20 complex interacts with the UL37 tegument protein, and this interaction is essential for virion intracellular envelopment and egress. To investigate the potential role of the UL37 deamidase functions, the recombinant virus FC819S and VC2C819S were constructed with a C819S substitution to inactivate the UL37 predicted deamidase active site on an HSV-1(F) and HSV-1(VC2) genetic background, respectively. FC819S replicated to similar levels with HSV-1(F) and produced similar size viral plaques. In contrast, VC2C819S replication was enhanced, and viral plaques increased in size, approaching those of the wild-type HSV-1(F) virus. FC819S infection of cell cultures caused enhanced GM-CSF secretion in comparison to HSV-1(F) across several cell lines, including HEp2 cells and cancer cell lines, DU145 (prostate) and Panc 04.03 (pancreas), and primary mouse peritoneal cells. VC2 infection of these cell lines caused GM-CSF secretion at similar levels to FC819S infection. However, the VC2C819S virus did not exhibit any further enhancement of GM-CSF secretion compared to the VC2 virus. These results suggest that the UL37 deamidation functions in conjunction with the gK/UL20 complex to facilitate virus replication and GM-CSF secretion.

Radix Isatidis polysaccharide (RIP) resists the infection of QX-type infectious bronchitis virus via the MDA5/TLR3/IRF7 signaling pathway

Although vaccines play a major role in the prevention of infectious bronchitis (IB), Anti-IB drugs still have great potential in poultry production. Radix Isatidis polysaccharide (RIP) is a crude extract of Banlangen with antioxidant, antibacterial, antiviral, and multiple immunomodulatory functions. The aim of this study was to explore the innate immune mechanisms responsible for RIP-mediated alleviation of infectious bronchitis virus (IBV)-induced kidney lesions in chickens. Specific-pathogen-free (SPF) chicken and chicken embryo kidney (CEK) cells cultures were pretreated with RIP and then infected with the QX-type IBV strain, Sczy3. Morbidity, mortality, and tissue mean lesion scores were calculated for IBV-infected chickens, and the viral loads, inflammatory factor gene mRNA expression levels, and innate immune pathway gene mRNA expression levels in infected chickens and CEK cell cultures were determined. The results show that RIP could alleviate IBV-induced kidney damage, decrease CEK cells susceptibility to IBV infection, and reduce viral loads. Additionally, RIP reduced the mRNA expression levels of the inflammatory factors IL-6, IL-8, and IL-1β by decreasing the mRNA expression level of NF-κB. Conversely, the expression levels of MDA5, TLR3, STING, Myd88, IRF7, and IFN-β were increased, indicating that RIP conferred resistance to QX-type IBV infection via the MDA5, TLR3, IRF7 signaling pathway. These results provide a reference for both further research into the antiviral mechanisms of RIP and the development of preventative and therapeutic drugs for IB.

First report of Duck Hepatitis A virus genotype 2 in India.

Sudden death of ducklings was reported in a duck farm located at Tiruvallur district in Tamil Nadu, India. Disease investigation began with post mortem findings of dead birds revealing enlarged pale-pink / pale-yellow liver with multifocal petechiae and ecchymosis. A positive amplification with duck hepatitis A virus specific primers by reverse transcription-polymerase chain reaction (RT-PCR) on the tissue samples collected from dead birds indicated infection by duck hepatitis A virus (DHAV), an avian picornavirus, known to cause acute and high-mortality in ducklings. The virus isolation was successful in 9-days old embryonated chicken eggs, in primary chicken embryo fibroblast (CEF) cells and from experimentally infected ducklings. The embryonic death on day 5 to 7 post inoculation in chicken embryos with signs of cutaneous hemorrhage, edema and greenish yellow liver together with histopathology of embryonic liver and kidney further confirmed DHAV infection. TEM analysis of the infected allantoic fluid and infected CEF cell culture supernatant showed the presence of spherical shaped, non-enveloped virion particles of ~ 20-38nm diameter, typical for DHAV. Experimental infection of ducklings with RT-PCR positive tissue supernatant caused 40% to 50% mortality with typical petechial hemorrhages on the surface of liver. Further, histopathological analysis and RT-PCR of the inoculated duckling's tissues confirmed the presence of DHAV. Nucleotide sequencing of the 5'UTR region and VP1 region confirmed duck hepatitis A virus genotype 2 (DHAV-2). To the best of our knowledge, this is the first report of laboratory confirmation of DHAV-2 in India. This study warrants the need for the extensive epidemiological surveillance to understand the prevalence of DHAV-2 in India and to take appropriate control measures to curtail the disease spread.

IDENTIFICATION OF MYCOPLASMA CONTAMINATION IN CELL CULTURE

Mycoplasmas are the smallest and simplest prokaryotes wich were found in the endosomes of mammalian cells. They are widespread contaminants in cell cultures. It was identified a mycoplasma infection in a human breast cancer cell line. It was used fluorescence microscopy and nuclear affinity 4,6-diamidino-2-phenylindole (DAPI) staining. For the treatment of mycoplasma infection in cell culture, two antibiotics of the macrolide series (Tiamulin) and tetracyclines (Minocycline) was used. The effectiveness of combined antibiotic therapy against mycoplasmas has been proven, which was confirmed by the microscopic method. Therefore, treatment with combined antibiotics can completely eradicate mycoplasma infection from cultured cells.

Direct interaction of the molecular chaperone GRP78/BiP with the Newcastle disease virus hemagglutinin-neuraminidase protein plays a vital role in viral attachment to and infection of culture cells.

Glucose Regulated Proteins/Binding protein (GRP78/Bip), a representative molecular chaperone, effectively influences and actively participates in the replication processes of many viruses. Little is known, however, about the functional involvement of GRP78 in the replication of Newcastle disease virus (NDV) and the underlying mechanisms. The method of this study are to establish protein interactomes between host cell proteins and the NDV Hemagglutinin-neuraminidase (HN) protein, and to systematically investigate the regulatory role of the GRP78-HN protein interaction during the NDV replication cycle. Our study revealed that GRP78 is upregulated during NDV infection, and its direct interaction with HN is mediated by the N-terminal 326 amino acid region. Knockdown of GRP78 by small interfering RNAs (siRNAs) significantly suppressed NDV infection and replication. Conversely, overexpression of GRP78 resulted in a significant increase in NDV replication, demonstrating its role as a positive regulator in the NDV replication cycle. We further showed that the direct interaction between GRP78 and HN protein enhanced the attachment of NDV to cells, and masking of GRP78 expressed on the cell surface with specific polyclonal antibodies (pAbs) inhibited NDV attachment and replication. These findings highlight the essential role of GRP78 in the adsorption stage during the NDV infection cycle, and, importantly, identify the critical domain required for GRP78-HN interaction, providing novel insights into the molecular mechanisms involved in NDV replication and infection.

Comparative evaluation of some techniques used for the detection of rabies virus.

Neurotropic viruses in the family Rhabdoviridae, genus Lyssavirus, are what cause rabies, an acute, progressive, and highly lethal encephalomyelitis. Evaluation of the used diagnostic techniques to determine the most simple; rapid and accurate test for rabies virus (RABV) recognition in different specimens aiming to reach a rapid diagnosis as a step aid in the disease control and to prevent or even minimize the suspected hazard. The used techniques included an infection trial of Swiss mice with the mice-adapted challenge rabies virus followed by the detection of the virus in the infected mices' brains. Virus detection was carried out through the application BHK21 cell line infection; fluorescent antibody technique; latex agglutination test (LAT); direct enzyme-linked immunosorbent assay (ELISA); rabies antigen detection kit ELISA; conventional polymerase chain reaction (PCR). It was found that virus inoculation in mice and BHK21 cell lines needs 5-7 days with positivity of 90% and 100%, respectively. Rapid antigen kit was able to detect rabies antigen in mice brains suspension and BHK21 infected fluid within 3-5 minutes with percentages of 60% and 55.5%, respectively. In 1-1.5 hours, the direct fluorescent antibody method (DFAT) detected 90% and 100% of the rabies antigen in BHK21 cell line infection and brain impressions, respectively. Latex agglutination showed clear results with 88.8% with BHK21 infected fluid within 3-5 minutes while it did not carry out on brain emulsions to prevent falsely positive results brought on by the presence of tissue fragments. Conventional one-step PCR revealed 100% positivity with either brain or cell culture preparations within 2 days. Direct ELISA showed 88.8% positivity with BHK21 infected fluid with 1 day of work. Mice inoculation test, cell culture infection; DFAT and PCR are the most accurate techniques for the detection of RABV with a positivity of 90%-100% followed by LAT and ELISA with a positivity of 88.8%, and lastly, rabies antigen ELISA kit (RAK) with a positivity of 55.5%-60% taking in consideration the required time for each. In addition, the positivity % of the applied tests revealed their sensitivity and specificity.

S-Nitrosoglutathione Reduces the Density of Staphylococcus aureus Biofilms Established on Human Airway Epithelial Cells.

Patients with chronic rhinosinusitis (CRS) often show persistent colonization by bacteria in the form of biofilms which are resistant to antibiotic treatment. One of the most commonly isolated bacteria in CRS is Staphylococcus aureus (S. aureus). Nitric oxide (NO) is a potent antimicrobial agent and disperses biofilms efficiently. We hypothesized that S-nitrosoglutathione (GSNO), an endogenous NO carrier/donor, synergizes with gentamicin to disperse and reduce the bacterial biofilm density. We prepared GSNO formulations which are stable up to 12 months at room temperature and show the maximum amount of NO release within 1 h. We examined the effects of this GSNO formulation on the S. aureus biofilm established on the apical surface of the mucociliary-differentiated airway epithelial cell cultures regenerated from airway basal (stem) cells from cystic fibrosis (CF) and CRS patients. We demonstrate that for CF cells, which are defective in producing NO, treatment with GSNO at 100 μM increased the NO levels on the apical surface and reduced the biofilm bacterial density by 2 log units without stimulating pro-inflammatory effects or inducing epithelial cell death. In combination with gentamicin, GSNO further enhanced the killing of biofilm bacteria. Compared to placebo, GSNO significantly increased the ciliary beat frequency (CBF) in both infected and uninfected CF cell cultures. The combination of GSNO and gentamicin also reduced the bacterial density of biofilms grown on sinonasal epithelial cells from CRS patients and improved the CBF. These findings demonstrate that GSNO in combination with gentamicin may effectively reduce the density of biofilm bacteria in CRS patients. GSNO treatment may also enhance the mucociliary clearance by improving the CBF.

Nirmatrelvir-resistant SARS-CoV-2 variants with high fitness in an infectious cell culture system

The oral protease inhibitor nirmatrelvir is of key importance for prevention of severe coronavirus disease 2019 (COVID-19). To facilitate resistance monitoring, we studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) escape from nirmatrelvir in cell culture. Resistant variants harbored combinations of substitutions in the SARS-CoV-2 main protease (Mpro). Reverse genetics revealed that E166V and L50F+E166V conferred high resistance in infectious culture, replicon, and Mpro systems. While L50F, E166V, and L50F+E166V decreased replication and Mpro activity, L50F and L50F+E166V variants had high fitness in the infectious system. Naturally occurring L50F compensated for fitness cost of E166V and promoted viral escape. Molecular dynamics simulations revealed that E166V and L50F+E166V weakened nirmatrelvir-Mpro binding. Polymerase inhibitor remdesivir and monoclonal antibody bebtelovimab retained activity against nirmatrelvir-resistant variants, and combination with nirmatrelvir enhanced treatment efficacy compared to individual compounds. These findings have implications for monitoring and ensuring treatments with efficacy against SARS-CoV-2 and emerging sarbecoviruses.

Efficient production of protein complexes in mammalian cells using a poxvirus vector.

The production of full length, biologically active proteins in mammalian cells is critical for a wide variety of purposes ranging from structural studies to preparation of subunit vaccines. Prior research has shown that Modified vaccinia virus Ankara encoding the bacteriophage T7 RNA polymerase (MVA-T7) is particularly suitable for high level expression of proteins upon infection of mammalian cells. The expression system is safe for users and 10-50 mg of full length, biologically active proteins may be obtained in their native state, from a few litres of infected cell cultures. Here we report further improvements which allow an increase in the ease and speed of recombinant virus isolation, the scale-up of protein production and the simultaneous synthesis of several polypeptides belonging to a protein complex using a single virus vector. Isolation of MVA-T7 viruses encoding foreign proteins was simplified by combining positive selection for virus recombinants and negative selection against parental virus, a process which eliminated the need for tedious plaque purification. Scale-up of protein production was achieved by infecting a BHK 21 suspension cell line and inducing protein expression with previously infected cells instead of virus, thus saving time and effort in handling virus stocks. Protein complexes were produced from infected cells by concatenating the Tobacco Etch Virus (TEV) N1A protease sequence with each of the genes of the complex into a single ORF, each gene being separated from the other by twin TEV protease cleavage sites. We report the application of these methods to the production of a complex formed on the one hand between the HIV-1 integrase and its cell partner LEDGF and on the other between the HIV-1 VIF protein and its cell partners APOBEC3G, CBFβ, Elo B and Elo C. The strategies developed in this study should be valuable for the overexpression and subsequent purification of numerous protein complexes.

Desloratadine, an FDA-approved cationic amphiphilic drug, inhibits SARS-CoV-2 infection in cell culture and primary human nasal epithelial cells by blocking viral entry

The 2019 global coronavirus (COVID-19) pandemic has brought the world to a grinding halt, highlighting the urgent need for therapeutic and preventive solutions to slow the spread of emerging viruses. The objective of this study was to assess the anti-SARS-CoV-2 effectiveness of 8 FDA-approved cationic amphiphilic drugs (CADs). SARS-CoV-2-infected Vero cells, Calu-3 cells and primary Human Nasal Epithelial Cells (HNEC) were used to investigate the effects of CADs and revealed their antiviral mode of action. Among the CADs tested, desloratadine, a commonly used antiallergic, well-tolerated with no major side effects, potently reduced the production of SARS-CoV-2 RNA in Vero-E6 cells. Interestingly, desloratadine was also effective against HCoV-229E and HCoV-OC43 showing that it possessed broad-spectrum anti-coronavirus activity. Investigation of its mode of action revealed that it targeted an early step of virus lifecycle and blocked SARS-CoV-2 entry through the endosomal pathway. Finally, the ex vivo kinetic of the antiviral effect of desloratadine was evaluated on primary Human Nasal Epithelial Cells (HNEC), showing a significant delay of viral RNA production with a maximal reduction reached after 72 h of treatment. Thus, this treatment could provide a substantial contribution to prophylaxis and systemic therapy of COVID-19 or other coronaviruses infections and requires further studies.

Potential Involvement of Varicella Zoster Virus in Alzheimer’s Disease/Dementia via Reactivation of Herpes Simplex Virus Type 1

AbstractBackgroundInfections are associated with cognitive decline and development of AD/dementia. Epidemiological studies have revealed an increased AD/dementia risk after shingles, caused by varicella zoster virus (VZV), and/or a decreased risk after vaccination against shingles. This suggests that VZV might reside latently in brain, and on reactivation might cause direct damage in brain, as is thought to be the case with herpes simplex virus type 1 (HSV1), a virus strongly implicated in AD. Alternatively, shingles‐induced inflammation could lead to neuroinflammation and reactivation of HSV1 in brain, i.e., to an indirect consequence – a suggestion previously made to account for the risk of AD/dementia conferred by infections (Itzhaki and Dobson 2002), and for the reduction in risk after certain vaccinations ‐ by preventing occurrence of infection and consequent reactivation. Our aim was to investigate these possibilities by infecting cell cultures with VZV.MethodExperiments were carried out infecting human‐induced neural stem cells (hiNSC) cultures, and cultures quiescently infected with HSV1 with VZV. Cultures were observed for the presence of AD‐related phenotypes such as beta amyloid and P‐tau accumulation, gliosis, and neuroinflammation.ResultCells infected with VZV do not show the main AD characteristics ‐ beta amyloid and P‐tau accumulation – which HSV1 does cause ‐ but do show gliosis and increased levels of several cytokines, suggesting that VZV’s action is indirect. However, very intriguingly, we found that VZV infection of cells quiescently infected with HSV1 leads to reactivation of HSV1 and consequent AD‐like changes, including beta amyloid and P‐tau accumulation, supporting the above infection‐reactivation concept.ConclusionOur results support a role for VZV in AD/dementia via reactivation of HSV1 in brain and in combination with some current preliminary results, indicate that the risk of cognitive decline and of dementia conferred by some infectious diseases might indeed result from their causing HSV1 reactivation in brain.

The SARS-CoV-2 spike S375F mutation characterizes the Omicron BA.1 variant.

Recent studies have revealed the unique virological characteristics of Omicron, particularly those of its spike protein, such as less cleavage efficacy in cells, reduced ACE2 binding affinity, and poor fusogenicity. However, it remains unclear which mutation(s) determine these three virological characteristics of Omicron spike. Here, we show that these characteristics of the Omicron spike protein are determined by its receptor-binding domain. Of interest, molecular phylogenetic analysis revealed that acquisition of the spike S375F mutation was closely associated with the explosive spread of Omicron in the human population. We further elucidated that the F375 residue forms an interprotomer pi-pi interaction with the H505 residue of another protomer in the spike trimer, conferring the attenuated cleavage efficiency and fusogenicity of Omicron spike. Our data shed light on the evolutionary events underlying the emergence of Omicron at the molecular level.

Efficacy of favipiravir and molnupiravir against novel SARS-CoV-2 variants in vitro and in vivo

The COVID-19 disease pandemic remains a significant global problem, resulting in hundreds of millions of cases and millions of deaths. The search for specific inhibitors of SARS-CoV-2 for the treatment of this infection remains relevant. Drugs such as Favipiravir and Molnupiravir, which exhibit specific antiviral activity against SARS-CoV-2, are already being used to treat patients. However, there is limited evidence of their effectiveness, especially against novel genetic variants of the COVID-19 pathogen. The aim of this study was to investigate the antiviral effect of these drugs using an in vitro experimental model of SARS-CoV-2 infection in Vero E6 cell culture and an animal model of infection using Syrian hamsters. It has been established that Molnupiravir has an inhibitory effect against variants of the SARS-CoV-2 with IC50 values from 16.51 to 7.88 μM in vitro, and reduces the infectious titer of the virus in the lungs of animals by ~1.5 Log10 in vivo, in while Favipiravir shows lower activity and severe toxicity. Dose selection and frequency of use remain unexplored.

Identification of herbal teas and their compounds eliciting antiviral activity against SARS-CoV-2 in vitro

BackgroundThe SARS-CoV-2/COVID-19 pandemic has inflicted medical and socioeconomic havoc, and despite the current availability of vaccines and broad implementation of vaccination programs, more easily accessible and cost-effective acute treatment options preventing morbidity and mortality are urgently needed. Herbal teas have historically and recurrently been applied as self-medication for prophylaxis, therapy, and symptom alleviation in diverse diseases, including those caused by respiratory viruses, and have provided sources of natural products as basis for the development of therapeutic agents. To identify affordable, ubiquitously available, and effective treatments, we tested herbs consumed worldwide as herbal teas regarding their antiviral activity against SARS-CoV-2.ResultsAqueous infusions prepared by boiling leaves of the Lamiaceae perilla and sage elicit potent and sustained antiviral activity against SARS-CoV-2 when applied after infection as well as prior to infection of cells. The herbal infusions exerted in vitro antiviral effects comparable to interferon-β and remdesivir but outperformed convalescent sera and interferon-α2 upon short-term treatment early after infection. Based on protein fractionation analyses, we identified caffeic acid, perilla aldehyde, and perillyl alcohol as antiviral compounds. Global mass spectrometry (MS) analyses performed comparatively in two different cell culture infection models revealed changes of the proteome upon treatment with herbal infusions and provided insights into the mode of action. As inferred by the MS data, induction of heme oxygenase 1 (HMOX-1) was confirmed as effector mechanism by the antiviral activity of the HMOX-1-inducing compounds sulforaphane and fraxetin.ConclusionsIn conclusion, herbal teas based on perilla and sage exhibit antiviral activity against SARS-CoV-2 including variants of concern such as Alpha, Beta, Delta, and Omicron, and we identified HMOX-1 as potential therapeutic target. Given that perilla and sage have been suggested as treatment options for various diseases, our dataset may constitute a valuable resource also for future research beyond virology.

A new host-targeted antiviral cyclolignan (SAU-22.107) for Dengue Virus infection in cell cultures. Potential action mechanisms based on cell imaging

Dengue virus (DENV) infection is the most arbovirosis in the world. However, medications have not been approved for its treatment. Drug discovery based on the host-targeted antiviral (HTA) constitutes a new promising strategy, considering their high genetic barrier to resistance and the low probability of selecting drug resistance strains. In this study, we have tested fifty-seven podophyllotoxin-related cyclolignans on DENV-2 infected cells and found the most promising compound was S.71. Using cellular and molecular biology experiments, we have discovered that the new lignan altered the distribution of microtubules, induced changes in cell morphology, and caused retraction of the rough endoplasmic reticulum. In addition, the compound alters the viral envelope protein and the double-stranded RNA, while there is a decrease in negative-strand RNA synthesis; especially when the compound was added between 6- and 12-hours post-infection. Altogether, S.71 decreases the viral yield through an HTA-related mechanism of action, possibly altering the DENV genome replication and/or polyprotein translation, through the alteration of microtubule distribution and endoplasmic reticulum deterioration. Finally, pharmacokinetic predictors show that S.71 falls within the standard ranges established for drugs.