Attenuated effector T cells are linked to control of chronic HBV infection.

Hepatitis B virus (HBV)-specific CD8+ T cells play a dominant role during acute-resolving HBV infection but are functionally impaired during chronic HBV infection in humans. These functional deficits have been linked with metabolic and phenotypic heterogeneity, but it has remained unclear to what extent different subsets of HBV-specific CD8+ T cells still suppress viral replication. We addressed this issue by deep profiling, functional testing and perturbation of HBV-specific CD8+ T cells during different phases of chronic HBV infection. Our data revealed a mechanism of effector CD8+ T cell attenuation that emerges alongside classical CD8+ T cell exhaustion. Attenuated HBV-specific CD8+ T cells were characterized by cytotoxic properties and a dampened effector differentiation program, determined by antigen recognition and TGFß signaling, and were associated with viral control during chronic HBV infection. These observations identify a distinct subset of CD8+ T cells linked with immune efficacy in the context of a chronic human viral infection with immunotherapeutic potential.

Viral escape mutations do not account for non-protection from SIVmac239 challenge in RhCMV/SIV vaccinated rhesus macaques.

Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV.

Modelling Role of Protective and Nonprotective HLA Allele Inducing Different HIV Infection Outcomes.

Human immunodeficiency virus (HIV) infects CD4+ cells and causes progressive immune function failure, and CD8+ cells lyse infected CD4+ cell via recognising peptide presented by human leukocyte antigens (HLA). Variations in HLA allele lead to observed different HIV infection outcomes. Within-host HIV dynamics involves virus replication within infected cells and lysing of infected cells by CD8+ cells, but how variations in HLA alleles determine different infection outcomes was far from clear. Here, we used mathematical modelling and parameter inference with a new analysis of published virus inhibition assay data to estimate CD8+ cell lysing efficiency, and found that lysing efficiency fall in the gap between low bound (0.1-0.2 day-1 (Elemans et al. in PLoS Comput Biol 8(2):e1002381, 2012)) and upper boundary (6.5-8.4 day-1 (Wick et al. in J Virol 79(21):13579-13586, 2005)). Our outcomes indicate that both lysing efficiency and viral inoculum size jointly determine observed different infection outcomes. Low lysing rate associated with non-protective HLA alleles leads to monostable viral kinetic to high viral titre and oscillatory viral kinetics. High lysing rate associated with protective HLA alleles leads monostable viral kinetic to low viral titre and bistable viral kinetics; at a specific interval of CD8+ cell counts, small viral inoculum sizes are inhibited but not large viral inoculum sizes remain infectious. Further, with CD8+ cell recruitment, HIV kinetics always exhibit oscillatory kinetics, but lysing rate is negatively correlated with range of CD8+ cell count. Our finding highlights role of HLA allele determining different infection outcomes, thereby providing a potential mechanistic explanation for observed good and bad HIV infection outcomes induced by protective HLA allele.

Immunotoxin-mediated depletion of Gag-specific CD8+ T cells undermines natural control of SIV.

Antibody-mediated depletion studies have demonstrated that CD8+ T cells are required for effective immune control of SIV. However, this approach is potentially confounded by several factors, including reactive CD4+ T cell proliferation, and provides no information on epitope specificity, a likely determinant of CD8+ T cell efficacy. We circumvented these limitations by selectively depleting CD8+ T cells specific for the Gag epitope CTPYDINQM (CM9) via the administration of immunotoxin-conjugated tetrameric complexes of CM9/Mamu-A*01. Immunotoxin administration effectively depleted circulating but not tissue-localized CM9-specific CD8+ T cells, akin to the bulk depletion pattern observed with antibodies directed against CD8. However, we found no evidence to indicate that circulating CM9-specific CD8+ T cells suppressed viral replication in Mamu-A*01+ rhesus macaques during acute or chronic progressive infection with a pathogenic strain of SIV. This observation extended to macaques with established infection during and after continuous antiretroviral therapy. In contrast, natural controller macaques experienced dramatic increases in plasma viremia after immunotoxin administration, highlighting the importance of CD8+ T cell-mediated immunity against CM9. Collectively, these data showed that CM9-specific CD8+ T cells were necessary but not sufficient for robust immune control of SIV in a nonhuman primate model and, more generally, validated an approach that could inform the design of next-generation vaccines against HIV-1.

Role of inflammatory cytokine burst in neuro-invasion of Japanese Encephalitis virus infection: an immunotherapeutic approaches.

Japanese Encephalitis remains a significant global health concern, contributing to millions of deaths annually worldwide. Microglial cells, as key innate immune cells within the central nervous system (CNS), exhibit intricate cellular structures and possess molecular phenotypic plasticity, playing pivotal roles in immune responses during CNS viral infections. Particularly under viral inflammatory conditions, microglial cells orchestrate innate and adaptive immune responses to mitigate viral invasion and dampen inflammatory reactions. This review article comprehensively summarizes the pathophysiology of viral invasion into the CNS and the cellular interactions involved, elucidating the roles of various immune mediators, including pro-inflammatory cytokines, in neuroinflammation. Leveraging this knowledge, strategies for modulating inflammatory responses and attenuating hyperactivation of glial cells to mitigate viral replication within the brain are discussed. Furthermore, current chemotherapeutic and antiviral drugs are examined, elucidating their mechanisms of action against viral replication. This review aims to provide insights into therapeutic interventions for Japanese Encephalitis and related viral infections, ultimately contributing to improved outcomes for affected individuals.

RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks.

The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-ß expression during virus infection, the expression level of IFN-ß in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.

Singapore grouper iridovirus VP146 modulates the cGAS-STING signaling pathway to escape the interferon immune response.

Singapore grouper iridovirus (SGIV) is a large double-stranded DNA virus that has caused significant economic losses to the grouper aquaculture industry. So far, the structure and function of SGIV proteins have been successively reported. In the present paper, the protein of SGIV VP146 was cloned and identified. VP146 was whole-cell distributed in GS cells. VP146 promoted SGIV replication and inhibited the transcription of interferon-related genes as well as pro-inflammatory cytokines in GS cells. In addition, VP146 was involved in the regulation of the cGAS-STING signaling pathway, and decreased cGAS-STING induced the promoter of ISRE and NF-κB. VP146 interacted with the proteins of cGAS, STING, TBK1, and IRF3 from grouper, but did not affect the binding of grouper STING to grouper TBK1 and grouper IRF3. Interestingly, grouper STING was able to affect the intracellular localization of VP146. Four segment structural domains of grouper STING were constructed, and grouper STING-CTT could affect the intracellular localization of VP146. VP146 had no effect on the self-binding of EcSITNG, nor on the binding of EcSTING to EcTBK1 and EcIRF3. Together, the results demonstrated that SGIV VP146 modulated the cGAS-STING signaling pathway to escape the interferon immune response.

Unraveling the dynamics of hepatitis C virus adaptive mutations and their impact on antiviral responses in primary human hepatocytes.

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative capacity in various liver cell lines, this study investigated virus and host determinants that underlie enhanced viral fitness. Characterization of a panel of molecular p100 clones revealed that cell culture adaptive mutations optimize a range of virus-host interactions, resulting in expanded cell tropism, altered dependence on the cellular co-factor micro-RNA 122 and increased rates of virus spread. On the host side, comparative transcriptional profiling of hepatoma cells infected either with p100pop or its progenitor virus revealed that enhanced replicative fitness correlated with activation of endoplasmic reticulum stress signaling and the unfolded protein response. In contrast, infection of primary human hepatocytes with p100pop led to a mild attenuation of virion production which correlated with a greater induction of cell-intrinsic antiviral defense responses. In summary, long-term passage experiments in cells where selective pressure from innate immunity is lacking improves multiple virus-host interactions, enhancing HCV replicative fitness. However, this study further indicates that HCV has evolved to replicate at low levels in primary human hepatocytes to minimize innate immune activation, highlighting that an optimal balance between replicative fitness and innate immune induction is key to establish persistence. IMPORTANCE: Hepatitis C virus (HCV) infection remains a global health burden with 58 million people currently chronically infected. However, the detailed molecular mechanisms that underly persistence are incompletely defined. We utilized a long-term cell culture-adapted HCV, exhibiting enhanced replicative fitness in different human liver cell lines, in order to identify molecular principles by which HCV optimizes its replication fitness. Our experimental data revealed that cell culture adaptive mutations confer changes in the host response and usage of various host factors. The latter allows functional flexibility at different stages of the viral replication cycle. However, increased replicative fitness resulted in an increased activation of the innate immune system, which likely poses boundary for functional variation in authentic hepatocytes, explaining the observed attenuation of the adapted virus population in primary hepatocytes.

PGAM5 degrades PDCoV N protein and activates type I interferon to antagonize viral replication.

IMPORTANCE: As a member of the δ-coronavirus family, porcine deltacoronavirus (PDCoV) is a vital reason for diarrhea in piglets, which can contribute to high morbidity and mortality rates. Initially identified in Hong Kong in 2012, the virus has rapidly spread worldwide. During PDCoV infection, the virus employs evasion mechanisms to evade host surveillance, while the host mounts corresponding responses to impede viral replication. Our research has revealed that PDCoV infection down-regulates the expression of PGAM5 to promote virus replication. In contrast, PGAM5 degrades PDCoV N through autophagy by interacting with the cargo receptor P62 and the E3 ubiquitination ligase STUB1. Additionally, PGAM5 interacts with MyD88 and TRAF3 to activate the IFN signal pathway, resulting in the inhibition of viral replication.

PKR-mediated stress response enhances dengue and Zika virus replication.

IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.

Deletions of MGF110-9L and MGF360-9L from African swine fever virus are highly attenuated in swine and confer protection against homologous challenge.

African swine fever, caused by a large icosahedral DNA virus (African swine fever virus, ASFV), is a highly contagious disease in domestic and feral swine, thus posing a significant economic threat to the global swine industry. Currently, there are no effective vaccines or the available methods to control ASFV infection. Attenuated live viruses with deleted virulence factors are considered to be the most promising vaccine candidates; however, the mechanism by which these attenuated viruses confer protection is unclear. Here, we used the Chinese ASFV CN/GS/2018 as a backbone and used homologous recombination to generate a virus in which MGF110-9L and MGF360-9L, two genes antagonize host innate antiviral immune response, were deleted (ASFV-ΔMGF110/360-9L). This genetically modified virus was highly attenuated in pigs and provided effective protection of pigs against parental ASFV challenge. Importantly, we found ASFV-ΔMGF110/360-9L infection induced higher expression of Toll-like receptor 2 (TLR2) mRNA compared with parental ASFV as determined by RNA-Seq and RT-PCR analysis. Further immunoblotting results showed that parental ASFV and ASFV-ΔMGF110/360-9L infection inhibited Pam3CSK4-triggered activating phosphorylation of proinflammatory transcription factor NF-κB subunit p65 and phosphorylation of NF-κB inhibitor IκBα levels, although NF-κB activation was higher in ASFV-ΔMGF110/360-9L-infected cells compared with parental ASFV-infected cells. Additionally, we show overexpression of TLR2 inhibited ASFV replication and the expression of ASFV p72 protein, whereas knockdown of TLR2 had the opposite effect. Our findings suggest that the attenuated virulence of ASFV-ΔMGF110/360-9L might be mediated by increased NF-κB and TLR2 signaling.

The Host Response to Influenza A Virus Interferes with SARS-CoV-2 Replication during Coinfection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) represent two highly transmissible airborne pathogens with pandemic capabilities. Although these viruses belong to separate virus families-SARS-CoV-2 is a member of the family Coronaviridae, while IAV is a member of the family Orthomyxoviridae-both have shown zoonotic potential, with significant animal reservoirs in species in close contact with humans. The two viruses are similar in their capacity to infect human airways, and coinfections resulting in significant morbidity and mortality have been documented. Here, we investigate the interaction between SARS-CoV-2 USA-WA1/2020 and influenza H1N1 A/California/04/2009 virus during coinfection. Competition assays in vitro were performed in susceptible cells that were either interferon type I/III (IFN-I/-III) nonresponsive or IFN-I/-III responsive, in addition to an in vivo golden hamster model. We find that SARS-CoV-2 infection does not interfere with IAV biology in vivo, regardless of timing between the infections. In contrast, we observe a significant loss of SARS-CoV-2 replication following IAV infection. The latter phenotype correlates with increased levels of IFN-I/-III and immune priming that interferes with the kinetics of SARS-CoV-2 replication. Together, these data suggest that cocirculation of SARS-CoV-2 and IAV is unlikely to result in increased severity of disease. IMPORTANCE The human population now has two circulating respiratory RNA viruses with high pandemic potential, namely, SARS-CoV-2 and influenza A virus. As both viruses infect the airways and can result in significant morbidity and mortality, it is imperative that we also understand the consequences of getting coinfected. Here, we demonstrate that the host response to influenza A virus uniquely interferes with SARS-CoV-2 biology although the inverse relationship is not evident. Overall, we find that the host response to both viruses is comparable to that to SARS-CoV-2 infection alone.

A Simplified SARS-CoV-2 Mouse Model Demonstrates Protection by an Oral Replicon-Based mRNA Vaccine.

A mouse model of SARS-CoV-2 that can be developed in any molecular biology lab with standard facilities will be valuable in evaluating drugs and vaccines. Here we present a simplified SARS-CoV-2 mouse model exploiting the rapid adenoviral purification method. Mice that are sensitive to SARS-CoV-2 infection were generated by transducing human angiotensin-converting enzyme 2 (hACE2) by an adenovirus. The expression kinetics of the hACE2 in transduced mice were assessed by immunohistochemistry, RT-PCR, and qPCR. Further, the ability of the hACE2 to support viral replication was determined in vitro and in vivo. The hACE2 expression in the lungs of mice was observed for at least nine days after transduction. The murine macrophages expressing hACE2 supported viral replication with detection of high viral titers. Next, in vivo studies were carried out to determine viral replication and lung disease following SARS-CoV-2 challenge. The model supported viral replication, and the challenged mouse developed lung disease characteristic of moderate interstitial pneumonia. Further, we illustrated the utility of the system by demonstrating protection using an oral mRNA vaccine. The multicistronic vaccine design enabled by the viral self-cleaving peptides targets receptor binding domain (RBD), heptad repeat domain (HR), membrane glycoprotein (M) and epitopes of nsp13 of parental SARS-CoV-2. Further, Salmonella and Semliki Forest virus replicon were exploited, respectively, for gene delivery and mRNA expression. We recorded potent cross-protective neutralizing antibodies in immunized mice against the SARS-CoV-2 delta variant. The vaccine protected the mice against viral replication and SARS-CoV-2-induced weight loss and lung pathology. The findings support the suitability of the model for preclinical evaluation of anti-SARS-CoV-2 therapies and vaccines. In addition, the findings provide novel insights into mRNA vaccine design against infectious diseases not limiting to SARS-CoV-2.

Sustainable Antiviral Efficacy of Rejuvenated HIV-Specific Cytotoxic T Lymphocytes Generated from Induced Pluripotent Stem Cells.

Persistence of HIV latently infected cells is a barrier to HIV cure. The "kick and kill" strategy for a cure includes clearance of the viral reservoir by HIV-specific cytotoxic T lymphocytes (CTLs). However, exhaustion and senescence of T cells accelerates during HIV infection, and does not fully recover, despite complete viral suppression under antiretroviral therapy. We previously established an induced pluripotent stem cell (iPSC) from a parental HIV-specific CTL clone and generated an iPSC-derived rejuvenated HIV-specific CTL clone (iPSC-CTL), which exhibited an early memory phenotype, high proliferation capacity and effector functions in vitro. Here, we assessed the antiviral efficacy of the HIV-specific iPSC-CTL by single- and multiple-round viral suppression assays (VSAs). The HIV-specific iPSC-CTL suppressed viral replication in an HLA-dependent manner with equivalent efficacy to the parental CTL clone in single-round VSA. In multiple-round VSA, however, the ability of the iPSC-CTL to suppress viral replication was longer than that of the parental CTL clone. These results indicate that HIV-specific iPSC-CTL can sustainably exert suppressive pressure on viral replication, suggesting a novel approach to facilitate clearance of the HIV reservoir via adoptive transfer of rejuvenated CTLs. IMPORTANCE Elimination of latently HIV-infected cells is required for HIV cure. In the "kick and kill" strategy proposed for a cure to HIV, the host immune system, including HIV-specific cytotoxic T lymphocytes (CTLs), play a central role in eliminating HIV antigen-expressing cells following reactivation by latency-reversing agents (LRAs). However, CTL dysfunction due to exhaustion and senescence in chronic HIV infection can be an obstacle to this strategy. Adoptive transfer with effective HIV-specific CTLs may be a solution of this problem. We previously generated an induced pluripotent stem cell (iPSC)-derived rejuvenated HIV-specific CTL clone (iPSC-CTL) with high functional and proliferative capacity. The present study demonstrates that iPSC-CTL can survive and suppress HIV replication in vitro longer than the parental CTL clone, indicating the potential of iPSC-CTL to sustainably exert suppressive pressure on viral replication. Adoptive transfer with rejuvenated HIV-specific CTLs in combination with LRAs may be a new intervention strategy for HIV cure/remission.

The Spread of SARS-CoV-2 Variant Omicron with a Doubling Time of 2.0-3.3 Days Can Be Explained by Immune Evasion.

Omicron, the novel highly mutated SARS-CoV-2 Variant of Concern (VOC, Pango lineage B.1.1.529) was first collected in early November 2021 in South Africa. By the end of November 2021, it had spread and approached fixation in South Africa, and had been detected on all continents. We analyzed the exponential growth of Omicron over four-week periods in the two most populated of South Africa's provinces, Gauteng and KwaZulu-Natal, arriving at the doubling time estimates of, respectively, 3.3 days (95% CI: 3.2-3.4 days) and 2.7 days (95% CI: 2.3-3.3 days). Similar or even shorter doubling times were observed in other locations: Australia (3.0 days), New York State (2.5 days), UK (2.4 days), and Denmark (2.0 days). Log-linear regression suggests that the spread began in Gauteng around 11 October 2021; however, due to presumable stochasticity in the initial spread, this estimate can be inaccurate. Phylogenetics-based analysis indicates that the Omicron strain started to diverge between 6 October and 29 October 2021. We estimated that the weekly growth of the ratio of Omicron to Delta is in the range of 7.2-10.2, considerably higher than the growth of the ratio of Delta to Alpha (estimated to be in in the range of 2.5-4.2), and Alpha to pre-existing strains (estimated to be in the range of 1.8-2.7). High relative growth does not necessarily imply higher Omicron infectivity. A two-strain SEIR model suggests that the growth advantage of Omicron may stem from immune evasion, which permits this VOC to infect both recovered and fully vaccinated individuals. As we demonstrated within the model, immune evasion is more concerning than increased transmissibility, because it can facilitate larger epidemic outbreaks.

Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells.

Influenza neuraminidase (NA) is implicated in various aspects of the virus replication cycle and therefore is an attractive target for vaccination and antiviral strategies. Here we investigated the potential for NA-specific antibodies to interfere with A(H1N1)pdm09 replication in primary human airway epithelial (HAE) cells. Mouse polyclonal anti-NA sera and a monoclonal antibody could block initial viral entry into HAE cells as well as egress from the cell surface. NA-specific polyclonal serum also reduced virus replication across multiple rounds of infection. Restriction of virus entry correlated with the ability of the serum or monoclonal antibody to mediate neuraminidase inhibition (NI). Finally, human sera with NI activity against the N1 of A(H1N1)pdm09 could decrease H6N1 virus infection of HAE cells, highlighting the potential contribution of anti-NA antibodies in the control of influenza virus infection in humans.

Macrophages activated by hepatitis B virus have distinct metabolic profiles and suppress the virus via IL-1ß to downregulate PPARα and FOXO3.

Macrophages display phenotypic plasticity and can be induced by hepatitis B virus (HBV) to undergo either M1-like pro-inflammatory or M2-like anti-inflammatory polarization. Here, we report that M1-like macrophages stimulated by HBV exhibit a strong HBV-suppressive effect, which is diminished in M2-like macrophages. Transcriptomic analysis reveals that HBV induces the expression of interleukin-1ß (IL-1ß) in M1-like macrophages, which display a high oxidative phosphorylation (OXPHOS) activity distinct from that of conventional M1-like macrophages. Further analysis indicates that OXPHOS attenuates the expression of IL-1ß, which suppresses the expression of peroxisome proliferator-activated receptor α (PPARα) and forkhead box O3 (FOXO3) in hepatocytes to suppress HBV gene expression and replication. Moreover, multiple HBV proteins can induce the expression of IL-1ß in macrophages. Our results thus indicate that macrophages can respond to HBV by producing IL-1ß to suppress HBV replication. However, HBV can also metabolically reprogram macrophages to enhance OXPHOS to minimize this host antiviral response.

Reduced Replication of Highly Pathogenic Avian Influenza Virus in Duck Endothelial Cells Compared to Chicken Endothelial Cells Is Associated with Stronger Antiviral Responses.

Highly pathogenic avian influenza viruses (HPAIVs) cause fatal systemic infections in chickens, which are associated with endotheliotropism. HPAIV infections in wild birds are generally milder and not endotheliotropic. Here, we aimed to elucidate the species-specific endotheliotropism of HPAIVs using primary chicken and duck aortic endothelial cells (chAEC and dAEC respectively). Viral replication kinetics and host responses were assessed in chAEC and dAEC upon inoculation with HPAIV H5N1 and compared to embryonic fibroblasts. Although dAEC were susceptible to HPAIV upon inoculation at high multiplicity of infection, HPAIV replicated to lower levels in dAEC than chAEC during multi-cycle replication. The susceptibility of duck embryonic endothelial cells to HPAIV was confirmed in embryos. Innate immune responses upon HPAIV inoculation differed between chAEC, dAEC, and embryonic fibroblasts. Expression of the pro-inflammatory cytokine IL8 increased in chicken cells but decreased in dAEC. Contrastingly, the induction of antiviral responses was stronger in dAEC than in chAEC, and chicken and duck fibroblasts. Taken together, these data demonstrate that although duck endothelial cells are permissive to HPAIV infection, they display markedly different innate immune responses than chAEC and embryonic fibroblasts. These differences may contribute to the species-dependent differences in endotheliotropism and consequently HPAIV pathogenesis.

Elimination of Aicardi-Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication.

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi-Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR-Cas9 gene KO or lentiviral viral protein X-mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2-infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.

Modeling Innate Antiviral Immunity in Physiological Context.

An effective innate antiviral response is critical for the mitigation of severe disease and host survival following infection. In vivo, the innate antiviral response is triggered by cells that detect the invading pathogen and then communicate through autocrine and paracrine signaling to stimulate the expression of genes that inhibit viral replication, curtail cell proliferation, or modulate the immune response. In other words, the innate antiviral response is complex and dynamic. Notably, in the laboratory, culturing viruses and assaying viral life cycles frequently utilizes cells that are derived from tissues other than those that support viral replication during natural infection, while the study of viral pathogenesis often employs animal models. In recapitulating the human antiviral response, it is important to consider that variation in the expression and function of innate immune sensors and antiviral effectors exists across species, cell types, and cell differentiation states, as well as when cells are placed in different contexts. Thus, to gain novel insight into the dynamics of the host response and how specific sensors and effectors impact infection kinetics by a particular virus, the model system must be selected carefully. In this review, we briefly introduce key signaling pathways involved in the innate antiviral response and highlight how these differ between systems. We then review the application of tissue-engineered or 3D models for studying the antiviral response, and suggest how these in vitro culture systems could be further utilized to assay physiologically-relevant host responses and reveal novel insight into virus-host interactions.