Phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response.

Stress granules (SGs), the main component is GTPase-activating protein-binding protein 1 (G3BP1), which are assembled during viral infection and function to sequester host and viral mRNAs and proteins, are part of the antiviral responses. In this study, we found that porcine deltacoronavirus (PDCoV) infection induced stable formation of robust SGs in cells through a PERK (protein kinase R-like endoplasmic reticulum kinase)-dependent mechanism. Overexpression of SGs marker proteins G3BP1 significantly reduced PDCoV replication in vitro, while inhibition of endogenous G3BP1 enhanced PDCoV replication. Moreover, PDCoV infected LLC-PK1 cells raise the phosphorylation level of G3BP1. By overexpression of the G3BP1 phosphorylated protein or the G3BP1 dephosphorylated protein, we found that phosphorylation of G3BP1 is involved in the regulation of PDCoV-induced inflammatory response. Taken together, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets for antiviral target.

2-Bromopalmitate depletes lipid droplets to inhibit viral replication.

The global impact of emerging viral infections emphasizes the urgent need for effective broad-spectrum antivirals. The cellular organelle, lipid droplet (LD), is utilized by many types of viruses for replication, but its reduction does not affect cell survival. Therefore, LD is a potential target for developing broad-spectrum antivirals. In this study, we found that 2-bromopalmitate (2 BP), a previously defined palmitoylation inhibitor, depletes LD across all studied cell lines and exerts remarkable antiviral effects on different coronaviruses. We comprehensively utilized 2 BP, alongside other palmitoylation inhibitors such as cerulenin and 2-fluoro palmitic acid (2-FPA), as well as the enhancer palmostatin B and evaluated their impact on LD and the replication of human coronaviruses (hCoV-229E, hCoV-Oc43) and murine hepatitis virus (MHV-A59) at non-cytotoxic concentrations. While cerulenin and 2-FPA exhibited moderate inhibition of viral replication, 2 BP exhibited a much stronger suppressive effect on MHV-A59 replication, although they share similar inhibitory effects on palmitoylation. As expected, palmostatin B significantly enhanced viral replication, it failed to rescue the inhibitory effects of 2 BP, whereas it effectively counteracted the effects of cerulenin and 2-FPA. This suggests that the mechanism that 2 BP used to inhibit viral replication is beyond palmitoylation inhibition. Further investigations unveil that 2 BP uniquely depletes LDs, a phenomenon not exhibited by 2-FPA and cerulenin. Importantly, the depletion of LDs was closely associated with the inhibition of viral replication because the addition of oleic acid to 2 BP significantly rescued LD depletion and its inhibitory effects on MHV-A59. Our findings indicate that the inhibitory effects of 2 BP on viral replication primarily stem from LD disruption rather than palmitoylation inhibition. Intriguingly, fatty acid (FA) assays demonstrated that 2 BP reduces the FA level in mitochondria while concurrently increasing FA levels in the cytoplasm. These results highlight the crucial role of LDs in viral replication and uncover a novel biological activity of 2 BP. These insights contribute to the development of broad-spectrum antiviral strategies. IMPORTANCE: In our study, we conducted a comparative investigation into the antiviral effects of palmitoylation inhibitors including 2-bromopalmitate (2-BP), 2-fluoro palmitic acid (2-FPA), and cerulenin. Surprisingly, we discovered that 2-BP has superior inhibitory effects on viral replication compared to 2-FPA and cerulenin. However, their inhibitory effects on palmitoylation were the same. Intrigued by this finding, we delved deeper into the underlying mechanism of 2-BP's potent antiviral activity, and we unveiled a novel biological activity of 2-BP: depletion of lipid droplets (LDs). Importantly, we also highlighted the crucial role of LDs in viral replication. Our insights shed new light on the antiviral mechanism of LD depletion paving the way for the development of broad-spectrum antiviral strategies by targeting LDs.

Gut microbiota-derived butyrate promotes coronavirus TGEV infection through impairing RIG-I-triggered local type I interferon responses via class I HDAC inhibition.

Gut microbiota-derived metabolites are important for the replication and pathogenesis of many viruses. However, the roles of bacterial metabolites in swine enteric coronavirus (SECoV) infection remain poorly understood. Recent studies show that SECoVs infection in vivo significantly alters the composition of short-chain fatty acids (SCFAs)-producing gut microbiota. This prompted us to investigate whether and how SCFAs impact SECoV infection. Employing alphacoronavirus transmissible gastroenteritis virus (TGEV), a major cause of diarrhea in piglets, as a model, we found that SCFAs, particularly butyrate, enhanced TGEV infection both in porcine intestinal epithelial cells and swine testicular (ST) cells at the late stage of viral infection. This effect depended on the inhibited productions of virus-induced type I interferon (IFN) and downstream antiviral IFN-stimulated genes (ISGs) by butyrate. Mechanistically, butyrate suppressed the expression of retinoic acid-inducible gene I (RIG-I), a key viral RNA sensor, and downstream mitochondrial antiviral-signaling (MAVS) aggregation, thereby impairing type I IFN responses and increasing TGEV replication. Using pharmacological and genetic approaches, we showed that butyrate inhibited RIG-I-induced type I IFN signaling by suppressing class I histone deacetylase (HDAC). In summary, we identified a novel mechanism where butyrate enhances TGEV infection by suppressing RIG-I-mediated type I IFN responses. Our findings highlight that gut microbiota-derived metabolites like butyrate can be exploited by SECoV to dampen innate antiviral immunity and establish infection in the intestine.IMPORTANCESwine enteric coronaviruses (SECoVs) infection in vivo alters the composition of short-chain fatty acids (SCFAs)-producing gut microbiota, but whether microbiota-derived SCFAs impact coronavirus gastrointestinal infection is largely unknown. Here, we demonstrated that SCFAs, particularly butyrate, substantially increased alphacoronavirus TGEV infection at the late stage of infection, without affecting viral attachment or internalization. Furthermore, enhancement of TGEV by butyrate depended on impeding virus-induced type I interferon (IFN) responses. Mechanistically, butyrate suppressed the cytoplasmic viral RNA sensor RIG-I expression and downstream type I IFN signaling activation by inhibiting class I HDAC, thereby promoting TGEV infection. Our work reveals novel functions of gut microbiota-derived SCFAs in enhancing enteric coronavirus infection by impairing RIG-I-dependent type I IFN responses. This implies that bacterial metabolites could be therapeutic targets against SECoV infection by modulating antiviral immunity in the intestine.

Cleavage of HDAC6 to dampen its antiviral activity by nsp5 is a common strategy of swine enteric coronaviruses.

HDAC6, a structurally and functionally unique member of the histone deacetylase (HDAC) family, is an important host factor that restricts viral infection. The broad-spectrum antiviral activity of HDAC6 makes it a potent antiviral agent. Previously, we found that HDAC6 functions to antagonize porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. However, the final outcome is typically a productive infection that materializes as cells succumb to viral infection, indicating that the virus has evolved sophisticated mechanisms to combat the antiviral effect of HDAC6. Here, we demonstrate that PDCoV nonstructural protein 5 (nsp5) can cleave HDAC6 at glutamine 519 (Q519), and cleavage of HDAC6 was also detected in the context of PDCoV infection. More importantly, the anti-PDCoV activity of HDAC6 was damaged by nsp5 cleavage. Mechanistically, the cleaved HDAC6 fragments (amino acids 1-519 and 520-1159) lost the ability to degrade PDCoV nsp8 due to their impaired deacetylase activity. Furthermore, nsp5-mediated cleavage impaired the ability of HDAC6 to activate RIG-I-mediated interferon responses. We also tested three other swine enteric coronaviruses (transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and swine acute diarrhea syndrome-coronavirus) and found that all these coronaviruses have adopted similar mechanisms to cleave HDAC6 in both an overexpression system and virus-infected cells, suggesting that cleavage of HDAC6 is a common strategy utilized by swine enteric coronaviruses to antagonize the host's antiviral capacity. Together, these data illustrate how swine enteric coronaviruses antagonize the antiviral function of HDAC6 to maintain their infection, providing new insights to the interaction between virus and host.IMPORTANCEViral infections and host defenses are in constant opposition. Once viruses combat or evade host restriction, productive infection is achieved. HDAC6 is a broad-spectrum antiviral protein that has been demonstrated to inhibit many viruses, including porcine deltacoronavirus (PDCoV). However, whether HDAC6 is reciprocally targeted and disabled by viruses remains unclear. In this study, we used PDCoV as a model and found that HDAC6 is targeted and cleaved by nsp5, a viral 3C-like protease. The cleaved HDAC6 loses its deacetylase activity as well as its ability to degrade viral proteins and activate interferon responses. Furthermore, this cleavage mechanism is shared among other swine enteric coronaviruses. These findings shed light on the intricate interplay between viruses and HDAC6, highlighting the strategies employed by viruses to evade host antiviral defenses.

Porcine delta coronavirus inhibits NHE3 activity of porcine intestinal epithelial cells through miR-361-3p/NHE3 regulatory axis.

Porcine deltacoronavirus (PDCoV) infection in piglets can cause small intestinal epithelial necrosis and atrophic enteritis, which leads to severe damages to host cells, and result in diarrhea. In this study, we investigated the relationship between miR-361, SLC9A3(Solute carrier family 9, subfamily A, member 3), and NHE3(sodium-hydrogen exchanger member 3) in in porcine intestinal epithelial cells (IPI-2I) cells after PDCoV infection. Our results showed that the ssc-miR-361-3p expression inhibits the mRNA level of SLC9A3 gene which lead to the descending of NHE3 protein expression, and the NHE3 activity was suppressed. NHE3 activity was suppressed via down-regulation expression of SLC9A3 mRNA by transfection with siRNA. Ssc-miR-361-3p mimics and inhibitors were used to change the expression of ssc-miR-361-3p in IPI-2I cells. Ssc-miR-361-3p overexpression reduced the mRNA level of SLC9A3 gene, the level of NHE3 protein expression and NHE3 activity in IPI-2I cells, while ssc-miR-361-3p inhibits NHE3. Furthermore, luciferase reporter assay showed that SLC9A3 gene was a direct target of ssc-miR-361-3p. Ssc-miR-361-3p inhibition restored NHE3 activity in PDCoV infected IPI-2I cells by up-regulating SLC9A3 mRNA expression and NHE3 protein expression. These results demonstrate that the PDCoV infection can inhibit NHE3 activity through miR-361-3p/SLC9A3 regulatory axis. The relevant research is reported for the first time in PDCoV, which has significance in exploring the pathogenic mechanism of PDCoV and can provide a theoretical basis for its prevention and control. suggesting that NHE3 and ssc-miR-361-3p may be potential therapeutic targets for diarrhea in infected piglets.

DHA and EPA inhibit porcine coronavirus replication by alleviating ER stress.

IMPORTANCE: Porcine epidemic diarrhea caused by porcine coronaviruses remains a major threat to the global swine industry. Fatty acids are extensively involved in the whole life of the virus. In this study, we found that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) significantly reduced the viral load of porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine delta coronavirus (PDCoV) and acted on the replication of the viruses rather than attachment and entry. We further confirmed that DHA and EPA inhibited PEDV replication by alleviating the endoplasmic reticulum stress. Meanwhile, DHA and EPA alleviate PEDV-induced inflammation and reactive oxygen species (ROS) levels and enhance the cellular antioxidant capacity. These data indicate that DHA and EPA have antiviral effects on porcine coronaviruses and provide a molecular basis for the development of new fatty acid-based therapies to control porcine coronavirus infection and transmission.

On a path toward a broad-spectrum anti-viral: inhibition of HIV-1 and coronavirus replication by SR kinase inhibitor harmine.

IMPORTANCE: This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4+ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.

Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

Porcine deltacoronavirus resists antibody neutralization through cell-to-cell transmission.

ABSTRACTPorcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus that has been reported to infect a variety of animals and even humans. Cell-cell fusion has been identified as an alternative pathway for the cell-to-cell transmission of certain viruses, but the ability of PDCoV to exploit this transmission model, and the relevant mechanisms, have not been fully elucidated. Herein, we provide evidence that cell-to-cell transmission is the main mechanism supporting PDCoV spread in cell culture and that this efficient spread model is mediated by spike glycoprotein-driven cell-cell fusion. We found that PDCoV efficiently spread to non-susceptible cells via cell-to-cell transmission, and demonstrated that functional receptor porcine aminopeptidase N and cathepsins in endosomes are involved in the cell-to-cell transmission of PDCoV. Most importantly, compared with non-cell-to-cell infection, the cell-to-cell transmission of PDCoV was resistant to neutralizing antibodies and immune sera that potently neutralized free viruses. Taken together, our study revealed key characteristics of the cell-to-cell transmission of PDCoV and provided new insights into the mechanism of PDCoV infection.

Pangolin merbecovirus gets down to (poly)basics.

Trafficking of live mammals is considered a major risk for emergence of zoonotic viruses. SARS-CoV-2-related coronaviruses have previously been identified in pangolins, the world's most smuggled mammal. A new study identifies a MERS-related coronavirus in trafficked pangolins with broad mammalian tropism and a newly acquired furin cleavage site in Spike.

A bat MERS-like coronavirus circulates in pangolins and utilizes human DPP4 and host proteases for cell entry.

It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.

Porcine deltacoronavirus uses heparan sulfate as an attachment receptor.

Porcine deltacoronavirus (PDCoV) is a newly emerging swine enteropathogenic coronavirus with extensive tissue tropism and cross-species transmission potential. Heparan sulfate (HS) is a complex polysaccharide ubiquitously expressed on cell surfaces and the extracellular matrix and acts as an attachment factor for many viruses. However, whether PDCoV uses HS as an attachment receptor is unclear. In this study, we found that treatment with heparin sodium or heparinase Ⅱ significantly inhibited PDCoV binding and infection among LLC-PK1 and IPI-2I cells. Attenuation of HS sulfuration by sodium chlorate also impeded PDCoV binding and infection. Moreover, we demonstrated that HS functioned independently of amino peptidase N (APN), a functional PDCoV receptor, in PDCoV infection. Molecular docking revealed that the S1 subunit of the PDCoV spike protein might be a putative region for HS binding. Taken together, these results firstly confirmed that HS is an attachment receptor for PDCoV infection, providing new insight into better understanding the mechanisms of PDCoV-host interactions.

Exploration of PDCoV-induced apoptosis through mitochondrial dynamics imbalance and the antagonistic effect of SeNPs.

Porcine Deltacoronavirus (PDCoV), an enveloped positive-strand RNA virus that causes respiratory and gastrointestinal diseases, is widely spread worldwide, but there is no effective drug or vaccine against it. This study investigated the optimal Selenium Nano-Particles (SeNPs) addition concentration (2 - 10 µg/mL) and the mechanism of PDCoV effect on ST (Swine Testis) cell apoptosis, the antagonistic effect of SeNPs on PDCoV. The results indicated that 4 µg/mL SeNPs significantly decreased PDCoV replication on ST cells. SeNPs relieved PDCoV-induced mitochondrial division and antagonized PDCoV-induced apoptosis via decreasing Cyt C release and Caspase 9 and Caspase 3 activation. The above results provided an idea and experimental basis associated with anti-PDCoV drug development and clinical use.

Stromal Antigen 2 Deficiency Induces Interferon Responses and Restricts Porcine Deltacoronavirus Infection.

Porcine deltacoronavirus (PDCoV) is a recently discovered enteropathogenic coronavirus and has caused significant economic impacts on the pork industry. Although studies have partly uncovered the molecular mechanism of PDCoV-host interaction, it requires further research. In this study, we explored the roles of Stromal Antigen 2 (STAG2) in PDCoV infection. We found that STAG2-deficient cells inhibited infection with vesicular stomatitis virus (VSV) and PDCoV, whereas restoration of STAG2 expression in STAG2-depleted (STAG2-/-) IPEC-J2 cells line restored PDCoV infection, suggesting that STAG2 is involved in the PDCoV replication. Furthermore, we found that STAG2 deficiency results in robust interferon (IFN) expression. Subsequently, we found that STAG2 deficiency results in the activation of JAK-STAT signaling and the expression of IFN stimulated gene (ISG), which establish an antiviral state. Taken together, the depletion of STAG2 activates the JAK-STAT signaling and induces the expression of ISG, thereby inhibiting PDCoV replication. Our study provides new insights and potential therapeutic targets for unraveling the mechanism of PDCoV replication.

Autophagy Modulators in Coronavirus Diseases: A Double Strike in Viral Burden and Inflammation.

Coronaviruses are the etiologic agents of several diseases. Coronaviruses of critical medical importance are characterized by highly inflammatory pathophysiology, involving severe pulmonary impairment and infection of multiple cell types within the body. Here, we discuss the interplay between coronaviruses and autophagy regarding virus life cycle, cell resistance, and inflammation, highlighting distinct mechanisms by which autophagy restrains inflammatory responses, especially those involved in coronavirus pathogenesis. We also address different autophagy modulators available and the rationale for drug repurposing as an attractive adjunctive therapy. We focused on pharmaceuticals being tested in clinical trials with distinct mechanisms but with autophagy as a common target. These autophagy modulators act in cell resistance to virus infection and immunomodulation, providing a double-strike to prevent or treat severe disease development and death from coronaviruses diseases.

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus.

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike-biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.

Cross-reactive memory T cells associate with protection against SARS-CoV-2 infection in COVID-19 contacts.

Cross-reactive immune responses to SARS-CoV-2 have been observed in pre-pandemic cohorts and proposed to contribute to host protection. Here we assess 52 COVID-19 household contacts to capture immune responses at the earliest timepoints after SARS-CoV-2 exposure. Using a dual cytokine FLISpot assay on peripheral blood mononuclear cells, we enumerate the frequency of T cells specific for spike, nucleocapsid, membrane, envelope and ORF1 SARS-CoV-2 epitopes that cross-react with human endemic coronaviruses. We observe higher frequencies of cross-reactive (p = 0.0139), and nucleocapsid-specific (p = 0.0355) IL-2-secreting memory T cells in contacts who remained PCR-negative despite exposure (n = 26), when compared with those who convert to PCR-positive (n = 26); no significant difference in the frequency of responses to spike is observed, hinting at a limited protective function of spike-cross-reactive T cells. Our results are thus consistent with pre-existing non-spike cross-reactive memory T cells protecting SARS-CoV-2-naïve contacts from infection, thereby supporting the inclusion of non-spike antigens in second-generation vaccines.

Viral burden and diversity in acute respiratory tract infections in hospitalized children in wet and dry zones of Sri Lanka.

The present study was done to identify the viral diversity, seasonality and burden associated with childhood acute respiratory tract infection (ARTI) in Sri Lanka. Nasopharyngeal aspirates (NPA) of hospitalized children (1 month-5 years) with ARTI were collected in 2 centers (wet and dry zones) from March 2013 to August 2014. Respiratory viral antigen detection by immunofluorescence assay (IFA) was used to identify the infecting viruses. IFA negative 100 NPA samples were tested for human metapeumovirus (hMPV), human bocavirus and corona viruses by polymerase chain reaction. Of the 443 and 418 NPAs, 37.2% and 39.4% were positive for any of the 8 different respiratory viruses tested from two centers studied. Viral co-infection was detected with respiratory syncytial virus (RSV) in both centers. Peak viral detection was noted in the wet zone from May-July 2013 and 2014 and in the dry zone from December-January 2014 suggesting a local seasonality for viral ARTI. RSV showed a clear seasonality with a direct correlation of monthly RSV infections with rainy days in the wet zone and an inverse correlation with temperature in both centers. The case fatality rate was 2.7% for RSV associated ARTI. The overall disability adjusted life years was 335.9 and for RSV associated ARTI it was 241.8. RSV was the commonly detected respiratory virus with an annual seasonality and distribution in rainy seasons in the dry and wet zones of Sri Lanka. Identifying the virus and seasonality will contribute to employ preventive measures and reduce the empirical use of antibiotics in resource limited settings.

CRISPR Tackles Emerging Viral Pathogens.

Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics.