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Respiratory syncytial virus (RSV) causes severe infections in infants, immunocompromised or elderly individuals resulting in annual epidemics of respiratory disease. Currently, limited clinical surveillance and the lack of predictable seasonal dynamics limits the public health response. Wastewater-based epidemiology (WBE) has recently been used globally as a key metric in determining prevalence of SARS-CoV-2 in the community but its application to other respiratory viruses is limited. In this study, we present an integrated genomic WBE approach, applying RT-qPCR and partial G-gene sequencing to track RSV levels and variants in the community. We report increasing detection of RSV in wastewater concomitant with increasing numbers of positive clinical cases. Analysis of wastewater-derived RSV sequences permitted identification of distinct circulating lineages within and between seasons. Altogether, our genomic WBE platform has the potential to complement ongoing global surveillance and aid the management of RSV by informing the timely deployment of pharmaceutical and non-pharmaceutical interventions.
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Patients with decompensated liver cirrhosis, in particular those classified as Childs-Pugh class C, are at increased risk of severe coronavirus disease-2019 (COVID-19) upon infection with severe acute respiratory coronavirus 2 (SARS-CoV-2). The biological mechanisms underlying this are unknown. We aimed to examine the levels of serum intrinsic antiviral proteins as well as alterations in the innate antiviral immune response in patients with decompensated liver cirrhosis. Serum from 53 SARS-CoV-2 unexposed and unvaccinated individuals, with decompensated liver cirrhosis undergoing assessment for liver transplantation, were screened using SARS-CoV-2 pseudoparticle and SARS-CoV-2 virus assays. The ability of serum to inhibit interferon (IFN) signalling was assessed using a cell-based reporter assay. Severity of liver disease was assessed using two clinical scoring systems, the Child-Pugh class and the MELD-Na score. In the presence of serum from SARS-CoV-2 unexposed patients with decompensated liver cirrhosis there was no association between SARS-CoV-2 pseudoparticle infection or live SARS-CoV-2 virus infection and severity of liver disease. Type I IFNs are a key component of the innate antiviral response. Serum from patients with decompensated liver cirrhosis contained elevated levels of auto-antibodies capable of binding IFN-α2b compared to healthy controls. High MELD-Na scores were associated with the ability of these auto-antibodies to neutralize type I IFN signalling by IFN-α2b but not IFN-ß1a. Our results demonstrate that neutralizing auto-antibodies targeting IFN-α2b are increased in patients with high MELD-Na scores. The presence of neutralizing type I IFN-specific auto-antibodies may increase the likelihood of viral infections, including severe COVID-19, in patients with decompensated liver cirrhosis.
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COVID-19 , Interferon Tipo I , Hepatopatias , Transplante de Fígado , Humanos , Anticorpos , Cirrose HepáticaRESUMO
Antiviral defenses can sense viral RNAs and mediate their destruction. This presents a challenge for host cells since they must destroy viral RNAs while sparing the host mRNAs that encode antiviral effectors. Here, we show that highly upregulated interferon-stimulated genes (ISGs), which encode antiviral proteins, have distinctive nucleotide compositions. We propose that self-targeting by antiviral effectors has selected for ISG transcripts that occupy a less self-targeted sequence space. Following interferon (IFN) stimulation, the CpG-targeting antiviral effector zinc-finger antiviral protein (ZAP) reduces the mRNA abundance of multiple host transcripts, providing a mechanistic explanation for the repression of many (but not all) interferon-repressed genes (IRGs). Notably, IRGs tend to be relatively CpG rich. In contrast, highly upregulated ISGs tend to be strongly CpG suppressed. Thus, ZAP is an example of an effector that has not only selected compositional biases in viral genomes but also appears to have notably shaped the composition of host transcripts in the vertebrate interferome.
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Fosfatos de Dinucleosídeos , Fatores Reguladores de Interferon/genética , RNA Viral , Proteínas de Ligação a RNA/metabolismo , Células A549 , Linhagem Celular , Humanos , Interferon beta/farmacologia , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Fenômenos Fisiológicos Virais , VírusRESUMO
The emergence of SARS-CoV-2 variants has exacerbated the COVID-19 global health crisis. Thus far, all variants carry mutations in the spike glycoprotein, which is a critical determinant of viral transmission being responsible for attachment, receptor engagement and membrane fusion, and an important target of immunity. Variants frequently bear truncations of flexible loops in the N-terminal domain (NTD) of spike; the functional importance of these modifications has remained poorly characterised. We demonstrate that NTD deletions are important for efficient entry by the Alpha and Omicron variants and that this correlates with spike stability. Phylogenetic analysis reveals extensive NTD loop length polymorphisms across the sarbecoviruses, setting an evolutionary precedent for loop remodelling. Guided by these analyses, we demonstrate that variations in NTD loop length, alone, are sufficient to modulate virus entry. We propose that variations in NTD loop length act to fine-tune spike; this may provide a mechanism for SARS-CoV-2 to navigate a complex selection landscape encompassing optimisation of essential functionality, immune-driven antigenic variation and ongoing adaptation to a new host.
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COVID-19 , SARS-CoV-2 , COVID-19/genética , Humanos , Filogenia , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Wastewater Based Epidemiology (WBE) has become an integral part of the public health effort to track the levels of SARS-CoV-2 within communities. Detection of SARS-CoV-2 in wastewater can be challenging due to relatively low levels of virus within the sample. The wastewater matrix is also comprised of commercial and domestically derived contaminants, as well as RNases, all of which can adversely affect RT-qPCR analysis. To improve SARS-CoV-2 detection within wastewater samples we investigated both the effect of template dilution (as a means to reduce RT-qPCR inhibition) and sample stabilisation via addition of DNA/RNA Shield™ and/or RNA Later™ (to prevent RNA degradation via RNases) as a means to improve viral fragment detection. Using both methodologies, a significant improvement in SARS-CoV-2 detection from wastewater samples was observed. No adverse effects of stabilising agent addition on downstream Next-Generation Sequencing workflows were detected.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Excipientes , Águas Residuárias , RNA , RNA Viral/genéticaRESUMO
IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α-pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.
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Citocinas/deficiência , Resistência à Doença/genética , Interferon-alfa/metabolismo , Infecções por Paramyxoviridae/imunologia , Transdução de Sinais/imunologia , Ubiquitinas/deficiência , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Chlorocebus aethiops , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Vírus da Parainfluenza 2 Humana/imunologia , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 5/imunologia , Infecções por Paramyxoviridae/virologia , Processamento de Proteína Pós-Traducional/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , Enzimas Ativadoras de Ubiquitina/genética , Células VeroRESUMO
Host IFNL4 haplotype status contributes to the development of chronic hepatitis C virus (HCV) infection in individuals who are acutely infected with the virus. In silico studies revealed that specific amino acid variants at multiple sites on the HCV polyprotein correlate with functional single-nucleotide polymorphisms (SNPs) in the IFNL4 locus. Thus, SNPs at the IFNL4 locus may select variants that influence virus replication and thereby the outcome of infection. Here, we examine the most significantly IFNL4-associated amino acid variants that lie in the 'lambda (L) 2 loop' of the HCV NS5B RNA polymerase. L2 loop variants were introduced into both sub-genomic replicon and full-length infectious clones of HCV and viral replication was examined in the presence and absence of exogenous IFNλ4. Our data demonstrate that while mutation of the NS5B L2 loop affects replication, individual IFNL4-associated variants have modest but consistent effects on replication in both the presence and absence of IFNλ4. Given the strong genetic association between these variants and IFNL4, these data suggest a nuanced effect of each individual position on viral replication, the combined effect of which might mediate resistance to the effects of IFNλ4.
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Hepacivirus/genética , Interleucinas/genética , Replicação Viral/genética , RNA Polimerases Dirigidas por DNA/genética , Haplótipos , Hepatite C Crônica/virologia , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
As antimicrobial signalling molecules, type III or lambda interferons (IFNλs) are critical for defence against infection by diverse pathogens, including bacteria, fungi and viruses. Counter-intuitively, expression of one member of the family, IFNλ4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural frameshift mutation that abrogates IFNλ4 production improves HCV clearance. To further understand how genetic variation between and within species affects IFNλ4 function, we screened a panel of all known extant coding variants of human IFNλ4 for their antiviral potential and identify three that substantially affect activity: P70S, L79F and K154E. The most notable variant was K154E, which was found in African Congo rainforest 'Pygmy' hunter-gatherers. K154E greatly enhanced in vitro activity in a range of antiviral (HCV, Zika virus, influenza virus and encephalomyocarditis virus) and gene expression assays. Remarkably, E154 is the ancestral residue in mammalian IFNλ4s and is extremely well conserved, yet K154 has been fixed throughout evolution of the hominid genus Homo, including Neanderthals. Compared to chimpanzee IFNλ4, the human orthologue had reduced activity due to amino acid K154. Comparison of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFNλ4 correlates with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data show that the human-specific K154 negatively affects IFNλ4 activity through a novel means by reducing its secretion and potency. We thus demonstrate that attenuated activity of IFNλ4 is conserved among humans and postulate that differences in IFNλ4 activity between species contribute to distinct host-specific responses to-and outcomes of-infection, such as HCV infection. The driver of reduced IFNλ4 antiviral activity in humans remains unknown but likely arose between 6 million and 360,000 years ago in Africa.
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Antivirais/uso terapêutico , Infecções por Cardiovirus/tratamento farmacológico , Hepatite C/tratamento farmacológico , Interleucinas/genética , Polimorfismo de Nucleotídeo Único , Infecção por Zika virus/tratamento farmacológico , Animais , Evolução Biológica , Infecções por Cardiovirus/genética , Infecções por Cardiovirus/virologia , Células Cultivadas , Vírus da Encefalomiocardite/efeitos dos fármacos , Vírus da Encefalomiocardite/isolamento & purificação , Regulação da Expressão Gênica , Hepacivirus/efeitos dos fármacos , Hepacivirus/isolamento & purificação , Hepatite C/genética , Hepatite C/virologia , Humanos , Pan troglodytes , Especificidade da Espécie , Zika virus/efeitos dos fármacos , Zika virus/isolamento & purificação , Infecção por Zika virus/genética , Infecção por Zika virus/virologiaAssuntos
COVID-19 , SARS-CoV-2 , Animais , Técnicas de Cultura de Células , Humanos , Estágios do Ciclo de VidaRESUMO
Mumps is caused by the mumps virus (MuV), a member of the Paramyxoviridae family of enveloped, non-segmented, negative-sense RNA viruses. Mumps is characterized by painful inflammatory symptoms, such as parotitis and orchitis. The virus is highly neurotropic, with laboratory evidence of central nervous system (CNS) infection in approximately half of cases. Symptomatic CNS infection occurs less frequently; nonetheless, prior to the introduction of routine vaccination, MuV was a leading cause of aseptic meningitis and viral encephalitis in many developed countries. Despite being one of the oldest recognized diseases, with a worldwide distribution, surprisingly little attention has been given to its study. Cases of aseptic meningitis associated with some vaccine strains and a global resurgence of cases, including in highly vaccinated populations, has renewed interest in the virus, particularly in its pathogenesis and the need for development of clinically relevant models of disease. In this review we summarize the current state of knowledge on the virus, its pathogenesis and its clinical and pathological outcomes.
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Vírus da Caxumba/patogenicidade , Caxumba/patologia , Caxumba/virologia , Patologia Molecular/métodos , Animais , Biópsia , Modelos Animais de Doenças , Genótipo , Interações Hospedeiro-Patógeno , Humanos , Caxumba/epidemiologia , Caxumba/prevenção & controle , Vacina contra Caxumba/uso terapêutico , Vírus da Caxumba/genética , Valor Preditivo dos Testes , Prognóstico , Virologia/métodos , VirulênciaRESUMO
Chronic infection of the liver by hepatitis C virus (HCV) induces a range of host factors including IFN-stimulated genes such as ISG15. ISG15 functions as an antiviral factor that limits virus replication. Previous studies have suggested that ISG15 could influence HCV replication in both a positive and a negative manner. In this report, we determined the effect of ISG15 on HCV RNA replication in two independent cell lines that support viral genome synthesis by inhibiting ISG15 expression through small interfering RNA, short-hairpin RNA and CRISPR/Cas9 gene knockout approaches. Our results demonstrated that ISG15 impairs HCV RNA replication in both the presence and absence of IFN stimulation, consistent with an antiviral role for ISG15 during HCV infection. ISG15 conjugation to protein substrates typically requires the E3 ligase, HERC5. Our results showed that the inhibitory effect of ISG15 on HCV RNA replication does not require its conjugation to substrates by HERC5.
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Citocinas/imunologia , Hepacivirus/genética , Hepatite C/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , RNA Viral/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/imunologia , Citocinas/genética , Hepacivirus/fisiologia , Hepatite C/genética , Hepatite C/virologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA Viral/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genética , Replicação ViralRESUMO
As of 8 July 2022, the World Health Organization (WHO) have reported 1010 probable cases of acute hepatitis of unknown aetiology in children worldwide, including approximately 250 cases in the United Kingdom (UK). Clinical presentations have often been severe, with liver transplantation a frequent clinical outcome. Human adenovirus F41 (HAdV-F41) has been detected in most children with acute hepatitis, but its role in the pathogenesis of this infection has yet to be established. Wastewater-based epidemiology (WBE) has become a well-established tool for monitoring the community spread of SARS-CoV-2, as well as other pathogens and chemicals. In this study, we adopted a WBE approach to monitoring levels of HAdV-F40/41 in wastewater before and during an acute hepatitis outbreak in Northern Ireland. We report increasing detection of HAdV-F40/41 in wastewater, concomitant with increasing numbers of clinical cases. Amplicon whole genome sequencing further classified the wastewater-derived HAdV as belonging to the F41 genotype which in turn was homologous to clinically derived sequences. We propose that WBE has the potential to inform community surveillance of HAdV-F41 and can further contribute to the ongoing global discussion supporting HAdV-F41 involvement in acute hepatitis cases.
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Adenovírus Humanos , COVID-19 , Hepatite , Criança , Humanos , Águas Residuárias , SARS-CoV-2 , Doença AgudaRESUMO
Flavivirids (family Flaviviridae) are a group of positive-strand ribonucleic acid (RNA) viruses that pose serious risks to human and animal health on a global scale. Here, we use flavivirid-derived deoxyribonucleic acid (DNA) sequences, identified in animal genomes, to reconstruct the long-term evolutionary history of family Flaviviridae. We demonstrate that flavivirids are >100 million years old and show that this timing can be combined with dates inferred from co-phyletic analysis to produce a cohesive overview of their evolution, distribution, and diversity wherein the main flavivirid subgroups originate in early animals and broadly co-diverge with major animal phyla. In addition, we reveal evidence that the 'classical flaviviruses' of vertebrates, most of which are transmitted via blood-feeding arthropod vectors, originally evolved in haematophagous arachnids and later acquired the capacity to be transmitted by insects. Our findings imply that the biological properties of flavivirids have been acquired gradually over the course of animal evolution. Thus, broad-scale comparative analysis will likely reveal fundamental insights into their biology. We therefore published our results via an open, extensible, database (Flavivirid-GLUE), which we constructed to facilitate the wider utilisation of genomic data and evolution-related domain knowledge in flavivirid research.
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A thin, 30 µm, flexible, robust low-density polyethylene, LDPE, film, loaded with 30 wt% P25 TiO2, is extruded and subsequently rendered highly active photocatalytically by exposing it to UVA (352 nm, 1.5 mW cm-2) for 144 h. The film was tested for anti-viral activity using four different viruses, namely, two strains of Influenza A Virus (IAV), WSN, and a recombinant PR8, encephalomyocarditis virus (EMCV), and SARS-CoV-2 (SARS2). The film was irradiated with either UVA radiation (352 nm, 1.5 mW cm-2; although only 0.25 mW cm-2 for SARS2) or with light from a cool white fluorescent lamp (UVA irradiance: 365 nm, 0.047 mW cm-2). In all cases the films exhibited an average virus inactivation rate of >1.5log/h. In the case of SARS2, the rates were > 2log/h, with the rate determined using a dedicated, low intensity UVA source (0.25 mW cm-2) only 1.3 x's faster than that for a cool white lamp (UVA irradiance = 0.047 mW cm-2), which suggests that SARS2 is particularly prone to photocatalytic inactivation even under low UV irradiation conditions, such as found in a room lit with just white fluorescent tubes. This is the first example of a flexible, very thin, photocatalytic plastic film, produced by a scalable process (extrusion), for virus inactivation. The potential of such a film for use as a disposable, self-sterilising thin plastic material alternative to the common, non-photocatalytic, inert equivalent used currently for curtains, aprons and table coverings in healthcare is discussed briefly.
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COVID-19 , Titânio , Catálise , Humanos , Plásticos , Polietileno , SARS-CoV-2 , Raios UltravioletaRESUMO
Natural hepatitis C virus (HCV) infection is restricted to humans, whereas other primates such as rhesus macaques are non-permissive for infection. To identify human and rhesus macaque genes that differ or share the ability to inhibit HCV replication, we conducted a medium-throughput screen of lentivirus-expressed host genes that disrupt replication of HCV subgenomic replicon RNA expressing secreted Gaussia luciferase. A combined total of >800 interferon-stimulated genes (ISGs) were screened. Our findings confirmed established anti-HCV ISGs, such as IRF1, PKR and DDX60. Novel species−specific inhibitors were also identified and independently validated. Using a cell-based system that recapitulates productive HCV infection, we identified that over-expression of the 'Rho Guanine Nucleotide Exchange Factor 3' gene (ARHGEF3) from both species inhibits full-length virus replication. Additionally, replication of two mosquito-borne flaviviruses, yellow fever virus (YFV) and Zika virus (ZIKV), were also reduced in cell lines over-expressing ARHGEF3 compared to controls. In conclusion, we ascribe novel antiviral activity to the cellular gene ARHGEF3 that inhibits replication of HCV and other important human viral pathogens belonging to the Flaviviridae, and which is conserved between humans and rhesus macaques.
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Hepatite C , Infecção por Zika virus , Zika virus , Animais , Antivirais/farmacologia , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Humanos , Interferons/farmacologia , Macaca mulatta , Fatores de Troca de Nucleotídeo Guanina Rho , Replicação Viral , Infecção por Zika virus/tratamento farmacológicoRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and caused >5 million deaths by February 2022. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFNλ1) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) culture model derived from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFNλ1 expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFNλ1 secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFNλ treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFNλ response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFNλ as a potential pharmaceutical against SARS-CoV-2 infection.
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COVID-19 , Infecções por Vírus Respiratório Sincicial , Antivirais/farmacologia , Células Epiteliais/metabolismo , Humanos , Interferons/metabolismo , Interferons/farmacologia , Janus Quinases/metabolismo , SARS-CoV-2 , Fatores de Transcrição STAT/metabolismo , Transdução de SinaisRESUMO
Hepatitis C virus (HCV) is a highly diverse pathogen that frequently establishes a chronic long-term infection, but the origins and drivers of HCV diversity in the human population remain unclear. Previously unidentified strains of HCV genotype 6 (gt6) were recently discovered in chronically infected individuals of the Li ethnic group living in Baisha County, Hainan Island, China. The Li community, who were early settlers on Hainan Island, has a distinct host genetic background and cultural identity compared to other ethnic groups on the island and mainland China. In this report, we generated 33 whole virus genome sequences to conduct a comprehensive molecular epidemiological analysis of these novel gt6 strains in the context of gt6 isolates present in Southeast Asia. With the exception of one gt6a isolate, the Li gt6 sequences formed three novel clades from two lineages which constituted 3 newly assigned gt6 subtypes and 30 unassigned strains. Using Bayesian inference methods, we dated the most recent common ancestor for all available gt6 whole virus genome sequences to approximately 2767 bce (95 per cent highest posterior density (HPD) intervals, 3670-1397 bce), which is far earlier than previous estimates. The substitution rate was 1.20 × 10-4 substitutions/site/year (s/s/y), and this rate varied across the genome regions, from 1.02 × 10-5 s/s/y in the 5'untranslated region (UTR) region to 3.07 × 10-4 s/s/y in E2. Thus, our study on an isolated ethnic minority group within a small geographical area of Hainan Island has substantially increased the known diversity of HCV gt6, already acknowledged as the most diverse HCV genotype. The extant HCV gt6 sequences from this study were probably transmitted to the Li through at least three independent events dating perhaps from around 4,000 years ago. This analysis describes deeper insight into basic aspects of HCV gt6 molecular evolution including the extensive diversity of gt6 sequences in the isolated Li ethnic group.
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SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality positively associated with increasing host age and presence of co-morbidities. SARS-CoV-2 continues to adapt to the human population, resulting in several variants of concern (VOC) with novel properties, such as Alpha and Delta. However, factors driving SARS-CoV-2 fitness and evolution in paediatric cohorts remain poorly explored. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change in primary airway cell cultures derived from children. Through viral whole-genome sequencing, we explored changes in genetic diversity over time of two pre-VOC clinical isolates of SARS-CoV-2 during passage in paediatric well-differentiated primary nasal epithelial cell (WD-PNEC) cultures and in parallel, in unmodified Vero-derived cell lines. We identified a consistent, rich genetic diversity arising in vitro, variants of which could rapidly rise to near fixation within two passages. Within isolates, SARS-CoV-2 evolution was dependent on host cells, with paediatric WD-PNECs showing a reduced diversity compared to Vero (E6) cells. However, mutations were not shared between strains. Furthermore, comparison of both Vero-grown isolates on WD-PNECs disclosed marked growth attenuation mapping to the loss of the polybasic cleavage site (PBCS) in Spike, while the strain with mutations in Nsp12 (T293I), Spike (P812R) and a truncation of Orf7a remained viable in WD-PNECs. Altogether, our work demonstrates that pre-VOC SARS-CoV-2 efficiently infects paediatric respiratory epithelial cells, and its evolution is restrained compared to Vero (E6) cells, similar to the case of adult cells. We highlight the significant genetic plasticity of SARS-CoV-2 while uncovering an influential role for collaboration between viral and host cell factors in shaping viral evolution and ultimately fitness in human respiratory epithelium.
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Evolução Molecular , Mucosa Respiratória/virologia , SARS-CoV-2/genética , Animais , Células Cultivadas , Criança , Chlorocebus aethiops , Genótipo , Humanos , Mutação , Nariz/citologia , Nariz/virologia , Fenótipo , SARS-CoV-2/classificação , SARS-CoV-2/crescimento & desenvolvimento , Células Vero , Sequenciamento Completo do GenomaRESUMO
Interferon lambdas (IFNλ) (also known as type III IFNs) are critical cytokines that combat infection predominantly at barrier tissues, such as the lung, liver, and gastrointestinal tract. Humans have four IFNλs (1-4), where IFNλ1-3 show ~80%-95% homology, and IFNλ4 is the most divergent displaying only ~30% sequence identity. Variants in IFNλ4 in humans are associated with the outcome of infection, such as with hepatitis C virus. However, how IFNλ4 variants impact cytokine signalling in other tissues and how well this is conserved is largely unknown. In this study, we address whether differences in antiviral signalling exist between IFNλ4 variants in human hepatocyte and intestinal cells, comparing them to IFNλ3. We demonstrate that compared to IFNλ3, wild-type human IFNλ4 induces a signalling response with distinct magnitudes and kinetics, which is modified by naturally occurring variants P70S and K154E in both cell types. IFNλ4's distinct antiviral response was more rapid yet transient compared to IFNλ1 and 3. Additionally, divergent antiviral kinetics were also observed using non-human primate IFNλs and cell lines. Furthermore, an IFNλ4-like receptor-interacting interface failed to alter IFNλ1's kinetics. Together, our data provide further evidence that major functional differences exist within the IFNλ gene family. These results highlight the possible tissue specialisation of IFNλs and encourage further investigation of the divergent, non-redundant activities of IFNλ4 and other IFNλs.