RESUMO
After contracting COVID-19, a substantial number of individuals develop a Post-COVID-Condition, marked by neurologic symptoms such as cognitive deficits, olfactory dysfunction, and fatigue. Despite this, biomarkers and pathophysiological understandings of this condition remain limited. Employing magnetic resonance imaging, we conduct a comparative analysis of cerebral microstructure among patients with Post-COVID-Condition, healthy controls, and individuals that contracted COVID-19 without long-term symptoms. We reveal widespread alterations in cerebral microstructure, attributed to a shift in volume from neuronal compartments to free fluid, associated with the severity of the initial infection. Correlating these alterations with cognition, olfaction, and fatigue unveils distinct affected networks, which are in close anatomical-functional relationship with the respective symptoms.
Assuntos
COVID-19 , Disfunção Cognitiva , Fadiga , Imageamento por Ressonância Magnética , Transtornos do Olfato , SARS-CoV-2 , Humanos , COVID-19/complicações , COVID-19/diagnóstico por imagem , COVID-19/fisiopatologia , COVID-19/patologia , Disfunção Cognitiva/diagnóstico por imagem , Disfunção Cognitiva/fisiopatologia , Disfunção Cognitiva/virologia , Masculino , Fadiga/fisiopatologia , Feminino , Pessoa de Meia-Idade , Transtornos do Olfato/diagnóstico por imagem , Transtornos do Olfato/virologia , Transtornos do Olfato/fisiopatologia , Adulto , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Encéfalo/fisiopatologia , Síndrome de COVID-19 Pós-Aguda , IdosoRESUMO
PURPOSE: This study aims to describe clinical, virological and radiological characteristics as well as treatment strategies and outcomes of immunocompromised patients with persistent SARS-CoV-2 replication. METHODS: We performed a retrospective cohort study of immunocompromised patients at the University Medical Center Freiburg between 01/2022 and 05/2023. Patients with substantial immunosuppression and persistent SARS-CoV-2 detection (Ct-value < 30 after 14 days) were included. RESULTS: 36 patients in our cohort reported mainly fever, dyspnoea or continuous cough. Viral load was significantly higher in concurrent samples taken from the lower respiratory tract (Ct-value = 26) than from the upper respiratory tract (Ct-value = 34). Time of detectable viral RNA after start of antiviral treatment was shorter in patients receiving two antivirals (median 15 days vs. 31 days with one antiviral agent). Short-course antiviral therapy (≤ 5 days) was less efficient in reduction of symptoms and viral load than prolonged therapy > 10 days. In 30% (8/27) of patients with repeated CT scans, we found the emergence of chronic pulmonary changes, which were more frequently in patients with B cell depletion (37%, 7/19) compared to patients with organ transplantation (12%, 2/17). CONCLUSION: Ongoing SARS-CoV-2 replication in the lower respiratory tract is a relevant differential diagnosis in patients with severe immunosuppression and continuous cough, fever or dyspnoea even if nasopharyngeal swabs test negative for SARS-CoV-2. Especially in B cell-depleted patients, this may lead to inflammatory or fibrotic-like pulmonary changes, which are partially reversible after inhibition of viral replication. Antiviral therapy seems to be most effective in combination and over a prolonged period of time of > 10 days. TRIAL REGISTRATION NUMBER: DRKS 00027299.
RESUMO
BACKGROUND: Increasing evidence suggests that some patients suffer from persistent symptoms for months after recovery from acute COVID-19.âHowever, the clinical phenotype and its pathogenesis remain unclear. We here present data on complaints and results of a diagnostic workup of patients presenting to the post-COVID clinic at the University Medical Center Freiburg. METHODS: Retrospective data analysis of persistently symptomatic patients presenting to our clinic at least 6 months after onset of acute COVID-19.âAll patients were assessed by a doctor and routine laboratory analysis was carried out. Quality of life was assessed using SF-36 questionnaire. In case of specific persisting symptoms, further organ-specific diagnostic evaluation was performed, and patients were referred to respective departments/specialists. FINDINGS: 132 Patients (58 male, 74 female; mean age 53.8 years) presented to our clinic at least 6 months after COVID-19. 79 (60â%) had been treated as outpatients and 53 (40â%) as inpatients. Most common complaints were persistent fatigue (82â%) and dyspnea on exertion (61â%). Further common complaints were impairments of concentration (54â%), insomnia (43â%), and impairments of smell or taste (35â%). Quality of life was reduced in all sections of the SF-36 questionnaire, yielding a reduced working capacity. Significant pathological findings in laboratory, echocardiographic and radiological work-up were rare. Impairments in lung function tests were more common in previously hospitalized patients. CONCLUSION: Patients presenting 6 months after onset of acute COVID-19 suffer from a diverse spectrum of symptoms with impaired quality of life, also referred to as Long COVID or Post-Acute Sequelae of SARS-CoV-2 infection (PASC). Further research is needed to determine the frequency of these post-COVID syndromes and their pathogenesis, natural course and treatment options. Evaluation and management should be multi-disciplinary.
Assuntos
COVID-19 , Masculino , Feminino , Humanos , SARS-CoV-2 , Pacientes Ambulatoriais , Qualidade de Vida , Estudos Retrospectivos , Seguimentos , Centros Médicos Acadêmicos , Síndrome de COVID-19 Pós-AgudaRESUMO
Continuously emerging variants of concern (VOCs) sustain the SARS-CoV-2 pandemic. The SARS-CoV-2 Omicron/B.1.1.529 VOC harbours multiple mutations in the spike protein associated with high infectivity and efficient evasion from humoral immunity induced by previous infection or vaccination. By performing in-depth comparisons of the SARS-CoV-2-specific T-cell epitope repertoire after infection and messenger RNA vaccination, we demonstrate that spike-derived epitopes were not dominantly targeted in convalescent individuals compared to non-spike epitopes. In vaccinees, however, we detected a broader spike-specific T-cell response compared to convalescent individuals. Booster vaccination increased the breadth of the spike-specific T-cell response in convalescent individuals but not in vaccinees with complete initial vaccination. In convalescent individuals and vaccinees, the targeted T-cell epitopes were broadly conserved between wild-type SARS-CoV-2 variant B and Omicron/B.1.1.529. Hence, our data emphasize the relevance of vaccine-induced spike-specific CD8+ T-cell responses in combating VOCs including Omicron/B.1.1.529 and support the benefit of boosting convalescent individuals with mRNA vaccines.
Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Epitopos de Linfócito T/genética , Humanos , RNA Mensageiro/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
BACKGROUND: Increasing evidence suggests that some patients suffer from persistent symptoms for months after recovery from acute COVID-19. However, the clinical phenotype and its pathogenesis remain unclear. We here present data on complaints and results of a diagnostic workup of patients presenting to the post-COVID clinic at the University Medical Center Freiburg. METHODS: Retrospective data analysis of persistently symptomatic patients presenting to our clinic at least 6 months after onset of acute COVID-19. All patients were assessed by a doctor and routine laboratory analysis was carried out. Quality of life was assessed using SF-36 questionnaire. In case of specific persisting symptoms, further organ-specific diagnostic evaluation was performed, and patients were referred to respective departments/specialists. FINDINGS: 132 Patients (58 male, 74 female; mean age 53.8 years) presented to our clinic at least 6 months after COVID-19. 79 (60â%) had been treated as outpatients and 53 (40â%) as inpatients. Most common complaints were persistent fatigue (82â%) and dyspnea on exertion (61â%). Further common complaints were impairments of concentration (54â%), insomnia (43â%), and impairments of smell or taste (35â%). Quality of life was reduced in all sections of the SF-36 questionnaire, yielding a reduced working capacity. Significant pathological findings in laboratory, echocardiographic and radiological work-up were rare. Impairments in lung function tests were more common in previously hospitalized patients. CONCLUSION: Patients presenting 6 months after onset of acute COVID-19 suffer from a diverse spectrum of symptoms with impaired quality of life, also referred to as Long COVID or Post-Acute Sequelae of SARS-CoV-2 infection (PASC). Further research is needed to determine the frequency of these post-COVID syndromes and their pathogenesis, natural course and treatment options. Evaluation and management should be multi-disciplinary.
Assuntos
COVID-19/complicações , SARS-CoV-2/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anosmia , Anticorpos Antivirais/sangue , COVID-19/epidemiologia , Dispneia , Fadiga , Feminino , Seguimentos , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Estudos Retrospectivos , Distúrbios do Início e da Manutenção do Sono , Inquéritos e Questionários , Distúrbios do Paladar , Adulto Jovem , Síndrome de COVID-19 Pós-AgudaRESUMO
Some patients complain of persisting symptoms after acute COVID-19. There is no universal definition yet for these post-acute sequelae, also termed Long COVID. Although their prevalence remains to be established, a delayed recovery seems to be more common than after other acute infectious diseases. Common complaints include fatigue with exercise intolerance, dyspnea on exertion, chest pain, and neuropsychiatric symptoms. Female sex, comorbidities, and severity of the acute disease have been identified as risk factors for persisting symptoms. It is, however, important to highlight that they are not limited to patients after severe COVID-19. Whilst their pathogenesis and prognosis is largely unknown, diagnostic evaluation should focus on exclusion of objective organ dysfunctions. Due to the variable presentation, management is interdisciplinary and may include physiotherapy, rehabilitation programmes, and psychological support. This article aims to summarize the current - limited - evidence on persisting symptoms after COVID-19.
Assuntos
COVID-19/complicações , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/etiologia , COVID-19/terapia , Humanos , Prevalência , Fatores de Risco , Síndrome de COVID-19 Pós-AgudaRESUMO
Lysine acetylation is a post-translational modification known to regulate protein functions. Here we identify several acetylation sites of the influenza A virus nucleoprotein (NP), including the lysine residues K77, K113 and K229. Viral growth of mutant virus encoding K229R, mimicking a non-acetylated NP lysine residue, is severely impaired compared to wildtype or the mutant viruses encoding K77R or K113R. This attenuation is not the result of decreased polymerase activity, altered protein expression or disordered vRNP co-segregation but rather caused by impaired particle release. Interestingly, release deficiency is also observed mimicking constant acetylation at this site (K229Q), whereas virus encoding NP-K113Q could not be generated. However, mimicking NP hyper-acetylation at K77 and K229 severely diminishes viral polymerase activity, while mimicking NP hypo-acetylation at these sites has no effect on viral replication. These results suggest that NP acetylation at K77, K113 and K229 impacts multiple steps in viral replication of influenza A viruses.
Assuntos
Vírus da Influenza A/genética , Lisina/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas do Core Viral/genética , Replicação Viral/genética , Acetilação , Animais , Cães , Células HEK293 , Humanos , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/metabolismo , Células Madin Darby de Rim Canino , Mutação , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismoRESUMO
Zoonotic transmission of influenza A viruses can give rise to devastating pandemics, but currently it is impossible to predict the pandemic potential of circulating avian influenza viruses. Here, we describe a new mouse model suitable for such risk assessment, based on the observation that the innate restriction factor MxA represents an effective species barrier that must be overcome by zoonotic viruses. Our mouse lacks functional endogenous Mx genes but instead carries the human MX1 locus as a transgene. Such transgenic mice were largely resistant to highly pathogenic avian H5 and H7 influenza A viruses, but were almost as susceptible to infection with influenza viruses of human origin as nontransgenic littermates. Influenza A viruses that successfully established stable lineages in humans have acquired adaptive mutations which allow partial MxA escape. Accordingly, an engineered avian H7N7 influenza virus carrying a nucleoprotein with signature mutations typically found in human virus isolates was more virulent in transgenic mice than parental virus, demonstrating that a few amino acid changes in the viral target protein can mediate escape from MxA restriction in vivo. Similar mutations probably need to be acquired by emerging influenza A viruses before they can spread in the human population.
Assuntos
Vírus da Influenza A/imunologia , Proteínas de Resistência a Myxovirus/imunologia , Nucleoproteínas/genética , Animais , Resistência à Doença/genética , Resistência à Doença/imunologia , Feminino , Humanos , Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H7N7/imunologia , Vírus da Influenza A Subtipo H7N7/patogenicidade , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Proteínas de Resistência a Myxovirus/genéticaRESUMO
To establish a new lineage in the human population, avian influenza A viruses (AIV) must overcome the intracellular restriction factor MxA. Partial escape from MxA restriction can be achieved when the viral nucleoprotein (NP) acquires the critical human-adaptive amino acid residues 100I/V, 283P, and 313Y. Here, we show that introduction of these three residues into the NP of an avian H5N1 virus renders it genetically unstable, resulting in viruses harboring additional single mutations, including G16D. These substitutions restored genetic stability yet again yielded viruses with varying degrees of attenuation in mammalian and avian cells. Additionally, most of the mutant viruses lost the capacity to escape MxA restriction, with the exception of the G16D virus. We show that MxA escape is linked to attenuation by demonstrating that the three substitutions promoting MxA escape disturbed intracellular trafficking of incoming viral ribonucleoprotein complexes (vRNPs), thereby resulting in impaired nuclear import, and that the additional acquired mutations only partially compensate for this import block. We conclude that for adaptation to the human host, AIV must not only overcome MxA restriction but also an associated block in nuclear vRNP import. This inherent difficulty may partially explain the frequent failure of AIV to become pandemic.
Assuntos
Substituição de Aminoácidos , Virus da Influenza A Subtipo H5N1/genética , Proteínas de Resistência a Myxovirus/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Células A549 , Animais , Aves/virologia , Linhagem Celular , Cães , Células HEK293 , Humanos , Virus da Influenza A Subtipo H5N1/patogenicidade , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação , Proteínas do Nucleocapsídeo , Conformação Proteica , Transporte Proteico , Proteínas de Ligação a RNA/química , Proteínas do Core Viral/químicaRESUMO
Phosphorylation at the highly conserved serine residues S23 to S25 in the nuclear export protein (NEP) of influenza A viruses was suspected to regulate its nuclear export activity or polymerase activity-enhancing function. Mutation of these phosphoacceptor sites to either alanine or aspartic acid showed only a minor effect on both activities but revealed the presence of other phosphoacceptor sites that might be involved in regulating NEP activity.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Vírus da Influenza A/crescimento & desenvolvimento , Infecções por Orthomyxoviridae/virologia , Serina/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , DNA Viral/genética , DNA Polimerase Dirigida por DNA/metabolismo , Células HEK293 , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Camundongos , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/metabolismo , Fosforilação , Homologia de Sequência de Aminoácidos , Virulência , Replicação ViralRESUMO
The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase activity in human cells in a concentration-dependent manner. While low NEP levels enhance the polymerase activity, high levels are inhibitory. To gain insights into the underlying mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP resides in the C-terminal moiety and that removal of the last three amino acids completely abrogates this activity. Moreover, compared to full-length NEP, the C-terminal moiety alone exhibited significantly higher activity and seemed to be deregulated, since even the highest concentration did not result in an inhibition of polymerase activity. To determine transient interactions between the N- and C-terminal domains in cis, we fused both ends of NEP to a split click beetle luciferase and performed fragment complementation assays. With decreasing temperature, increased luciferase activity was observed, suggesting that intramolecular binding between the C- and N-terminal domains is preferentially stabilized at low temperatures. This stabilizing effect was significantly reduced with the adaptive mutation M16I or a combination of adaptive mutations (M16I, Y41C, and E75G), which further increased polymerase activity also at 34°C. We therefore propose a model in which the N-terminal moiety of NEP exerts an inhibitory function by back-folding to the C-terminal domain. In this model, adaptive mutations in NEP decrease binding between the C- and N-terminal domains, thereby allowing the protein to "open up" and become active already at a low temperature.
Assuntos
Núcleo Celular/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Mutação , Proteínas não Estruturais Virais/genética , Células HEK293 , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Temperatura , Proteínas não Estruturais Virais/química , Replicação ViralRESUMO
The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored.
Assuntos
Proteínas de Ligação ao GTP/genética , Evasão da Resposta Imune/genética , Vírus da Influenza A Subtipo H1N1/genética , Mutação , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Resistência à Doença/genética , Evolução Molecular , Proteínas de Ligação ao GTP/química , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Resistência a Myxovirus , Infecções por Orthomyxoviridae/imunologia , Pandemias , Estrutura Secundária de Proteína , Seleção Genética , Zoonoses/virologiaRESUMO
In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration.
Assuntos
Núcleo Celular/metabolismo , Cromatina/metabolismo , Carioferinas/metabolismo , RNA Viral/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Linhagem Celular , Núcleo Celular/virologia , Cães , Ácidos Graxos Insaturados/farmacologia , Carioferinas/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Orthomyxoviridae/genética , Orthomyxoviridae/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Replicação Viral/genética , Proteína Exportina 1RESUMO
To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/ß receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.
Assuntos
Virus da Influenza A Subtipo H5N1/enzimologia , Vírus da Influenza A Subtipo H7N7/enzimologia , Influenza Humana/enzimologia , Mutação , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Animais , Cães , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H7N7/imunologia , Vacinas contra Influenza/biossíntese , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/genética , Influenza Humana/imunologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/imunologia , Vacinas Atenuadas/biossíntese , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A(1-25)) with that of FluB (PB1-B(1-25)) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B(1-25) peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A(1-25) peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.