RESUMO
Susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the outcome of coronavirus disease 2019 (COVID-19) have been linked to underlying health conditions and the age of affected individuals. Here, we assessed the effect of age on SARS-CoV-2 infection using a ferret model. For this, young (6-month-old) and aged (18- to 39-month-old) ferrets were inoculated intranasally with various doses of SARS-CoV-2. By using infectious virus shedding in respiratory secretions and seroconversion, we estimated that the infectious dose of SARS-CoV-2 in aged animals is â¼32 PFU per animal, while in young animals it was estimated to be â¼100 PFU. We showed that viral replication in the upper respiratory tract and shedding in respiratory secretions is enhanced in aged ferrets compared to young animals. Similar to observations in humans, this was associated with higher transcription levels of two key viral entry factors, ACE2 and TMPRSS2, in the upper respiratory tract of aged ferrets. IMPORTANCE In humans, ACE2 and TMPRSS2 are expressed in various cells and tissues, and differential expression has been described in young and old people, with a higher level of expressing cells being detected in the nasal brushing of older people than young individuals. We described the same pattern occurring in ferrets, and we demonstrated that age affects susceptibility of ferrets to SARS-CoV-2. Aged animals were more likely to get infected when exposed to lower infectious dose of the virus than young animals, and the viral replication in the upper respiratory tract and shedding are enhanced in aged ferrets. Together, these results suggest that the higher infectivity and enhanced ability of SARS-CoV-2 to replicate in aged individuals is associated, at least in part, with transcription levels of ACE2 and TMPRSS2 at the sites of virus entry. The young and aged ferret model developed here may represent a great platform to assess age-related differences in SARS-CoV-2 infection dynamics and replication.
Assuntos
COVID-19/virologia , Suscetibilidade a Doenças , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Fatores Etários , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Biomarcadores , COVID-19/genética , COVID-19/imunologia , Modelos Animais de Doenças , Furões , Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Especificidade de Órgãos , RNA Viral , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Carga ViralRESUMO
Senecavirus A (SVA) is a picornavirus that circulates in swine populations worldwide causing vesicular disease (VD) in affected animals. Here we developed a reverse genetics system for SVA based on the well-characterized wild-type SVA strain SD15-26 (wt SVA SD15-26). The full-length cDNA genome of SVA was cloned into a plasmid under a T7 RNA polymerase promoter. Following in vitro transcription, the genomic viral RNA was transfected into BHK-21 cells and rescue of infectious virus (rSVA SD15-26) was shown by inoculation of highly susceptible H1299 cells. In vitro characterization of the rSVA SD15-26 showed similar replication properties and protein expression levels as the wt SVA SD15-26. A pathogenesis study was conducted in 15-week-old finishing pigs to evaluate the pathogenicity and infection dynamics of the rSVA SD15-26 virus in comparison to the wt SVA SD15-26. Animals from both rSVA- and wt SVA SD15-26-inoculated groups presented characteristic SVA clinical signs (lethargy and lameness) followed by the development of vesicular lesions on the snout and/or feet. The clinical outcome of infection, including disease onset, severity and duration was similar in rSVA- and the wt SVA SD15-26-inoculated animals. All animals inoculated with rSVA or with wt SVA SD15-26 presented a short-term viremia, and animals from both groups shed similar amounts of virus in oral and nasal secretion, and faeces. Our data demonstrates that the rSVA SD5-26 clone is fully virulent and pathogenic in pigs, presenting comparable pathogenesis and infection dynamics to the wt SVA SD15-26 strain. The infectious clone generated here is a useful platform to study virulence determinants of SVA, and to dissect other aspects of SVA infection biology, pathogenesis and persistence.
Assuntos
Infecções por Picornaviridae , Picornaviridae/patogenicidade , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Cricetinae , Humanos , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologia , Suínos , Viremia/virologia , VirulênciaRESUMO
Senecavirus A (SVA) is an emerging picornavirus that causes vesicular disease (VD) in swine. The virus has been circulating in swine in the United Stated (USA) since at least 1988, however, since 2014 a marked increase in the number of SVA outbreaks has been observed in swine worldwide. The factors that led to the emergence of SVA remain unknown. Evolutionary changes that accumulated in the SVA genome over the years may have contributed to the recent increase in disease incidence. Here we compared full-genome sequences of historical SVA strains (identified before 2010) from the USA and global contemporary SVA strains (identified after 2011). The results from the genetic analysis revealed 6.32â% genetic divergence between historical and contemporary SVA isolates. Selection pressure analysis revealed that the SVA polyprotein is undergoing selection, with four amino acid (aa) residues located in the VP1 (aa 735), 2A (aa 941), 3C (aa 1547) and 3D (aa 1850) coding regions being under positive/diversifying selection. Several aa substitutions were observed in the structural proteins (VP1, VP2 and VP3) of contemporary SVA isolates when compared to historical SVA strains. Some of these aa substitutions led to changes in the surface electrostatic potential of the structural proteins. This work provides important insights into the molecular evolution and epidemiology of SVA.
Assuntos
Doenças Transmissíveis Emergentes , Infecções por Picornaviridae/veterinária , Picornaviridae/genética , Doenças dos Suínos/virologia , Substituição de Aminoácidos/genética , Animais , Doenças Transmissíveis Emergentes/veterinária , Doenças Transmissíveis Emergentes/virologia , Surtos de Doenças , Evolução Molecular , Variação Genética , Genoma Viral , Filogenia , Infecções por Picornaviridae/epidemiologia , Suínos , Doenças dos Suínos/epidemiologia , Estados Unidos/epidemiologia , Proteínas Virais/genética , Proteínas Estruturais Virais/genéticaRESUMO
Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here, we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression, or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression, and parturition stressors did not lead to overt/recrudescent clinical disease, but intermittent viremia and virus shedding were detected up to day 60 postinfection (p.i.) in all treatment groups following stress stimulation. Notably, real-time PCR and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for double-stranded RNA (dsRNA) demonstrated the presence of double-stranded viral RNA in tonsillar cells. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 p.i., confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.IMPORTANCE Persistent viral infections have significant implications for disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally or naturally infected animals long after resolution of the clinical disease. Here, we showed that SVA establishes persistent infection in SVA-infected animals, with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as sources of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology, suggesting that persistently infected pigs may function as reservoirs for SVA.
Assuntos
Portador Sadio/veterinária , Transmissão Vertical de Doenças Infecciosas/veterinária , Infecções por Picornaviridae/veterinária , Picornaviridae/patogenicidade , Doenças dos Suínos/transmissão , Animais , Portador Sadio/patologia , Portador Sadio/transmissão , Portador Sadio/virologia , Doença Crônica , Feminino , Tonsila Palatina/virologia , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/transmissão , Infecções por Picornaviridae/virologia , Recidiva , Estresse Fisiológico , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Viremia/patologia , Viremia/transmissão , Viremia/veterinária , Viremia/virologia , Eliminação de Partículas ViraisRESUMO
Senecavirus A (SVA), an emerging picornavirus of swine, causes vesicular disease (VD) that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Many aspects of SVA interactions with the host and the host immune responses to infection, however, remain unknown. In the present study, humoral and cellular immune responses to SVA were evaluated following infection in pigs. We show that SVA infection elicited an early and robust virus-neutralizing (VN) antibody response, which coincided and was strongly correlated with VP2- and VP3-specific IgM responses. Notably, the neutralizing antibody (NA) responses paralleled the reduction of viremia and resolution of the disease. Analysis of the major porcine T-cell subsets revealed that during the acute/clinical phase of SVA infection (14 days postinfection [p.i.]), T-cell responses were characterized by an increased frequency of αß T cells, especially CD4+ T cells, which were first detected by day 7 p.i. and increased in frequency until day 14 p.i. Additionally, the frequency of CD8+ and double-positive CD4+ CD8+ T cells (effector/memory T cells) expressing interferon gamma (IFN-γ) or proliferating in response to SVA antigen stimulation increased after day 10 p.i. Results presented here show that SVA elicits B- and T-cell activation early upon infection, with IgM antibody levels being correlated with early neutralizing activity against the virus and peak B- and T-cell responses paralleling clinical resolution of the disease. The work provides important insights into the immunological events that follow SVA infection in the natural host.IMPORTANCE Senecavirus A (SVA) has recently emerged in swine, causing outbreaks of vesicular disease (VD) in major swine-producing countries around the world, including the United States, Brazil, China, Thailand, and Colombia. Notably, SVA-induced disease is clinically indistinguishable from other high-consequence VDs of swine, such as FMD, swine vesicular disease, vesicular stomatitis, and vesicular exanthema of swine. Despite the clinical relevance of SVA-induced VD, many aspects of the virus infection biology remain unknown. Here, we assessed host immune responses to SVA infection. The results show that SVA infection elicits early B- and T-cell responses, with the levels of VN antibody and CD4+ T-cell responses paralleling the reduction of viremia and resolution of the disease. SVA-specific CD8+ T cells are detected later during infection. A better understanding of SVA interactions with the host immune system may allow the design and implementation of improved control strategies for this important pathogen of swine.
Assuntos
Imunidade Adaptativa , Picornaviridae , Doença Vesicular Suína/patologia , Linfócitos T/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Febre Aftosa/patologia , Interações Hospedeiro-Patógeno , Imunidade Celular , Imunidade Humoral , Suínos , Viremia/imunologia , Viremia/veterináriaRESUMO
Here we describe the identification and genetic characterization of a porcine hepe-astrovirus, or bastrovirus, obtained from feces from pigs in the United States. The genome of the new bastrovirus is 5,955 nt long and contains two open reading frames (ORFs). ORF1 encodes a protein containing three domains, viral methyltransferase, RNA helicase and RNA-dependent RNA polymerase (RdRp), and is closely related to the RdRp of hepatitis E virus. The ORF2 protein shares similarities with the astrovirus capsid precursor protein. Although structural features of bastroviruses may resemble those of astroviruses, distinct characteristics of the newly identified bastrovirus include the presence of an RNA helicase domain in ORF1 and the lack of ORF1b. In addition to genetic characterization, screening of 368 porcine samples (oral fluids, oral swabs or fecal swabs) collected in the United States (US) using a porcine-bastrovirus-specific real-time PCR assay revealed that 31% of those samples were positive. These results suggest a broad distribution of bastroviruses in the swine population in the US. This represents the first description of bastrovirus in swine in the US.
Assuntos
Infecções por Astroviridae/veterinária , Astroviridae/genética , Astroviridae/isolamento & purificação , Doenças dos Suínos/virologia , Animais , Astroviridae/classificação , Infecções por Astroviridae/epidemiologia , Infecções por Astroviridae/virologia , Genoma Viral , Fases de Leitura Aberta , Filogenia , RNA Polimerase Dependente de RNA/genética , Suínos , Doenças dos Suínos/epidemiologia , Estados Unidos/epidemiologia , Proteínas Virais/genéticaRESUMO
Passive immunity is critical for protection of neonatal piglets against porcine epidemic diarrhea virus (PEDV). Here, we investigated the immunogenicity of an orf virus (ORFV) vector expressing the full-length spike (S) protein of PEDV (ORFV-PEDV-S) in pregnant gilts and its ability to confer passive immunity and protection in piglets. Three doses of ORFV-PEDV-S were given to two groups of PEDV-negative pregnant gilts, with the last dose being administered two weeks prior to farrowing. One of the two groups immunized with the ORFV-PEDV-S recombinant virus was also exposed to live PEDV orally on day 31 post-immunization (pi). Antibody responses were assessed in serum, colostrum and milk of immunized gilts, and passive transfer of antibodies was evaluated in piglet sera. The protective efficacy of ORFV-PEDV-S was evaluated after challenge of the piglets with PEDV. PEDV-specific IgG, IgA and neutralizing antibody (NA) responses were detected in ORFV-PEDV-S-immunized and ORFV-PEDV-S-immunized/PEDV-exposed gilts. PEDV NA, IgG and IgA were detected in the serum of piglets born to immunized gilts, demonstrating the transfer of antibodies through colostrum and milk. Piglets born to immunized gilts showed reduced morbidity and a marked reduction in mortality after PEDV challenge in comparison to control piglets. Piglets born to gilts that received ORFV-PEDV-S and were exposed to live PEDV showed stronger NA responses and lower clinical scores when compared to piglets born to gilts immunized with ORFV-PEDV-S alone. These results demonstrate the potential of ORFV as a vaccine delivery platform capable of eliciting passive immunity against PEDV.
Assuntos
Anticorpos Antivirais/sangue , Infecções por Coronavirus/prevenção & controle , Imunidade Materno-Adquirida , Vírus do Orf/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Doenças dos Suínos/prevenção & controle , Animais , Animais Recém-Nascidos , Anticorpos Neutralizantes/sangue , Colostro , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Vetores Genéticos/imunologia , Imunização Passiva/métodos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Leite , Vírus do Orf/genética , Vírus da Diarreia Epidêmica Suína/patogenicidade , Gravidez , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologiaRESUMO
Papillomaviruses are a diverse group of viruses that are known to infect a wide range of animal species. Bovine papillomaviruses (BPVs) are divided into at least 21 genotypes (BPV1 to BPV21), with most BPV isolates/strains described to date belonging to one of four genera, including Deltapapillomavirus, Xipapillomavirus, Epsilonpapillomavirus and Dyoxipapillomavirus. Here, we describe the identification and genetic characterization of a new BPV type in the genus Dyokappapapillomavirus. A farm in the state of New York, USA, reported chronic cases of vulvovaginitis in Holstein cows in 2016. Biopsies and/or swab samples collected from the vaginal mucosa were subjected to diagnostic investigation. Conventional diagnostic assays yielded negative results, and vaginal swab samples were subjected to viral metagenomic sequencing. Notably, BLAST searches revealed a papillomavirus genome with 7480 bp in length (67% nt sequence identity to BPV16). Additionally, phylogenetic analysis of the L1 gene of the papillomavirus identified here (tentatively named BPV22) revealed that it clusters with members of the genus Dyokappapapillomavirus. Interestingly, the recently identified BPV16, which was detected in fibropapilloma lesions in cattle also clusters within the Dyokappapapillomavirus group. Each virus, however, forms a separate branch in the phylogenetic tree. These results indicate that the putative BPV22 represents the second BPV within the genus Dyokappapapillomavirus.
Assuntos
Doenças dos Bovinos/virologia , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/veterinária , Animais , Bovinos , Feminino , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Filogenia , Vulvovaginite/veterinária , Vulvovaginite/virologiaRESUMO
Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and pathogenesis, however, remain unknown. Here the pathogenesis of SVA was investigated in finishing pigs. Animals were inoculated via the oronasal route with SVA strain SD15-26 and monitored for clinical signs and lesions associated with SVA infection. Viraemia was assessed in serum and virus shedding monitored in oral and nasal secretions and faeces by real-time reverse transcriptase quantitative PCR (RT-qPCR) and/or virus isolation. Additionally, viral load and tissue distribution were assessed during acute infection and following convalescence from disease. Clinical signs characterized by lethargy and lameness were first observed on day 4 post-inoculation (pi) and persisted for approximately 2-10 days. Vesicular lesions were first observed on day 4 pi on the snout and/or feet, affecting the coronary bands, dewclaws, interdigital space and heel/sole of SVA-infected animals. A short-term viraemia was observed between days 3 and 10 pi, whereas virus shedding was detected between days 1 and 28 pi in oral and nasal secretions and faeces. Notably, RT-qPCR and in situ hybridization (ISH) performed on tissues collected on day 38 pi revealed the presence of SVA RNA in the tonsils of all SVA-infected animals. Serological responses to SVA were characterized by early neutralizing antibody responses (day 5 pi), which coincided with decreased levels of viraemia, virus shedding and viral load in tissues. This study provides significant insights into the pathogenesis and infectious dynamics of SVA in swine.
Assuntos
Picornaviridae/patogenicidade , Doenças dos Suínos/virologia , Viremia/veterinária , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Picornaviridae/genética , Picornaviridae/isolamento & purificação , Picornaviridae/fisiologia , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/patologia , Carga Viral , Viremia/sangue , Viremia/patologia , Viremia/virologia , Virulência , Eliminação de Partículas ViraisRESUMO
The parapoxvirus Orf virus (ORFV), has long been recognized for its immunomodulatory properties in permissive and non-permissive animal species. Here, a new recombinant ORFV expressing the full-length spike (S) protein of Porcine epidemic diarrhea virus (PEDV) was generated and its immunogenicity and protective efficacy were evaluated in pigs. The PEDV S was inserted into the ORFV121 gene locus, an immunomodulatory gene that inhibits activation of the NF-κB signalling pathway and contributes to ORFV virulence in the natural host. The recombinant ORFV-PEDV-S virus efficiently and stably expressed the PEDV S protein in cell culture in vitro. Three intramuscular (IM) immunizations with the recombinant ORFV-PEDV-S in 3-week-old pigs elicited robust serum IgG, IgA and neutralizing antibody responses against PEDV. Additionally, IM immunization with the recombinant ORFV-PEDV-S virus protected pigs from clinical signs of porcine epidemic diarrhoea (PED) and reduced virus shedding in faeces upon challenge infection. These results demonstrate the suitability of ORFV121 gene locus as an insertion site for heterologous gene expression and delivery by ORFV-based viral vectors. Additionally, the results provide evidence of the potential of ORFV as a vaccine delivery vector for enteric viral diseases of swine. This study may have important implications for future development of ORFV-vectored vaccines for swine.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Diarreia Epidêmica Suína/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Doenças dos Suínos/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Imunização , Parapoxvirus/genética , Parapoxvirus/metabolismo , Vírus da Diarreia Epidêmica Suína/genética , Glicoproteína da Espícula de Coronavírus/administração & dosagem , Glicoproteína da Espícula de Coronavírus/genética , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Senecavirus A (SVA) is an emerging picornavirus that has been recently associated with an increased number of outbreaks of vesicular disease and neonatal mortality in swine. Many aspects of SVA infection biology and epidemiology remain unknown. Here, we present a diagnostic investigation conducted in swine herds affected by vesicular disease and increased neonatal mortality. Clinical and environmental samples were collected from affected and unaffected herds and were screened for the presence of SVA by real-time reverse transcriptase PCR and virus isolation. Notably, SVA was detected and isolated from vesicular lesions and tissues of affected pigs, environmental samples, mouse feces, and mouse small intestine. SVA nucleic acid was also detected in houseflies collected from affected farms and from a farm with no history of vesicular disease. Detection of SVA in mice and housefly samples and recovery of viable virus from mouse feces and small intestine suggest that these pests may play a role on the epidemiology of SVA. These results provide important information that may allow the development of improved prevention and control strategies for SVA.
Assuntos
Surtos de Doenças , Microbiologia Ambiental , Moscas Domésticas/virologia , Camundongos/virologia , Picornaviridae/isolamento & purificação , Doença Vesicular Suína/epidemiologia , Doença Vesicular Suína/virologia , Animais , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SuínosRESUMO
Orf virus (ORFV) has been used as a vaccine delivery vector for multiple animal species. Several strategies are being used to improve the immunogenicity and efficacy of ORFV vectors, including the use of poxviral promoter(s) with strong early and late activity capable of driving the expression of the heterologous genes for a prolonged time and eliciting a potent immune response. Here, we used RNA-sequencing (RNA-Seq) approach to analyze the transcriptome of ORFV during infection in primary ovine cells. Based on the transcriptional profile of individual ORFV genes, we identified ORFV promoters with strong early and late activity and have shown that they can be used to express heterologous genes in ORFV vectors. Our results show that the intergenic regulatory sequence containing core promoter sequences present upstream of ORF112 (p112) and ORF116 (p116) lead to markedly higher transgene expression than conventional poxviral promoters. Thus, these promoters are valuable alternatives to express transgenes in poxviral vectors.
RESUMO
PURPOSE OF REVIEW: Passive administration of broadly neutralizing antibodies (bNAbs) is being evaluated as a therapeutic approach to prevent or treat HIV infections. However, a number of challenges face the widespread implementation of passive transfer for HIV. To reduce the need of recurrent administrations of bNAbs, gene-based delivery approaches have been developed which overcome the limitations of passive transfer. RECENT FINDINGS: The use of DNA and mRNA for the delivery of bNAbs has made significant progress. DNA-encoded monoclonal antibodies (DMAbs) have shown great promise in animal models of disease and the underlying DNA-based technology is now being tested in vaccine trials for a variety of indications. The COVID-19 pandemic greatly accelerated the development of mRNA-based technology to induce protective immunity. These advances are now being successfully applied to the delivery of monoclonal antibodies using mRNA in animal models. Delivery of bNAbs using viral vectors, primarily adeno-associated virus (AAV), has shown great promise in preclinical animal models and more recently in human studies. Most recently, advances in genome editing techniques have led to engineering of monoclonal antibody expression from B cells. These efforts aim to turn B cells into a source of evolving antibodies that can improve through repeated exposure to the respective antigen. SUMMARY: The use of these different platforms for antibody delivery has been demonstrated across a wide range of animal models and disease indications, including HIV. Although each approach has unique strengths and weaknesses, additional advances in efficiency of gene delivery and reduced immunogenicity will be necessary to drive widespread implementation of these technologies. Considering the mounting clinical evidence of the potential of bNAbs for HIV treatment and prevention, overcoming the remaining technical challenges for gene-based bNAb delivery represents a relatively straightforward path towards practical interventions against HIV infection.
Assuntos
COVID-19 , Infecções por HIV , HIV-1 , Animais , Humanos , Infecções por HIV/prevenção & controle , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV , Anticorpos Neutralizantes , Pandemias , HIV-1/genética , COVID-19/terapia , Anticorpos Monoclonais/genéticaRESUMO
A serological COVID-19 Multiplex Assay was developed and validated using serum samples from convalescent patients and those collected prior to the 2020 pandemic. After initial testing of multiple potential antigens, the SARS-CoV-2 nucleocapsid protein (NP) and receptor-binding domain (RBD) of the spike protein were selected for the human COVID-19 Multiplex Assay. A comparison of synthesized and mammalian expressed RBD proteins revealed clear advantages of mammalian expression. Antibodies directed against NP strongly correlated with SARS-CoV-2 virus neutralization assay titers (rsp = 0.726), while anti-RBD correlation was moderate (rsp = 0.436). Pan-Ig, IgG, IgA, and IgM against NP and RBD antigens were evaluated on the validation sample sets. Detection of NP and RBD specific IgG and IgA had outstanding performance (AUC > 0.90) for distinguishing patients from controls, but the dynamic range of the IgG assay was substantially greater. The COVID-19 Multiplex Assay was utilized to identify seroprevalence to SARS-CoV-2 in people living in a low-incidence community in Ithaca, NY. Samples were taken from a cohort of healthy volunteers (n = 332) in early June 2020. Only two volunteers had a positive result on a COVID-19 PCR test performed prior to serum sampling. Serological testing revealed an exposure rate of at least 1.2% (NP) or as high as 5.7% (RBD), higher than the measured incidence rate of 0.16% in the county at that time. This highly sensitive and quantitative assay can be used for monitoring community exposure rates and duration of immune response following both infection and vaccination.
Assuntos
Anticorpos Antivirais/química , Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/sangue , COVID-19/epidemiologia , Teste Sorológico para COVID-19/normas , Proteínas do Nucleocapsídeo de Coronavírus/química , Monitoramento Epidemiológico , Feminino , Humanos , Imunoglobulina A/química , Imunoglobulina A/imunologia , Imunoglobulina G/química , Imunoglobulina G/imunologia , Imunoglobulina M/química , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , New York/epidemiologia , Fosfoproteínas/química , Fosfoproteínas/imunologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , SARS-CoV-2/classificação , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses challenges to public health given its zoonotic potential. Thus effective IAV-S vaccines are needed and highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs, with higher levels of neutralizing antibodies (NA) being detected in OV-HA-NP-immunized animals pre-challenge infection. Although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S-specific proliferating CD8+ T cells upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in incremental protection against challenge infection as evidenced by a reduced secondary antibody response (NA and HI antibodies) following IAV-S challenge and reduced virus shedding in nasal secretions (lower viral RNA loads and frequency of animals shedding viral RNA and infectious virus), when compared to animals in the OV-HA group. Interestingly, broader cross neutralization activity was also observed in serum of OV-HA-NP-immunized animals against a panel of contemporary IAV-S isolates representing the major genetic clades circulating in swine. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza/imunologia , Vírus do Orf , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Sintéticas/imunologia , Animais , Anticorpos Antivirais/imunologia , Vetores Genéticos/imunologia , Vírus da Influenza A , SuínosRESUMO
The parapoxvirus orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host innate and pro-inflammatory responses to infection. Using the ORFV IA82 strain as the parental virus, recombinant viruses with individual deletions in the genes encoding the IMPs chemokine binding protein (CBP; ORFV112), inhibitor of granulocyte-monocyte colony-stimulating factor and IL-2 (GIF, ORFV117) and interleukin 10 homologue (vIL-10; ORFV127) were generated and characterized in vitro and in vivo. The replication properties of the individual gene deletion viruses in cell culture was not affected comparing with the parental virus. To investigate the effect of the individual gene deletions in ORFV infection and pathogenesis, groups of four lambs were inoculated with each virus and were monitored thereafter. Lambs inoculated with either recombinant or with the parental ORFV developed characteristic lesions of contagious ecthyma. The onset, nature and severity of the lesions in the oral commissure were similar in all inoculated groups from the onset (3 days post-inoculation [pi]) to the peak of clinical lesions (days 11-13 pi). Nonetheless, from days 11-13 pi onwards, the oral lesions in lambs inoculated with the recombinant viruses regressed faster than the lesions produced by the parental virus. Similarly, the amount of virus shed in the lesions were equivalent among lambs of all groups up to day 15 pi, yet they were significantly higher in the parental virus group from day 16-21 pi. In conclusion, individual deletion of these IMP genes from the ORFV genome resulted in slight reduction in virulence in vivo, as evidenced by a reduction in the duration of the clinical disease and virus shedding.
Assuntos
Genes Virais/imunologia , Vírus do Orf/genética , Vírus do Orf/patogenicidade , Doenças dos Ovinos/virologia , Fatores Etários , Animais , Citocinas/genética , Citocinas/imunologia , Ectima Contagioso/imunologia , Ectima Contagioso/virologia , Genoma Viral , Mutação , Vírus do Orf/imunologia , Ovinos/virologia , Doenças dos Ovinos/imunologia , Transdução de Sinais , Virulência , Replicação Viral/genética , Eliminação de Partículas Virais , Sequenciamento Completo do GenomaRESUMO
Coronavirus disease 19 (COVID-19), has claimed millions of human lives worldwide since the emergence of the zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019. Notably, most severe and fatal SARS-CoV-2 infections in humans have been associated with underlying clinical conditions, including diabetes, hypertension and heart diseases. Here, we describe a case of severe SARS-CoV-2 infection in a domestic cat (Felis catus) that presented with hypertrophic cardiomyopathy (HCM), a chronic heart condition that has been described as a comorbidity of COVID-19 in humans and that is prevalent in domestic cats. The lung and heart of the affected cat presented clear evidence of SARS-CoV-2 replication, with histological lesions similar to those observed in humans with COVID-19 with high infectious viral loads being recovered from these organs. The study highlights the potential impact of comorbidities on the outcome of SARS-CoV-2 infection in animals and provides important information that may contribute to the development of a feline model with the potential to recapitulate the clinical outcomes of severe COVID-19 in humans.
Assuntos
COVID-19/virologia , Cardiomiopatia Hipertrófica/virologia , SARS-CoV-2/fisiologia , Animais , COVID-19/patologia , Cardiomiopatia Hipertrófica/patologia , Gatos , Coração/virologia , Pulmão/virologia , SARS-CoV-2/genética , Replicação ViralRESUMO
Senecavirus A (SVA), an oncolytic picornavirus used for cancer treatment in humans, has recently emerged as a vesicular disease (VD)-causing agent in swine worldwide. Notably, SVA-induced VD is indistinguishable from foot-and-mouth disease (FMD) and other high-consequence VDs of pigs. Here we investigated the role of apoptosis on infection and replication of SVA. Given the critical role of the nuclear factor-kappa B (NF-κB) signaling pathway on modulation of cell death, we first assessed activation of NF-κB during SVA infection. Results here show that while early during infection SVA induces activation of NF-κB, as evidenced by nuclear translocation of NF-κB-p65 and NF-κB-mediated transcription, late in infection a cleaved product corresponding to the C-terminus of NF-κB-p65 is detected in infected cells, resulting in lower NF-κB transcriptional activity. Additionally, we assessed the potential role of SVA 3C protease (3Cpro) in SVA-induced host-cell apoptosis and cleavage of NF-κB-p65. Transient expression of SVA 3Cpro was associated with cleavage of NF-κB-p65 and Poly (ADP-ribose) polymerase (PARP), suggesting its involvement in virus-induced apoptosis. Most importantly, we showed that while cleavage of NF-κB-p65 is secondary to caspase activation, the proteolytic activity of SVA 3Cpro is essential for induction of apoptosis. Experiments using the pan-caspase inhibitor Z-VAD-FMK confirmed the relevance of late apoptosis for SVA infection, indicating that SVA induces apoptosis, presumably, as a mechanism to facilitate virus release and/or spread from infected cells. Together, these results suggest an important role of apoptosis for SVA infection biology.
Assuntos
Apoptose , Cisteína Endopeptidases/metabolismo , Interações Hospedeiro-Patógeno , Infecções por Picornaviridae/virologia , Picornaviridae/enzimologia , Proteínas Virais/metabolismo , Proteases Virais 3C , Animais , Apoptose/genética , Linhagem Celular , Cisteína Endopeptidases/química , Citometria de Fluxo , Genes Reporter , Humanos , Mediadores da Inflamação/metabolismo , Modelos Moleculares , NF-kappa B/metabolismo , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/metabolismo , Conformação Proteica , Proteólise , Transdução de Sinais , Relação Estrutura-Atividade , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Proteínas Virais/químicaRESUMO
Senecavirus A (SVA) is an emerging picornavirus causing vesicular disease (VD) clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Currently there are no vaccines currently available for SVA. Here we developed a recombinant SVA strain (rSVAm SacII) using reverse genetics and assessed its immunogenicity and protective efficacy in pigs. In vivo characterization of the rSVAm SacII strain demonstrated that the virus is attenuated, as evidenced by absence of lesions, decreased viremia and virus shedding in inoculated animals. Notably, while attenuated, rSVA mSacII virus retained its immunogenicity as high neutralizing antibody (NA) responses were detected in inoculated animals. To assess the immunogenicity and protective efficacy of rSVA mSacII, 4-week-old piglets were sham-immunized or immunized with inactivated or live rSVA mSacII virus-based formulations. A single immunization with live rSVA mSacII virus via the intramuscular (IM) and intranasal (IN) routes resulted in robust NA responses with antibodies being detected between days 3-7 pi. Neutralizing antibody responses in animals immunized with the inactivated virus via the IM route were delayed and only detected after a booster on day 21 pi. Immunization with live virus resulted in recall T cell proliferation (CD4+, CD8+, and CD4+/CD8+ T cells), demonstrating efficient stimulation of cellular immunity. Notably, a single dose of the live attenuated vaccine candidate resulted in protection against heterologous SVA challenge, as demonstrated by absence of overt disease and reduced viremia, virus shedding and viral load in tissues. The live attenuated vaccine candidate developed here represents a promising alternative to prevent and control SVA in swine.
Assuntos
Infecções por Picornaviridae/veterinária , Picornaviridae/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Imunização , Infecções por Picornaviridae/prevenção & controle , Suínos , Linfócitos T/imunologia , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Fowlpox virus (FWPV), the type species of the genus Avipoxvirus family Poxviridae, is a large double-stranded DNA virus that causes fowlpox in chickens and turkeys. Notably, sequences of the avian retrovirus reticuloendotheliosis virus (REV) are frequently found integrated into the genome of FWPV. While some FWPV strains carry remnants of the REV long terminal repeats (LTRs), other strains have been shown to contain insertions of nearly the full-length REV provirus in their genome. In the present study we detected heterogeneous FWPV populations carrying the REV LTR or the near full-length REV provirus genome in a Merriam's wild turkey (Meleagris gallopavo merriami). The bird presented papules distributed throughout the non-feathered areas of the head. Avipoxvirus-like virions were observed in the lesions by transmission electron microscopy and the presence of FWPV was confirmed by DNA sequencing. Metagenomic sequencing performed on nucleic acid extracted from the skin lesions revealed two FWPV genome populations carrying either a 197-nt remnant of the REV LTR or a 7939-nt long fragment corresponding to the full-length REV provirus. Notably, PCR amplification using primers targeting FWPV sequences flanking the REV insertion site, confirmed the natural occurrence of the heterogeneous FWPV genome populations in one additional clinical sample from another turkey affected by fowlpox. Additionally, sequencing of a historical FWPV isolate obtained from chickens in the US in 2000 also revealed the presence of the two FWPV-REV genome populations. Results here demonstrate distinct FWPV populations containing variable segments of REV genome integrated into their genome. These distinct genome populations are likely a result of homologous recombination events that take place during FWPV replication.