RESUMO
The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria.IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production.
Assuntos
Substituição de Aminoácidos , Cisteína Endopeptidases/imunologia , Vírus da Febre Aftosa/imunologia , Mutação de Sentido Incorreto , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Proteases Virais 3C , Animais , Cisteína Endopeptidases/genética , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/genética , Células HEK293 , Humanos , Proteínas Virais/genética , Vacinas Virais/genéticaRESUMO
UNLABELLED: The pathogenesis of persistent foot-and-mouth disease virus (FMDV) infection was investigated in 46 cattle that were either naive or had been vaccinated using a recombinant, adenovirus-vectored vaccine 2 weeks before challenge. The prevalence of FMDV persistence was similar in both groups (62% in vaccinated cattle, 67% in nonvaccinated cattle), despite vaccinated cattle having been protected from clinical disease. Analysis of antemortem infection dynamics demonstrated that the subclinical divergence between FMDV carriers and animals that cleared the infection had occurred by 10 days postinfection (dpi) in vaccinated cattle and by 21 dpi in nonvaccinated animals. The anatomic distribution of virus in subclinically infected, vaccinated cattle was restricted to the pharynx throughout both the early and the persistent phases of infection. In nonvaccinated cattle, systemically disseminated virus was cleared from peripheral sites by 10 dpi, while virus selectively persisted within the nasopharynx of a subset of animals. The quantities of viral RNA shed in oropharyngeal fluid during FMDV persistence were similar in vaccinated and nonvaccinated cattle. FMDV structural and nonstructural proteins were localized to follicle-associated epithelium of the dorsal soft palate and dorsal nasopharynx in persistently infected cattle. Host transcriptome analysis of tissue samples processed by laser capture microdissection indicated suppression of antiviral host factors (interferon regulatory factor 7, CXCL10 [gamma interferon-inducible protein 10], gamma interferon, and lambda interferon) in association with persistent FMDV. In contrast, during the transitional phase of infection, the level of expression of IFN-λ mRNA was higher in follicle-associated epithelium of animals that had cleared the infection. This work provides novel insights into the intricate mechanisms of FMDV persistence and contributes to further understanding of this critical aspect of FMDV pathogenesis. IMPORTANCE: The existence of a prolonged, asymptomatic carrier state is a political impediment for control and potential eradication of foot-and-mouth disease (FMD). When FMD outbreaks occur, they are often extinguished by massive depopulation of livestock due to the fear that some animals may have undiagnosed subclinical infection, despite uncertainty over the biological relevance of FMD virus (FMDV) persistence. The work described here elucidates aspects of the FMDV carrier state in cattle which may facilitate identification and/or abrogation of asymptomatic FMDV infection. The divergence between animals that clear infection and those that develop persistent infection was demonstrated to occur earlier than previously established. The host antiviral response in tissues maintaining persistent FMDV was downregulated, whereas upregulation of IFN-λ mRNA was found in the epithelium of cattle that had recently cleared the infection. This suggests that the clearing of FMDV infection is associated with an enhanced mucosal antiviral response, whereas FMDV persistence is associated with suppression of the host antiviral response.
Assuntos
Portador Sadio/veterinária , Doenças dos Bovinos/virologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/transmissão , Faringe/virologia , Vacinação/veterinária , Vacinas Sintéticas/administração & dosagem , Animais , Portador Sadio/imunologia , Portador Sadio/virologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , RNA Viral/genética , Vacinas Virais/administração & dosagem , Replicação ViralRESUMO
BACKGROUND: Understanding the mechanisms of attenuation and virulence of foot-and-mouth disease virus (FMDV) in the natural host species is critical for development of next-generation countermeasures such as live-attenuated vaccines. Functional genomics analyses of FMDV have identified few virulence factors of which the leader proteinase (Lpro) is the most thoroughly investigated. Previous work from our laboratory has characterized host factors in cattle inoculated with virulent FMDV and attenuated mutant strains with transposon insertions within Lpro. METHODS: In the current study, the characteristics defining virulence of FMDV in cattle were further investigated by comparing the pathogenesis of a mutant, attenuated strain (FMDV-Mut) to the parental, virulent virus from which the mutant was derived (FMDV-WT). The only difference between the two viruses was an insertion mutation in the inter-AUG region of the leader proteinase of FMDV-Mut. All cattle were infected by simulated-natural, aerosol inoculation. RESULTS: Both viruses were demonstrated to establish primary infection in the nasopharyngeal mucosa with subsequent dissemination to the lungs. Immunomicroscopic localization of FMDV antigens indicated that both viruses infected superficial epithelial cells of the nasopharynx and lungs. The critical differences between the two viruses were a more rapid establishment of infection by FMDV-WT and quantitatively greater virus loads in secretions and infected tissues compared to FMDV-Mut. The slower replicating FMDV-Mut established a subclinical infection that was limited to respiratory epithelial sites, whereas the faster replication of FMDV-WT facilitated establishment of viremia, systemic dissemination of infection, and clinical disease. CONCLUSION: The mutant FMDV was capable of achieving all the same early pathogenesis landmarks as FMDV-WT, but was unable to establish systemic infection. The precise mechanism of attenuation remains undetermined; but current data suggests that the impaired replication of the mutant is more responsible for attenuation than differences in host immunological factors. These results complement previous studies by providing data of high-granularity describing tissue-specific tropism of FMDV and by demonstrating microscopic localization of virulent and attenuated clones of the same field-strain FMDV.
Assuntos
Doenças dos Bovinos/virologia , Vírus da Febre Aftosa/patogenicidade , Febre Aftosa/virologia , Virulência , Aerossóis , Animais , Bovinos , Células Epiteliais/patologia , Células Epiteliais/virologia , Febre Aftosa/imunologia , Febre Aftosa/patologia , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/crescimento & desenvolvimento , Vírus da Febre Aftosa/isolamento & purificação , Pulmão/virologia , Mutagênese Insercional , Nasofaringe/patologia , Nasofaringe/virologia , RNA Viral/isolamento & purificação , Vacinas Atenuadas/imunologia , Proteínas Estruturais Virais , Fatores de Virulência , Replicação ViralRESUMO
BACKGROUND: In order to investigate host factors associated with the establishment of persistent foot-and-mouth disease virus (FMDV) infection, the systemic response to vaccination and challenge was studied in 47 steers. Eighteen steers that had received a recombinant FMDV A vaccine 2 weeks earlier and 29 non-vaccinated steers were challenged by intra-nasopharyngeal deposition of FMDV A24. For up to 35 days after challenge, host factors including complete blood counts with T lymphocyte subsets, type I/III interferon (IFN) activity, neutralizing and total FMDV-specific antibody titers in serum, as well as antibody-secreting cells (in 6 non-vaccinated animals) were characterized in the context of viral infection dynamics. RESULTS: Vaccination generally induced a strong antibody response. There was a transient peak of FMDV-specific serum IgM in non-vaccinated animals after challenge, while IgM levels in vaccinated animals did not increase further. Both groups had a lasting increase of specific IgG and neutralizing antibody after challenge. Substantial systemic IFN activity in non-vaccinated animals coincided with viremia, and no IFN or viremia was detected in vaccinated animals. After challenge, circulating lymphocytes decreased in non-vaccinated animals, coincident with viremia, IFN activity, and clinical disease, whereas lymphocyte and monocyte counts in vaccinated animals were unaffected by vaccination but transiently increased after challenge. The CD4(+)/CD8(+) T cell ratio in non-vaccinated animals increased during acute infection, driven by an absolute decrease of CD8(+) cells. CONCLUSIONS: The incidence of FMDV persistence was 61.5 % in non-vaccinated and 54.5 % in vaccinated animals. Overall, the systemic factors examined were not associated with the FMDV carrier/non-carrier divergence; however, significant differences were identified between responses of non-vaccinated and vaccinated cattle.
Assuntos
Vírus da Febre Aftosa/fisiologia , Febre Aftosa/prevenção & controle , Febre Aftosa/virologia , Vacinas Virais/imunologia , Adenoviridae , Animais , Portador Sadio , Bovinos , Doenças dos Bovinos , Ensaio de Imunoadsorção Enzimática/veterinária , ELISPOT/veterinária , Feminino , Febre Aftosa/imunologia , Vetores Genéticos , Masculino , Vacinação , Vacinas SintéticasRESUMO
Foot-and-mouth disease (FMD) is a worldwide problem limiting the trade of animals and their products from affected countries. The rapid isolation, serotyping, and vaccine matching of FMD virus from disease outbreaks is critical for enabling the implementation of effective vaccination programs and to stop the spread of infection during outbreaks. Some primary cells have been shown to be highly susceptible to most strains of FMD virus (FMDV) but are difficult and expensive to prepare and maintain. Since the αVß6 integrin is a principal receptor for FMDV, we transduced a bovine kidney cell line to stably express both the αV and ß6 bovine integrin subunits. This stable cell line (LFBK-αVß6) showed ß6 expression and enhanced susceptibility to FMDV infection for ≥ 100 cell passages. LFBK-αVß6 cells were highly sensitive for detecting all serotypes of FMDV from experimentally infected animals, including the porcinophilic FMDV strain O/TAW/97. In comparison to other cell types that are currently used for virus isolation, LFBK-αVß6 cells were more effective at detecting FMDV in clinical samples, supporting their use as a more sensitive tool for virus isolation.
Assuntos
Células Epiteliais/virologia , Vírus da Febre Aftosa/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Receptores Virais/biossíntese , Receptores de Vitronectina/biossíntese , Animais , Bovinos , Técnicas de Cultura de Células/métodos , Linhagem Celular , Expressão Gênica , Instabilidade Genômica , Receptores Virais/genética , Receptores de Vitronectina/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transdução Genética , Cultura de Vírus/métodosRESUMO
Vaccination of domestic animals with chemically inactivated foot-and-mouth disease virus (FMDV) is widely practiced to control FMD. Currently, FMD vaccine manufacturing requires the growth of large volumes of virulent FMDV in biocontainment-level facilities. Here, two marker FMDV vaccine candidates (A(24)LL3D(YR) and A(24)LL3B(PVKV)3D(YR)) featuring the deletion of the leader coding region (L(pro)) and one of the 3B proteins were constructed and evaluated. These vaccine candidates also contain either one or two sets of mutations to create negative antigenic markers in the 3D polymerase (3D(pol)) and 3B nonstructural proteins. Two mutations in 3D(pol), H(27)Y and N(31)R, as well as RQKP(9-12)âPVKV substitutions, in 3B(2) abolish reactivity with monoclonal antibodies targeting the respective sequences in 3D(pol) and 3B. Infectious cDNA clones encoding the marker viruses also contain unique restriction endonuclease sites flanking the capsid-coding region that allow for easy derivation of custom designed vaccine candidates. In contrast to the parental A(24)WT virus, single A(24)LL3D(YR) and double A(24)LL3B(PVKV)3D(YR) mutant viruses were markedly attenuated upon inoculation of cattle using the natural aerosol or direct tongue inoculation. Likewise, pigs inoculated with live A(24)LL3D(YR) virus in the heel bulbs showed no clinical signs of disease, no fever, and no FMD transmission to in-contact animals. Immunization of cattle with chemically inactivated A(24)LL3D(YR) and A(24)LL3B(PVKV)3D(YR) vaccines provided 100% protection from challenge with parental wild-type virus. These attenuated, antigenically marked viruses provide a safe alternative to virulent strains for FMD vaccine manufacturing. In addition, a competitive enzyme-linked immunosorbent assay targeted to the negative markers provides a suitable companion test for differentiating infected from vaccinated animals.
Assuntos
Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vacinas Virais/efeitos adversos , Vacinas Virais/imunologia , Animais , Bovinos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Febre Aftosa/imunologia , Febre Aftosa/transmissão , Vírus da Febre Aftosa/genética , Deleção de Genes , Mutação de Sentido Incorreto , Análise de Sobrevida , Suínos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Marcadoras/administração & dosagem , Vacinas Marcadoras/efeitos adversos , Vacinas Marcadoras/genética , Vacinas Marcadoras/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-λ, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-λ3 (boIFN-λ3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-λ3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-λ3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-λ3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-λ3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle.
Assuntos
Antivirais , Doenças dos Bovinos/terapia , Febre Aftosa/terapia , Interferon gama/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Linhagem Celular , Cricetinae , Febre Aftosa/genética , Febre Aftosa/imunologia , Vírus da Febre Aftosa/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Leucócitos Mononucleares/metabolismo , Resultado do TratamentoRESUMO
Inactivated, wild-type foot-and-mouth disease virus (FMDV) vaccines are currently used to control FMD around the world. These traditional FMD vaccines are produced using large quantities of infectious, virulent, wild-type FMD viruses, with the associated risk of virus escape from manufacturing facilities or incomplete inactivation during the vaccine formulation process. While higher quality vaccines produced from wild-type FMDV are processed to reduce non-structural antigens, there is still a risk that small amounts of non-structural proteins may be present in the final product. A novel, antigenically marked FMD-LL3B3D vaccine platform under development by Zoetis, Inc. and the USDA-ARS, consists of a highly attenuated virus platform containing negative antigenic markers in the conserved non-structural proteins 3Dpol and 3B that render resultant vaccines fully DIVA compatible. This vaccine platform allows for the easy exchange of capsid coding sequences to create serotype-specific vaccines. Here we demonstrate the efficacy of the inactivated FMD-LL3B3D-A24 Cruzeiro vaccine in cattle against wild-type challenge with A24 Cruzerio. A proprietary adjuvant system was used to formulate the vaccines that conferred effective protection at low doses while maintaining the DIVA compatibility. In contrast to wild-type FMDV, the recombinant FMD-LL3B3D mutant viruses have been shown to induce no clinical signs of FMD and no shedding of virus in cattle or pigs when inoculated as a live virus. The FMD-LL3B3D vaccine platform, currently undergoing development in the US, provides opportunities for safer vaccine production with full DIVA compatibility in support of global FMDV control and eradication initiatives.
RESUMO
In this study, infrared thermography (IRT) was assessed as a means of detecting foot-and-mouth disease virus (FMDV)-infected cattle before and after the development of clinical signs. Preliminary IRT imaging demonstrated that foot temperatures increased in FMDV-infected animals. The maximum foot temperatures of healthy (n=53), directly inoculated (DI) (n=12), contact (CT) (n=6), and vaccine trial (VT) (n=21) cattle were measured over the course of FMD infection. A cut-off value was established at 34.4 degrees C (sensitivity=61.1%, specificity=87.7%) with the aim of detecting FMDV-infected animals both before and after clinical signs were observed. Seven of 12 (58%) DI and 3/6 (50%) CT animals showed maximum foot temperatures exceeding the 34.4 degrees C cut-off before the development of foot vesicles. In contrast, only 5/21 (24%) VT animals displayed pre-clinical foot temperatures above this cut-off possibly indicating partial vaccine protection of this group. These results show IRT as a promising screening technology to quickly identify potentially infected animals for confirmatory diagnostic testing during FMD outbreaks. Further evaluation of this technology is needed to determine the value of IRT in detecting animals with mild clinical signs or sub-clinical infections.
Assuntos
Doenças dos Bovinos/diagnóstico , Febre Aftosa/diagnóstico , Raios Infravermelhos , Termografia/veterinária , Animais , Bovinos , Doenças dos Bovinos/patologia , Febre/patologia , Febre/veterinária , Pé/patologia , Febre Aftosa/patologia , Vírus da Febre Aftosa/patogenicidade , Masculino , Sensibilidade e Especificidade , Termografia/métodosRESUMO
Foot-and-mouth disease virus (FMDV) is capable of infecting all cloven-hoofed domestic livestock species, including cattle, pigs, goats, and sheep. However, in contrast to cattle and pigs, the pathogenesis of FMDV in small ruminants has been incompletely elucidated. The objective of the current investigation was to characterize tissue- and cellular tropism of early and late stages of FMDV infection in sheep following three different routes of simulated natural virus exposure. Extensive post-mortem harvest of tissue samples at pre-determined time points during early infection (24 and 48 hours post infection) demonstrated that tissues specifically susceptible to primary FMDV infection included the paraepiglottic- and palatine tonsils, as well as the nasopharyngeal mucosa. Additionally, experimental aerosol inoculation of sheep led to substantial virus replication in the lungs at 24-48 hours post-inoculation. During persistent infection (35 days post infection), the paraepiglottic- and palatine tonsils were the only tissues from which infectious FMDV was recovered. This is strikingly different from cattle, in which persistent FMDV infection has consistently been located to the nasopharyngeal mucosa. Analysis of tissue sections by immunomicroscopy revealed a strict epithelial tropism during both early and late phases of infection as FMDV was consistently localized to cytokeratin-expressing epithelial cells. This study expands upon previous knowledge of FMDV pathogenesis in sheep by providing detailed information on the temporo-anatomic distribution of FMDV in ovine tissues. Findings are discussed in relation to similar investigations previously performed in cattle and pigs, highlighting similarities and differences in FMDV pathogenesis across natural host species.
Assuntos
Tonsila Faríngea/virologia , Vírus da Febre Aftosa/patogenicidade , Febre Aftosa/virologia , Tonsila Palatina/virologia , Ovinos/virologia , Tonsila Faríngea/patologia , Animais , Bovinos , Febre Aftosa/patologia , Vírus da Febre Aftosa/isolamento & purificação , Masculino , Tonsila Palatina/patologia , Mucosa Respiratória/patologia , Mucosa Respiratória/virologia , Especificidade da Espécie , Suínos , Virulência , Replicação ViralRESUMO
Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven hooved animals that continues to cause economic disruption in both endemic countries or when introduced into a formally FMD free country. Vaccines that protect against clinical disease and virus shedding are critical to control FMD. The replication deficient human adenovirus serotype 5 (Ad5) vaccine vector expressing empty FMD virus (FMDV) capsid, AdtFMD, is a promising new vaccine platform. With no shedding or spreading of viral vector detected in field trials, this vaccine is very safe to manufacture, as there is no requirement for high containment faciitites. Here, we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9â¯months following a single vaccination with the commercial AdtFMD vaccine, provisionally licensed for cattle in the United States. Further, we tested the effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome and humoral immunity. Results demonstrate that a single dose vaccination in cattle with the commercial vaccine vector expressing capsid proteins of the FMDV strain A24 Cruzeiro (Adt.A24), induced protection against clinical FMD at 6â¯months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9â¯months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Anti-FMDV antibody secreting cells are detected in peripheral blood early following vaccination, but are absent after 28â¯days post-vaccination. Thus, the decay in antibody mediated immunity over time is likely a function of FMDV-specific antibody half-life. These data reveal the short time span of anti-FMDV antibody secreting cells (ASCs) and important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.
Assuntos
Doenças dos Bovinos/prevenção & controle , Vírus da Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Vacinação/veterinária , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Virais/imunologia , Adenovírus Humanos/genética , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/imunologia , Bovinos , Doenças dos Bovinos/virologia , Vetores Genéticos , Imunidade Humoral/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Deletions within the 3A coding region of foot-and-mouth disease virus (FMDV) are associated with decreased virulence in cattle; however, the mechanisms are unknown. We compared experimental infection of cattle with virulent FMDV O1Campos (O1Ca) and a mutant derivative (O1Ca∆3A) lacking residues 87-106 of 3A. Unexpectedly, primary infection of the nasopharyngeal mucosa was similar for both viruses. However, while O1Ca caused viremia and fulminant clinical disease, O1Ca∆3A infection was subclinical and aviremic. There were no differences in expression of anti-viral cytokines in nasopharyngeal tissues between the groups, suggesting attenuation by O1Ca∆3A was a consequence of reduced replication efficiency in bovine cells, rather than a difference in the host response. These results demonstrated that although deletion in 3A of FMDV confers a clinically attenuated phenotype in cattle, the deletion does not prevent subclinical infection. These findings have implications for field scenarios involving outbreaks with apparently host-limited strains of FMDV.
Assuntos
Doenças dos Bovinos/virologia , Vírus da Febre Aftosa/fisiologia , Febre Aftosa/virologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Bovinos , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/patogenicidade , Deleção de Sequência , Virulência , Replicação ViralRESUMO
Vesicular stomatitis virus (VSV) causes sporadic outbreaks of vesicular disease in the southwestern United States. The intrinsic characteristics of epidemic strains associated with these outbreaks are poorly understood. In this study, we report the distinctive genomic and biological characteristics of an epidemic (NJ0612NME6) strain of VSV compared with an endemic (NJ0806VCB) strain. Genomic comparisons between the two strains revealed a total of 111 nucleotide differences (23 non-synonymous) with potentially relevant replacements located in the P, G, and L proteins. When tested in experimentally infected pigs, a natural host of VSV, the epidemic strain caused higher fever and an increased number of vesicular lesions compared to pigs infected with the endemic strain. Pigs infected with the epidemic strain showed decreased systemic antiviral activity (type I - IFN), lower antibody levels, higher levels of interleukin 6, and lower levels of tumor necrosis factor during the acute phase of disease compared to pigs infected with the endemic strain. Furthermore, we document the existence of an RNAemia phase in pigs experimentally infected with VSV and explored the cause for the lack of recovery of infectious virus from blood. Finally, the epidemic strain was shown to be more efficient in down-regulating transcription of IRF-7 in primary porcine macrophages. Collectively, the data shows that the epidemic strain of VSV we tested has an enhanced ability to modulate the innate immune response of the vertebrate host. Further studies are needed to examine other epidemic strains and what contributions a phenotype of increased virulence might have on the transmission of VSV during epizootics.
RESUMO
BACKGROUND: Immunophenotyping of blood lymphocytes by flow cytometry is important in infectious disease research. In animal experiments and other longitudinal studies, the processing, prompt staining, and analysis of fresh samples is a logistical challenge and daily assay variation can confound data interpretation. OBJECTIVE: This study examined the feasibility of cryopreservation and deferred analysis of bovine peripheral blood T lymphocytes from normal or infected animals. METHODS: Peripheral blood mononuclear cells were collected from 4 naïve Holstein steers and 4 steers infected with foot-and-mouth-disease virus serotype Asia1. Identical aliquots were labeled and analyzed immediately, labeled for deferred analysis, or stored at -70°C or over liquid nitrogen for up to 3 weeks before labeling. RESULTS: Freezing of unlabeled cells induced statistically significant changes in phenotypic recognition. In infected animals, the γδ T-cell population increased by 28% and CD8(+) αßT cells by 32%, while total CD3(+) cells decreased by 16%, and CD4(+) αßT cells decreased by 12%. Subsequent storage of frozen cells for the duration of the study, however, had no significant effect. There was less than 20% relative change in subpopulation sizes, and storage at -70°C or over liquid nitrogen was equivalent. CONCLUSIONS: Depending on the objectives and practical limitations of a study, deferred labeling of peripheral blood lymphocytes can be a viable option. Although frozen storage of lymphocytes can introduce some artifactual distortion of relative cell populations, frozen cells can be maintained in storage until all samples in a longitudinal study can be analyzed in batch under standardized conditions and without introducing further bias.
Assuntos
Doenças dos Bovinos/sangue , Bovinos/sangue , Criopreservação/veterinária , Febre Aftosa/sangue , Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doenças dos Bovinos/imunologia , Criopreservação/métodos , Criopreservação/normas , Estudos de Viabilidade , Citometria de Fluxo/veterinária , Vírus da Febre Aftosa/imunologia , Congelamento , Imunofenotipagem/veterinária , Leucócitos Mononucleares/imunologia , Estudos Longitudinais , Masculino , Manejo de Espécimes/veterináriaRESUMO
Abnormal body temperature is a major indicator of disease; infrared thermography (IRT) can assess changes in body surface temperature quickly and remotely. This technology can be applied to a myriad of diseases of various etiologies across a wide range of host species in veterinary medicine. It is used to monitor the physiologic status of individual animals, such as measuring feed efficiency or diagnosing pregnancy. Infrared thermography has applications in the assessment of animal welfare, and has been used to detect soring in horses and monitor stress responses. This review addresses the variety of uses for IRT in veterinary medicine, including disease detection, physiologic monitoring, welfare assessment, and potential future applications.
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Termografia/veterinária , Bem-Estar do Animal , Animais , Temperatura Corporal/fisiologia , Raios Infravermelhos , Monitorização Fisiológica/veterináriaRESUMO
Understanding the quantitative characteristics of a pathogen's capability to transmit during distinct phases of infection is important to enable accurate predictions of the spread and impact of a disease outbreak. In the current investigation, the potential for transmission of foot-and-mouth disease virus (FMDV) during the incubation (preclinical) period of infection was investigated in seven groups of pigs that were sequentially exposed to a group of donor pigs that were infected by simulated-natural inoculation. Contact-exposed pigs were comingled with infected donors through successive 8-h time slots spanning from 8 to 64 h post-inoculation (hpi) of the donor pigs. The transition from latent to infectious periods in the donor pigs was clearly defined by successful transmission of foot-and-mouth disease (FMD) to all contact pigs that were exposed to the donors from 24 hpi and later. This onset of infectiousness occurred concurrent with detection of viremia, but approximately 24 h prior to the first appearance of clinical signs of FMD in the donors. Thus, the latent period of infection ended approximately 24 h before the end of the incubation period. There were significant differences between contact-exposed groups in the time elapsed from virus exposure to the first detection of FMDV shedding, viremia, and clinical lesions. Specifically, the onset and progression of clinical FMD were more rapid in pigs that had been exposed to the donor pigs during more advanced phases of disease, suggesting that these animals had received a higher effective challenge dose. These results demonstrate transmission and dissemination of FMD within groups of pigs during the incubation period of infection. Furthermore, these findings suggest that under current conditions, shedding of FMDV in oropharyngeal fluids is a more precise proxy for FMDV infectiousness than clinical signs of infection. These findings may impact modeling of the propagation of FMD outbreaks that initiate in pig holdings and should be considered when designing FMD control strategies.
RESUMO
In order to investigate the mechanisms of persistent foot-and-mouth disease virus (FMDV) infection in cattle, transcriptome alterations associated with the FMDV carrier state were characterized using a bovine whole-transcriptome microarray. Eighteen cattle (8 vaccinated with a recombinant FMDV A vaccine, 10 non-vaccinated) were challenged with FMDV A24 Cruzeiro, and the gene expression profiles of nasopharyngeal tissues collected between 21 and 35 days after challenge were compared between 11 persistently infected carriers and 7 non-carriers. Carriers and non-carriers were further compared to 2 naïve animals that had been neither vaccinated nor challenged. At a controlled false-discovery rate of 10% and a minimum difference in expression of 50%, 648 genes were differentially expressed between FMDV carriers and non-carriers, and most (467) had higher expression in carriers. Among these, genes associated with cellular proliferation and the immune response-such as chemokines, cytokines and genes regulating T and B cells-were significantly overrepresented. Differential gene expression was significantly correlated between non-vaccinated and vaccinated animals (biological correlation +0.97), indicating a similar transcriptome profile across these groups. Genes related to prostaglandin E2 production and the induction of regulatory T cells were overexpressed in carriers. In contrast, tissues from non-carrier animals expressed higher levels of complement regulators and pro-apoptotic genes that could promote virus clearance. Based on these findings, we propose a working hypothesis for FMDV persistence in nasopharyngeal tissues of cattle, in which the virus may be maintained by an impairment of apoptosis and the local suppression of cell-mediated antiviral immunity by inducible regulatory T cells.
Assuntos
Doenças dos Bovinos/imunologia , Bovinos/virologia , Vírus da Febre Aftosa/imunologia , Febre Aftosa/imunologia , Imunidade Celular , Transcriptoma , Animais , Apoptose , Bovinos/genética , Bovinos/imunologia , Doenças dos Bovinos/genética , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/virologia , Febre Aftosa/genética , Febre Aftosa/prevenção & controle , Febre Aftosa/virologia , Perfilação da Expressão Gênica , Imunidade Humoral , Imunidade Inata , Nasofaringe/imunologia , Nasofaringe/metabolismo , Nasofaringe/virologia , Vacinação , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologiaRESUMO
African swine fever virus (ASFV) produces a contagious disease of domestic pigs that results in severe economic consequences to the swine industry. Control of the disease has been hampered by the unavailability of vaccines. We recently reported the development of two experimental vaccine strains (ASFV-G-Δ9GL and ASFV-G-ΔMGF) based on the attenuation of the highly virulent and epidemiologically relevant Georgia2007 isolate. Deletion of the 9GL gene or six genes of the MGF360/505 group produced two attenuated ASFV strains which were able to confer protection to animals when challenged with the virulent parental virus. Both viruses, although efficient in inducing protection, present concerns regarding their safety. In an attempt to solve this problem we developed a novel virus strain, ASFV-G-Δ9GL/ΔMGF, based on the deletion of all genes deleted in ASFV-G-Δ9GL and ASFV-G-ΔMGF. ASFV-G-Δ9GL/ΔMGF is the first derivative of a highly virulent ASFV field strain subjected to a double round of recombination events seeking to sequentially delete specific genes. ASFV-G-Δ9GL/ΔMGF showed a decreased ability to replicate in primary swine macrophage cultures relative to that of ASFV-G and ASFV-G-ΔMGF but similar to that of ASFV-G-Δ9GL. ASFV-G-Δ9GL/ΔMGF was attenuated when intramuscularly inoculated into swine, even at doses as high as 10(6) HAD50. Animals infected with doses ranging from 10(2) to 10(6) HAD50 did not present detectable levels of virus in blood at any time post-infection and they did not develop detectable levels of anti-ASFV antibodies. Importantly, ASFV-G-Δ9GL/ΔMGF does not induce protection against challenge with the virulent parental ASFV-G isolate. Results presented here suggest caution towards approaches involving genomic manipulations when developing rationally designed ASFV vaccine strains.
Assuntos
Vírus da Febre Suína Africana/genética , Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/patologia , Febre Suína Africana/virologia , Deleção de Sequência , Proteínas Virais/genética , Vacinas Virais/imunologia , Febre Suína Africana/prevenção & controle , Vírus da Febre Suína Africana/imunologia , Vírus da Febre Suína Africana/fisiologia , Animais , Anticorpos Antivirais/sangue , Células Cultivadas , Georgia , Injeções Intramusculares , Macrófagos/virologia , Recombinação Genética , Suínos , Resultado do Tratamento , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Virulência , Replicação ViralRESUMO
Since the early 2000s outbreaks of foot-and-mouth disease (FMD) have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea). One outbreak with FMD virus (FDMV) serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic.