RESUMEN
The pig is a natural host for influenza viruses and integrally involved in virus evolution through interspecies transmissions between humans and swine. Swine have many physiological, anatomical, and immunological similarities to humans, and are an excellent model for human influenza. Here, we employed single cell RNA-sequencing (scRNA-seq) and flow cytometry to characterize the major leukocyte subsets in bronchoalveolar lavage (BAL), twenty-one days after H1N1pdm09 infection or respiratory immunization with an adenoviral vector vaccine expressing hemagglutinin and nucleoprotein with or without IL-1ß. Mapping scRNA-seq clusters from BAL onto those previously described in peripheral blood facilitated annotation and highlighted differences between tissue resident and circulating immune cells. ScRNA-seq data and functional assays revealed lasting impacts of immune challenge on BAL populations. First, mucosal administration of IL-1ß reduced the number of functionally active Treg cells. Second, influenza infection upregulated IFI6 in BAL cells and decreased their susceptibility to virus replication in vitro. Our data provide a reference map of porcine BAL cells and reveal lasting immunological consequences of influenza infection and respiratory immunization in a highly relevant large animal model for respiratory virus infection.
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Subtipo H1N1 del Virus de la Influenza A , Vacunas contra la Influenza , Pulmón , Infecciones por Orthomyxoviridae , Análisis de la Célula Individual , Animales , Porcinos , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología , Pulmón/inmunología , Pulmón/virología , Vacunas contra la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Inmunización , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/virologíaRESUMEN
Coronaviruses infect a wide variety of host species, resulting in a range of diseases in both humans and animals. The coronavirus genome consists of a large positive-sense single-stranded molecule of RNA containing many RNA structures. One structure, denoted s2m and consisting of 41 nucleotides, is located within the 3' untranslated region (3' UTR) and is shared between some coronavirus species, including infectious bronchitis virus (IBV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, as well as other pathogens, including human astrovirus. Using a reverse genetic system to generate recombinant viruses, we investigated the requirement of the s2m structure in the replication of IBV, a globally distributed economically important Gammacoronavirus that infects poultry causing respiratory disease. Deletion of three nucleotides predicted to destabilize the canonical structure of the s2m or the deletion of the nucleotides corresponding to s2m impacted viral replication in vitro. In vitro passaging of the recombinant IBV with the s2m sequence deleted resulted in a 36-nucleotide insertion in place of the deletion, which was identified to be composed of a duplication of flanking sequences. A similar result was observed following serial passage of human astrovirus with a deleted s2m sequence. RNA modeling indicated that deletion of the nucleotides corresponding to the s2m impacted other RNA structures present in the IBV 3' UTR. Our results indicated for both IBV and human astrovirus a preference for nucleotide occupation in the genome location corresponding to the s2m, which is independent of the specific s2m sequence. IMPORTANCE Coronaviruses infect many species, including humans and animals, with substantial effects on livestock, particularly with respect to poultry. The coronavirus RNA genome consists of structural elements involved in viral replication whose roles are poorly understood. We investigated the requirement of the RNA structural element s2m in the replication of the Gammacoronavirus infectious bronchitis virus, an economically important viral pathogen of poultry. Using reverse genetics to generate recombinant IBVs with either a disrupted or deleted s2m, we showed that the s2m is not required for viral replication in cell culture; however, replication is decreased in tracheal tissue, suggesting a role for the s2m in the natural host. Passaging of these viruses as well as human astrovirus lacking the s2m sequence demonstrated a preference for nucleotide occupation, independent of the s2m sequence. RNA modeling suggested deletion of the s2m may negatively impact other essential RNA structures.
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Virus de la Bronquitis Infecciosa , Mamastrovirus , Mutagénesis Insercional , Animales , Humanos , Regiones no Traducidas 3'/genética , Pollos/virología , Virus de la Bronquitis Infecciosa/genética , Mamastrovirus/genética , Mutagénesis Insercional/genética , Enfermedades de las Aves de Corral/virología , ARN Viral/genética , Replicación Viral/genética , Estabilidad del ARN/genética , Eliminación de Secuencia/genéticaRESUMEN
The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious global pathogen prevalent in all types of poultry flocks. IBV is responsible for economic losses and welfare issues in domestic poultry, resulting in a significant risk to food security. IBV vaccines are currently generated by serial passage of virulent IBV field isolates through embryonated hens' eggs. The different patterns of genomic variation accumulated during this process means that the exact mechanism of attenuation is unknown and presents a risk of reversion to virulence. Additionally, the passaging process adapts the virus to replicate in chicken embryos, increasing embryo lethality. Vaccines produced in this manner are therefore unsuitable for in ovo application. We have developed a reverse genetics system, based on the pathogenic IBV strain M41, to identify genes which can be targeted for rational attenuation. During the development of this reverse genetics system, we identified four amino acids, located in nonstructural proteins (nsps) 10, 14, 15, and 16, which resulted in attenuation both in vivo and in ovo. Further investigation highlighted a role of amino acid changes, Pro85Leu in nsp 10 and Val393Leu in nsp 14, in the attenuated in vivo phenotype observed. This study provides evidence that mutations in nsps offer a promising mechanism for the development of rationally attenuated live vaccines against IBV, which have the potential for in ovo application. IMPORTANCE The Gammacoronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute, highly contagious, economically important disease of poultry. Vaccination is achieved using a mixture of live attenuated vaccines for young chicks and inactivated vaccines as boosters for laying hens. Live attenuated vaccines are generated through serial passage in embryonated hens' eggs, an empirical process which achieves attenuation but retains immunogenicity. However, these vaccines have a risk of reversion to virulence, and they are lethal to the embryo. In this study, we identified amino acids in the replicase gene which attenuated IBV strain M41, both in vivo and in ovo. Stability assays indicate that the attenuating amino acids are stable and unlikely to revert. The data in this study provide evidence that specific modifications in the replicase gene offer a promising direction for IBV live attenuated vaccine development, with the potential for in ovo application.
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Aminoácidos , Infecciones por Coronavirus , Virus de la Bronquitis Infecciosa , Enfermedades de las Aves de Corral , Proteínas no Estructurales Virales , Vacunas Virales , Aminoácidos/química , Aminoácidos/genética , Animales , Embrión de Pollo , Pollos , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Femenino , Virus de la Bronquitis Infecciosa/genética , Enfermedades de las Aves de Corral/prevención & control , Enfermedades de las Aves de Corral/virología , Vacunas Atenuadas/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Vacunas Virales/genéticaRESUMEN
BACKGROUND: Non-targeted whole genome sequencing is a powerful tool to comprehensively identify constituents of microbial communities in a sample. There is no need to direct the analysis to any identification before sequencing which can decrease the introduction of bias and false negatives results. It also allows the assessment of genetic aberrations in the genome (e.g., single nucleotide variants, deletions, insertions and copy number variants) including in noncoding protein regions. METHODS: The performance of four different random priming amplification methods to recover RNA viral genetic material of SARS-CoV-2 were compared in this study. In method 1 (H-P) the reverse transcriptase (RT) step was performed with random hexamers whereas in methods 2-4 RT incorporating an octamer primer with a known tag. In methods 1 and 2 (K-P) sequencing was applied on material derived from the RT-PCR step, whereas in methods 3 (SISPA) and 4 (S-P) an additional amplification was incorporated before sequencing. RESULTS: The SISPA method was the most effective and efficient method for non-targeted/random priming whole genome sequencing of SARS-CoV-2 that we tested. The SISPA method described in this study allowed for whole genome assembly of SARS-CoV-2 and influenza A(H1N1)pdm09 in mixed samples. We determined the limit of detection and characterization of SARS-CoV-2 virus which was 103 pfu/ml (Ct, 22.4) for whole genome assembly and 101 pfu/ml (Ct, 30) for metagenomics detection. CONCLUSIONS: The SISPA method is predominantly useful for obtaining genome sequences from RNA viruses or investigating complex clinical samples as no prior sequence information is needed. It might be applied to monitor genomic virus changes, virus evolution and can be used for fast metagenomics detection or to assess the general picture of different pathogens within the sample.
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COVID-19 , Subtipo H1N1 del Virus de la Influenza A , Virus ARN , Genoma Viral , Humanos , SARS-CoV-2/genética , Secuenciación Completa del GenomaRESUMEN
Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.
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Virus de la Bronquitis Infecciosa/genética , ARN Mensajero/genética , ARN Viral/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Transcripción Genética/genética , Regiones no Traducidas 5'/genética , Animales , Secuencia de Bases , Línea Celular , Pollos , Chlorocebus aethiops , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Sistemas de Lectura Abierta/genética , Enfermedades de las Aves de Corral/virología , Células Vero , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs; however, the molecular mechanism of attenuation is unknown. M41-CK, a virulent lab-adapted strain of IBV, was egg passaged over 100 times in four parallel independent replicates. All four final egg-passaged viruses were attenuated in vivo and exhibited similar growth phenotypes in adult chicken kidney cells and ex vivo tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, and the results showed that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hot spots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation; however, little is known about the evolution of the viral population during the process of attenuation. High-throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hot spots of variation in the attenuated viruses have the potential to be used in the rational attenuation of virulent IBV for next-generation vaccine design.
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Huevos/virología , Virus de la Bronquitis Infecciosa , Polimorfismo de Nucleótido Simple , Vacunas Virales , Animales , Línea Celular , Pollos , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Virus de la Bronquitis Infecciosa/genética , Virus de la Bronquitis Infecciosa/inmunología , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunologíaRESUMEN
African buffaloes (Syncerus caffer) are the principal "carrier" hosts of foot-and-mouth disease virus (FMDV). Currently, the epithelia and lymphoid germinal centers of the oropharynx have been identified as sites for FMDV persistence. We carried out studies in FMDV SAT1 persistently infected buffaloes to characterize the diversity of viruses in oropharyngeal epithelia, germinal centers, probang samples (oropharyngeal scrapings), and tonsil swabs to determine if sufficient virus variation is generated during persistence for immune escape. Most sequencing reads of the VP1 coding region of the SAT1 virus inoculum clustered around 2 subpopulations differing by 22 single-nucleotide variants of intermediate frequency. Similarly, most sequences from oropharynx tissue clustered into two subpopulations, albeit with different proportions, depending on the day postinfection (dpi). There was a significant difference between the populations of viruses in the inoculum and in lymphoid tissue taken at 35 dpi. Thereafter, until 400 dpi, no significant variation was detected in the viral populations in samples from individual animals, germinal centers, and epithelial tissues. Deep sequencing of virus from probang or tonsil swab samples harvested prior to postmortem showed less within-sample variability of VP1 than that of tissue sample sequences analyzed at the same time. Importantly, there was no significant difference in the ability of sera collected between 14 and 400 dpi to neutralize the inoculum or viruses isolated at later time points in the study from the same animal. Therefore, based on this study, there is no evidence of escape from antibody neutralization contributing to FMDV persistent infection in African buffalo.IMPORTANCE Foot-and-mouth disease virus (FMDV) is a highly contagious virus of cloven-hoofed animals and is recognized as the most important constraint to international trade in animals and animal products. African buffaloes (Syncerus caffer) are efficient carriers of FMDV, and it has been proposed that new virus variants are produced in buffalo during the prolonged carriage after acute infection, which may spread to cause disease in livestock populations. Here, we show that despite an accumulation of low-frequency sequence variants over time, there is no evidence of significant antigenic variation leading to immune escape. Therefore, carrier buffalo are unlikely to be a major source of new virus variants.
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Búfalos , Portador Sano/veterinaria , Evolución Molecular , Virus de la Fiebre Aftosa/crecimiento & desarrollo , Fiebre Aftosa/inmunología , Fiebre Aftosa/virología , Evasión Inmune , Animales , Proteínas de la Cápside/genética , Portador Sano/inmunología , Portador Sano/virología , Epitelio/virología , Virus de la Fiebre Aftosa/genética , Virus de la Fiebre Aftosa/inmunología , Inestabilidad Genómica , Centro Germinal/virología , Mutación , Orofaringe/virología , Análisis de Secuencia de ADNRESUMEN
In 2015, a mass die-off of ≈200,000 saiga antelopes in central Kazakhstan was caused by hemorrhagic septicemia attributable to the bacterium Pasteurella multocida serotype B. Previous analyses have indicated that environmental triggers associated with weather conditions, specifically air moisture and temperature in the region of the saiga antelope calving during the 10-day period running up to the event, were critical to the proliferation of latent bacteria and were comparable to conditions accompanying historically similar die-offs in the same areas. We investigated whether additional viral or bacterial pathogens could be detected in samples from affected animals using 3 different high-throughput sequencing approaches. We did not identify pathogens associated with commensal bacterial opportunisms in blood, kidney, or lung samples and thus concluded that P. multocida serotype B was the primary cause of the disease.
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Enfermedades de los Animales/mortalidad , Antílopes , Enfermedades de los Animales/epidemiología , Enfermedades de los Animales/historia , Enfermedades de los Animales/microbiología , Animales , Antílopes/microbiología , Infecciones Bacterianas/veterinaria , Femenino , Gammaproteobacteria/clasificación , Gammaproteobacteria/genética , Geografía Médica , Historia del Siglo XXI , Kazajstán/epidemiología , Masculino , Metagenómica , ARN Ribosómico 16S/genéticaRESUMEN
BACKGROUND: Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template. RESULTS: The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5' genomic termini and area immediately flanking the poly(C) region. CONCLUSIONS: We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.
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Virus de la Fiebre Aftosa/genética , Virus ARN/genética , Análisis de Secuencia de ARN/métodos , Virus de la Fiebre Aftosa/clasificación , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Datos de Secuencia Molecular , Poliadenilación , Virus ARN/clasificaciónRESUMEN
The possible emergence of drug-resistant avian flu raises concerns over the limited effectiveness of currently approved antivirals (neuraminidase inhibitors - NAIs) in the hypothetical event of a zoonotic spillover. Our study demonstrated that the recombinant avian A(H6N1) viruses showed reduced inhibition (RI) by multiple NAI drugs following the introduction of point mutations found predominantly in the neuraminidase gene (NA) of NAI-resistant human influenza strains (E119V, R292K and H274Y; N2 numbering). Moreover, A(H6N1)-H274Y showed increased replication efficiency in vitro, and a fitness advantage over wild-type (WT) when co-inoculated into embryonated hen's eggs. The results presented in our study together with the zoonotic potential of the A(H6N1) virus as evidenced by the human infection from 2013, highlight the need for enhanced monitoring of NAI resistance-associated signatures in circulating LPAI (low pathogenic avian influenza) globally.
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Gripe Aviar , Gripe Humana , Animales , Femenino , Humanos , Oseltamivir/farmacología , Pollos , Neuraminidasa/genética , Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Mutación , Resistencia a Medicamentos , Farmacorresistencia Viral/genéticaRESUMEN
African swine fever virus causes a lethal hemorrhagic disease of domestic pigs. The NAM P1/1995 isolate was originally described as B646L genotype XVIII; however, full genome sequencing revealed that this assignment was incorrect.
RESUMEN
African swine fever (ASF) is a global threat to animal health and food security. ASF is typically controlled by strict biosecurity, rapid diagnosis, and culling of affected herds. Much progress has been made in developing modified live virus vaccines against ASF. There is host variation in response to ASF infection in the field and under controlled conditions. To better understand the dynamics underlying this host differential morbidity, whole transcriptome profiling was carried out in twelve immunized and five sham immunized pigs. Seventeen MHC homozygous inbred Large white Babraham pigs were sampled at three time points before and after the challenge. The changes in the transcriptome profiles of infected animals were surveyed over time. In addition, the immunization effect on the host response was studied as well among the contrasts of all protection subgroups. The results showed two promising candidate genes to distinguish between recovered and non-recovered pigs after infection with a virulent African swine fever virus (ASFV) pre-infection: HTRA3 and GFPT2 (padj < 0.05). Variant calling on the transcriptome assemblies showed a two-base pair insertion into the ACOX3 gene closely located to HTRA3 that may regulate its expression as a putative genomic variant for ASF. Several significant DGEs, enriched gene ontology (GO) terms, and KEGG pathways at 1 day and 7 days post-infection, compared to the pre-infection, indicate a significant inflammation response immediately after ASF infection. The presence of the virus was confirmed by the mapping of RNA-Seq reads on two whole viral genome sequences. This was concordant with a higher virus load in the non-recovered animals 7 days post-infection. There was no transcriptome signature on the immunization at pre-infection and 1 day post-infection. More samples and data from additional clinical trials may support these findings.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Virus de la Fiebre Porcina Africana/genética , Transcriptoma , Perfilación de la Expresión Génica , InmunizaciónRESUMEN
African swine fever (ASF) is a devastating disease of domestic pigs that has spread across the globe since its introduction into Georgia in 2007. The etiological agent is a large double-stranded DNA virus with a genome of 170 to 180 kb in length depending on the isolate. Much of the differences in genome length between isolates are due to variations in the copy number of five different multigene families that are encoded in repetitive regions that are towards the termini of the covalently closed ends of the genome. Molecular epidemiology of African swine fever virus (ASFV) is primarily based on Sanger sequencing of a few conserved and variable regions, but due to the stability of the dsDNA genome changes in the variable regions occur relatively slowly. Observations in Europe and Asia have shown that changes in other genetic loci can occur and that this could be useful in molecular tracking. ASFV has been circulating in Western Africa for at least forty years. It is therefore reasonable to assume that changes may have accumulated in regions of the genome other than the standard targets over the years. At present only one full genome sequence is available for an isolate from Western Africa, that of a highly virulent isolate collected from Benin during an outbreak in 1997. In Cameroon, ASFV was first reported in 1981 and outbreaks have been reported to the present day and is considered endemic. Here we report three full genome sequences from Cameroon isolates of 1982, 1994 and 2018 outbreaks and identify novel single nucleotide polymorphisms and insertion-deletions that may prove useful for molecular epidemiology studies in Western Africa and beyond.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Fiebre Porcina Africana/epidemiología , Camerún/epidemiología , Sus scrofa/genética , Análisis de Secuencia , Análisis de Secuencia de ADNRESUMEN
Avian infectious bronchitis is an acute respiratory disease of poultry of particular concern for global food security. Investigation of infectious bronchitis virus (IBV), the causative agent of avian infectious bronchitis, via reverse genetics enables deeper understanding of virus biology and a rapid response to emerging variants. Classic methods of reverse genetics for IBV can be time consuming, rely on recombination for the introduction of mutations, and, depending on the system, can be subject to genome instability and unreliable success rates. In this study, we have applied data-optimized Golden Gate Assembly design to create a rapidly executable, flexible, and faithful reverse genetics system for IBV. The IBV genome was divided into 12 fragments at high-fidelity fusion site breakpoints. All fragments were synthetically produced and propagated in E. coli plasmids, amenable to standard molecular biology techniques for DNA manipulation. The assembly can be carried out in a single reaction, with the products used directly in subsequent viral rescue steps. We demonstrate the use of this system for generation of point mutants and gene replacements. This Golden Gate Assembly-based reverse genetics system will enable rapid response to emerging variants of IBV, particularly important to vaccine development for controlling spread within poultry populations.
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Virus de la Bronquitis Infecciosa , Genética Inversa , Virus de la Bronquitis Infecciosa/genética , Genética Inversa/métodos , Animales , Genoma Viral , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/veterinaria , Plásmidos/genética , Enfermedades de las Aves de Corral/virología , Escherichia coli/genéticaRESUMEN
Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.
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Genoma Viral , Dermatosis Nodular Contagiosa , Virus de la Dermatosis Nodular Contagiosa , Filogenia , Secuenciación Completa del Genoma , Virus de la Dermatosis Nodular Contagiosa/genética , Virus de la Dermatosis Nodular Contagiosa/clasificación , Virus de la Dermatosis Nodular Contagiosa/aislamiento & purificación , Animales , Dermatosis Nodular Contagiosa/virología , Dermatosis Nodular Contagiosa/epidemiología , Bovinos , África Central/epidemiología , África Occidental/epidemiología , Brotes de EnfermedadesRESUMEN
BACKGROUND: Prevention of transfusion-transmitted malaria in at-risk children and pregnant women in endemic areas with inexpensive chloroquine is no longer effective due to widespread drug resistance. There is an urgent need for devising new strategies for transfusion malarial safety. We investigated the frequency of transfusion transmission of malaria within the Ghanaian blood donation system using blood donations from 106 asymptomatic adult Ghanaian blood donors. STUDY DESIGN AND METHODS: Paired samples from 106 blood donations and recipients (before and after transfusion) were tested for anti-merozoite surface protein-1/2 using the commercial Lab21 malaria enzyme immunoassay (EIA), four antigen-specific in-house EIAs, and Plasmodium lactate dehydrogenase (pLDH) EIA. Additionally, Plasmodium DNA was screened for using a species-specific nested polymerase chain reaction (PCR) and a Pan-Plasmodium quantitative PCR. Donor-recipient parasite identity was defined by two concordant genotyping strategies. RESULTS: Plasmodium antibody prevalence was 100% in both donors and recipients, with at least one antigen. Parasitemia prevalence was 54.7% in both donors and recipients with median levels of 20 and 5.3 copies/µL, respectively, the difference being correlated to age (p = 0.0001). Multiple species infections were frequent (8.5%). Twenty-four units of parasitemic blood were transfused to nonparasitemic recipients, of which 10 (41.7%) became infected after transfusion. Molecular genotyping with 13 distinct markers (antigenic genes and microsatellite loci) identified three to nine parasitemic recipients after transfusion with level of allelic identity suggesting 14% to 28% definite or possible transfusion-related parasitemia. CONCLUSION: None of the currently available screening assays appear suitable to minimize transfusion malaria without compromising the blood supply in endemic areas.
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Donantes de Sangre , Malaria/epidemiología , Malaria/transmisión , Adulto , Antígenos de Protozoos/sangre , Estudios de Cohortes , Femenino , Genotipo , Humanos , Técnicas para Inmunoenzimas , Masculino , Persona de Mediana Edad , Prevalencia , Estudios RetrospectivosRESUMEN
BACKGROUND: Malaria parasites are frequently transmitted by unscreened blood transfusions in Africa. Pathogen reduction methods in whole blood would thus greatly improve blood safety. We aimed to determine the efficacy of riboflavin plus irradiation for treatment of whole blood infected with Plasmodium falciparum. STUDY DESIGN AND METHODS: Blood was inoculated with 10(4) or 10(5) parasites/mL and riboflavin treated with or without ultraviolet (UV) irradiation (40-160 J/mL red blood cells [mL(RBCs)]). Parasite genome integrity was assessed by quantitative amplification inhibition assays, and P. falciparum viability was monitored in vitro. RESULTS: Riboflavin alone did not affect parasite genome integrity or parasite viability. Application of UV after riboflavin treatment disrupted parasite genome integrity, reducing polymerase-dependent amplification by up to 2 logs (99%). At 80 J/mL(RBCs), riboflavin plus irradiation prevented recovery of viable parasites in vitro for 2 weeks, whereas untreated controls typically recovered to approximately 2% parasitemia after 4 days of in vitro culture. Exposure of blood to 160 J/mL(RBCs) was not associated with significant hemolysis. CONCLUSIONS: Riboflavin plus irradiation treatment of whole blood damages parasite genomes and drastically reduces P. falciparum viability in vitro. In the absence of suitable malaria screening assays, parasite inactivation should be investigated for prevention of transfusion-transmitted malaria in highly endemic areas.
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Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/efectos de la radiación , Riboflavina/farmacología , Rayos Ultravioleta , Seguridad de la Sangre , Humanos , Malaria/sangre , Malaria/parasitologíaRESUMEN
In the original publication [...].
RESUMEN
Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.
RESUMEN
Porcine respiratory disease is multifactorial and most commonly involves pathogen co-infections. Major contributors include swine influenza A (swIAV) and porcine reproductive and respiratory syndrome (PRRSV) viruses. Experimental co-infection studies with these two viruses have shown that clinical outcomes can be exacerbated, but how innate and adaptive immune responses contribute to pathogenesis and pathogen control has not been thoroughly evaluated. We investigated immune responses following experimental simultaneous co-infection of pigs with swIAV H3N2 and PRRSV-2. Our results indicated that clinical disease was not significantly exacerbated, and swIAV H3N2 viral load was reduced in the lung of the co-infected animals. PRRSV-2/swIAV H3N2 co-infection did not impair the development of virus-specific adaptive immune responses. swIAV H3N2-specific IgG serum titers and PRRSV-2-specific CD8ß+ T-cell responses in blood were enhanced. Higher proportions of polyfunctional CD8ß+ T-cell subset in both blood and lung washes were found in PRRSV-2/swIAV H3N2 co-infected animals compared to the single-infected groups. Our findings provide evidence that systemic and local host immune responses are not negatively affected by simultaneous swIAV H3N2/PRRSV-2 co-infection, raising questions as to the mechanisms involved in disease modulation.