RESUMEN
This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens.
Asunto(s)
Infecciones por Coronavirus , Virus de la Diarrea Epidémica Porcina , Enfermedades de los Porcinos , Virus de la Gastroenteritis Transmisible , Animales , Porcinos , Virus de la Gastroenteritis Transmisible/genética , Virus de la Gastroenteritis Transmisible/aislamiento & purificación , Virus de la Diarrea Epidémica Porcina/aislamiento & purificación , Virus de la Diarrea Epidémica Porcina/genética , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/veterinaria , Infecciones por Coronavirus/virología , Infecciones por Coronavirus/epidemiología , Enfermedades de los Porcinos/virología , Enfermedades de los Porcinos/diagnóstico , Estudios Retrospectivos , Gastroenteritis Porcina Transmisible/diagnóstico , Gastroenteritis Porcina Transmisible/virología , Gastroenteritis Porcina Transmisible/epidemiología , Reacción en Cadena de la Polimerasa/métodos , Deltacoronavirus/genética , Deltacoronavirus/aislamiento & purificación , Estados Unidos/epidemiologíaRESUMEN
The rapid spread of African swine fever virus (ASFV), causing severe and often lethal disease in domestic pigs and Eurasian wild boar, continues to be a threat to pig populations and dependent industries. Despite scientific achievements that have deepened our understanding of ASFV pathogenesis, alternative transmission routes for ASFV remain to be elucidated. We previously demonstrated the efficient transmission of ASFV from infected boars to naïve recipient gilts via artificial insemination, thereby highlighting the importance of surveillance of boar semen prior to its shipment. Since the accurate and reliable detection of even low amounts of ASFV in boar semen is key to disease prevention and control, we established a suitable diagnostic workflow to efficiently detect the ASFV genome in boar semen. Here, we assessed the sensitivity of various routine nucleic acid extraction kits as well as qPCR protocols in detecting the ASFV genome in the blood and semen of infected boars. The feasibility of the respective kits and methods for future use in boar studs was also considered. Variability in sensitivity mostly concerned samples with low to very low amounts of the ASFV genome. Ultimately, we defined a well-suited workflow for precisely detecting the ASFV genome in boar semen as early as 2 days post ASFV infection.
RESUMEN
The hemagglutinin-esterase-fusion (HEF) protein binds 9-O-acetylated sialic acids-containing glycans on the cell surface and drives influenza D virus (IDV) entry. The HEF is a primary determinant of the exceptional thermal and acid stability observed in IDV infection biology. Here, we expressed and purified the receptor binding domain (RBD) of the IDV HEF protein in Escherichia coli and characterized its receptor binding and antigenic properties. The data from these experiments indicate that (i) the RBD can bind with specificity to turkey red blood cells (RBC), and its binding can be specifically inhibited by IDV antibody; (ii) the RBD efficiently binds to the cell surface of MDCK cells expressing the receptor of IDV; and (iii) anti-RBD antibodies are capable of blocking RBD attachment to MDCK cells as well as of inhibiting the virus from agglutinating RBCs. These observations support the utility of this RBD in future receptor and entry studies of IDV.
Asunto(s)
Eritrocitos , Escherichia coli , Unión Proteica , Receptores Virales , Escherichia coli/genética , Escherichia coli/metabolismo , Animales , Perros , Receptores Virales/metabolismo , Receptores Virales/genética , Células de Riñón Canino Madin Darby , Hemaglutininas Virales/genética , Hemaglutininas Virales/inmunología , Hemaglutininas Virales/metabolismo , Proteínas Virales de Fusión/inmunología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismo , Expresión Génica , Anticuerpos Antivirales/inmunología , Humanos , Dominios Proteicos , DeltainfluenzavirusRESUMEN
The global distribution and ongoing evolution of type A swine influenza virus (IAV-S) continue to pose significant challenges against developing broadly protective vaccines to control swine influenza. This study focuses on the hemagglutinin (HA) consensus-based approach towards developing a more broadly protective swine influenza vaccine against various H3 strains circulating in domestic pig populations. By computationally analyzing >1000 swine H3 full-length HA sequences, we generated a consensus H3 and expressed it in the context of influenza A WSN/33 reverse genetics system. The derived recombinant chimeric swine influenza virus with the consensus H3 was inactivated and further evaluated as a potential universal vaccine in pigs. The consensus H3 vaccine elicited broadly active hemagglutination inhibition (HI) antibodies against divergent swine H3N2 influenza viruses including human H3N2 variant of concern, and strains belong to genetic clusters IV, IV-A, IV-B, IV-C, IV-D and IV-F. Importantly, vaccinated pigs were completely protected against challenge with a clinical swine H3N2 isolate in that neither viral shedding nor replication in lungs of vaccinated pigs were observed. These findings warrant further study of the consensus H3 vaccine platform for broad protection against diverse swine influenza viruses.
RESUMEN
Bovine respiratory disease (BRD) complex is a multifactorial respiratory disease of cattle. Seven-segmented influenza C (ICV) and D (IDV) viruses have been identified in cattle with BRD, however, molecular epidemiology and prevalence of IDV and ICV in the diseased population remain poorly characterized. Here, we conducted a molecular screening of 208 lung samples of bovine pneumonia cases for the presence of IDV and ICV. Our results demonstrated that both viruses were prevalent in BRD cases and the overall positivity rates of IDV and ICV were 20.88% and 5.99% respectively. Further analysis of three IDV strains isolated from lungs of cattle with BRD showed that these lung-tropic strains belonged to D/Michigan/2019 clade and diverged antigenically from the circulating dominant IDV clades D/OK and D/660. Our results reveal that IDV and ICV are associated with BRD complex and support a role for IDV and ICV in the etiology of BRD.
Asunto(s)
Complejo Respiratorio Bovino , Enfermedades de los Bovinos , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Thogotovirus , Virus , Bovinos , Animales , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/veterinaria , Prevalencia , Complejo Respiratorio Bovino/epidemiología , Enfermedades de los Bovinos/epidemiologíaRESUMEN
Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses. IMPORTANCE Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus. Here we demonstrated that the respiratory tract of guinea pigs possesses specific viral receptors for ICV. We also compared the replication kinetics and pathogenesis of huICV and swIDV, as these viruses share 50% sequence identity. The tissue tropism and pathology associated with huICV in guinea pigs are analogous to the mild respiratory disease caused by ICV in humans, thereby demonstrating the suitability of guinea pigs to study ICV. Our comparative analysis revealed that huICV and swIDV replicated differentially in the guinea pigs suggesting that the type-specific genetic differences can result in the disparity of the viral shedding and tissue tropism.
Asunto(s)
Modelos Animales de Enfermedad , Gammainfluenzavirus , Cobayas , Infecciones por Orthomyxoviridae , Thogotovirus , Animales , Humanos , Administración Intranasal , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Receptores ViralesRESUMEN
The rapid spread of the African swine fever virus (ASFV), causing severe disease with often high fatality rates in Eurasian suids, prevails as a threat for pig populations and dependent industries worldwide. Although advancing scientific progress continually enhances our understanding of ASFV pathogenesis, alternative transmission routes for ASFV have yet to be assessed. Here, we demonstrate that ASFV can efficiently be transferred from infected boars to naïve recipient gilts through artificial insemination (AI). In modern pig production, semen from boar studs often supplies many sow herds. Thus, the infection of a boar stud presents the risk of rapidly and widely distributing ASFV within or between countries. Daily blood and semen collection from four boars after intramuscular inoculation with ASFV strain 'Estonia 2014' resulted in the detection of ASFV genomes in the semen as early as 2 dpi, in blood at 1 dpi while semen quality remained largely unaffected. Ultimately, after insemination with extended semen, 7 of 14 gilts were ASFV positive by 7 days post insemination, and all gilts were ASFV positive by 35 days post insemination. Twelve out of 13 pregnant gilts aborted or resorbed at the onset of fever. A proportion of fetuses originating from the remaining gilt showed both abnormalities and replication of ASFV in fetal tissues. Thus, we present evidence for the efficient transmission of ASFV to gilts via AI and also to implanted embryos. These results underline the critical role that boar semen could play in ASFV transmission.
RESUMEN
Bats are a reservoir for many zoonotic viruses and host large numbers of genetically diverse species in the families Rhabdoviridae, Coronaviridae, and Paramyxoviridae. Viruses from these families have repeatedly spilled over to humans in recent decades, causing significant clinical disease and deaths. Here, metagenomic sequencing of a big brown bat (Eptesicus fuscus) submitted for rabies testing due to human exposure identified a novel paramyxovirus, Eptesicus fuscus orthorubulavirus (EfORV), in South Dakota, United States. The nearly complete 15,814-nucleotide genome shared 72% identity with that of human parainfluenza virus 4 (HPIV4), a virus that causes significant clinical disease, typically bronchiolitis and pneumonia, in children less than 2 years of age. Phylogenetic analysis confirmed a close evolutionary history between EfORV and HPIV4, reminiscent of other orthorubulaviruses with highly similar bat and mammalian species, including conspecific human and bat mumps virus, mammalian parainfluenza virus 5 and bat Alston virus, and porcine La Piedad Michoacán virus and bat Mapuera virus. These results support the idea that bats are a reservoir for diverse paramyxoviruses with closely shared evolutionary histories, compared with a number of significant human pathogens, and expand the range of bat paramyxoviruses to North America. Given the propensity of paramyxoviruses to overcome species barriers, additional surveillance and characterization of EfORV are warranted. IMPORTANCE Bats are a reservoir of large numbers of viruses. Among bat-borne zoonotic viruses, members of Coronaviridae and Paramyxoviridae have had the largest impact on human health. The repeated spillover of bat viruses to humans, often with devastating results, has led to increased surveillance and virus discovery efforts in hot spots for virus emergence, largely Asia and Africa. Apart from rabies virus, little surveillance of viruses in bats is performed in North America. Here, viral metagenomic sequencing identified a close relative to HPIV4 in a big brown bat found in a motel room in South Dakota. The virus, EfORV, was 72% identical to HPIV4, which causes clinically significant respiratory disease, mainly in children; it represents the first bat paramyxovirus identified in North America. Close genetic relationships between bat and mammalian orthorubulaviruses underscore the importance of bats as a reservoir for zoonotic viruses.
Asunto(s)
Quirópteros/virología , Paramyxoviridae/clasificación , Paramyxoviridae/aislamiento & purificación , Animales , Reservorios de Enfermedades/virología , Genoma Viral/genética , Humanos , Metagenómica , Virus de la Parainfluenza 4 Humana/clasificación , Virus de la Parainfluenza 4 Humana/genética , Paramyxoviridae/genética , South Dakota , Zoonosis/virologíaRESUMEN
A novel clade of RNA viruses was identified in the mammalian gastrointestinal tract by next-generation sequencing. Phylogenetically, these viruses are related to the genera Tombusviridae (plant viruses) and Flaviviridae, which includes mammalian, avian and insect hosts. Named in line with their characterization as stool-associated Tombus-like viruses, it is unclear if statoviruses infect mammals or are dietary in origin. Here, metagenomic sequencing of faecal material collected from a 10-week-old calf with enteric disease found that 20â% of the reads mapped to a de novo-assembled 4 kb contig with homology to statoviruses. Phylogenetic analysis of the statovirus genome found a clear evolutionary relationship with statovirus A, but, with only 47â% similarity, we propose that the statovirus sequence presents a novel species, statovirus F. A TaqMan PCR targeting statovirus F performed on faecal material found a cycle threshold of 11, suggesting a high titre of virus shed from the calf with enteric disease. A collection of 48 samples from bovine enteric disease diagnostic submissions were assayed by PCR to investigate statovirus F prevalence and 6 of 48 (12.5â%) were positive. An ELISA to detect antibodies to the coat protein found that antibodies to statovirus F were almost ubiquitous in bovine serum. Combined, the PCR and ELISA results suggest that statovirus F commonly infects cattle. Further research is needed to elucidate the aetiological significance of statovirus infection.
Asunto(s)
Enfermedades de los Bovinos/virología , Heces/virología , Tracto Gastrointestinal/virología , Enfermedades Intestinales/veterinaria , Enfermedades Intestinales/virología , Infecciones por Virus ARN/veterinaria , Virus ARN/clasificación , Virus ARN/aislamiento & purificación , Animales , Anticuerpos Antivirales/sangre , Bovinos , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenoma , Filogenia , Infecciones por Virus ARN/virología , Virus ARN/genética , Virus ARN/fisiología , Virus no Clasificados/clasificación , Virus no Clasificados/genética , Virus no Clasificados/aislamiento & purificación , Virus no Clasificados/fisiologíaRESUMEN
Bovine enteric disease has a complex etiology that can include viral, bacterial, and parasitic pathogens and is a significant source of losses due to morbidity and mortality. Boosepivirus was identified in calves with enteric disease with unclear etiology in Japan in 2009 and has not been reported elsewhere. Metagenomic sequencing and PCR here identified boosepivirus in bovine enteric disease diagnostic submissions from six states in the USA with 98% sequence identity to members of the species Boosepivirus B. In all cases, boosepivirus was identified as a coinfection with the established pathogens bovine coronavirus, bovine rotavirus, and cryptosporidia. Further research is needed to determine the clinical significance of boosepivirus infection.
Asunto(s)
Enfermedades de los Bovinos/virología , Infecciones por Picornaviridae/veterinaria , Picornaviridae/clasificación , Picornaviridae/aislamiento & purificación , Animales , Animales Recién Nacidos , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/epidemiología , Diarrea/diagnóstico , Diarrea/epidemiología , Diarrea/veterinaria , Diarrea/virología , Heces/virología , Genoma Viral/genética , Sistemas de Lectura Abierta , Filogenia , Picornaviridae/genética , Infecciones por Picornaviridae/diagnóstico , Infecciones por Picornaviridae/epidemiología , Infecciones por Picornaviridae/virología , ARN Viral/genética , Estados Unidos/epidemiología , Proteínas Virales/genéticaRESUMEN
Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.
Asunto(s)
Infecciones por Coronaviridae/veterinaria , Coronaviridae/aislamiento & purificación , Laboratorios/normas , Enfermedades de los Porcinos/virología , Animales , Prueba de COVID-19/veterinaria , Infecciones por Coronaviridae/diagnóstico , Infecciones por Coronaviridae/virología , Brotes de Enfermedades , Heces/virología , Estándares de Referencia , Estaciones del Año , Porcinos , Enfermedades de los Porcinos/diagnósticoRESUMEN
Immunization with an insect cell lysate/baculovirus mixture containing recombinant porcine epidemic diarrhea virus (PEDV) spike protein induced high levels of neutralizing antibodies in both mice and piglets. However, immunization of piglets with this vaccine resulted in enhancement of disease symptoms and virus replication in vaccine recipients exposed to PEDV challenge. Thus, these observations demonstrate a previously unrecognized challenge of PEDV vaccine research, which has important implications for coronavirus vaccine development.
RESUMEN
Veterinary diagnostic laboratories derive thousands of nucleotide sequences from clinical samples of swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), Senecavirus A and swine enteric coronaviruses. In addition, next generation sequencing has resulted in the rapid production of full-length genomes. Presently, sequence data are released to diagnostic clients but are not publicly available as data may be associated with sensitive information. However, these data can be used for field-relevant vaccines; determining where and when pathogens are spreading; have relevance to research in molecular and comparative virology; and are a component in pandemic preparedness efforts. We have developed a centralized sequence database that integrates private clinical data using PRRSV data as an exemplar, alongside publicly available genomic information. We implemented the Tripal toolkit, a collection of Drupal modules that are used to manage, visualize and disseminate biological data stored within the Chado database schema. New sequences sourced from diagnostic laboratories contain: genomic information; date of collection; collection location; and a unique identifier. Users can download annotated genomic sequences using a customized search interface that incorporates data mined from published literature; search for similar sequences using BLAST-based tools; and explore annotated reference genomes. Additionally, custom annotation pipelines have determined species, the location of open reading frames and nonstructural proteins and the occurrence of putative frame shifts. Eighteen swine pathogens have been curated. The database provides researchers access to sequences discovered by veterinary diagnosticians, allowing for epidemiological and comparative virology studies. The result will be a better understanding on the emergence of novel swine viruses and how these novel strains are disseminated in the USA and abroad. Database URLhttps://swinepathogendb.org.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Genómica , Humanos , Laboratorios , Sistemas de Lectura Abierta , Filogenia , Porcinos , Estados UnidosRESUMEN
Bats are a host and reservoir for a large number of viruses, many of which are zoonotic. In North America, the big brown bat (Eptesicus fuscus) is widely distributed and common. Big brown bats are a known reservoir for rabies virus, which, combined with their propensity to roost in human structures, necessitates testing for rabies virus following human exposure. The current pandemic caused by severe acute respiratory syndrome coronavirus 2, likely of bat origin, illustrates the need for continued surveillance of wildlife and bats for potentially emerging zoonotic viruses. Viral metagenomic sequencing was performed on 39 big brown bats and one hoary bat submitted for rabies testing due to human exposure in South Dakota. A new genotype of American bat vesiculovirus was identified in seven of 17 (41%) heart and lung homogenates at high levels in addition to two of 23 viscera pools. A second rhabdovirus, Sodak rhabdovirus 1 (SDRV1), was identified in four of 23 (17%) viscera pools. Phylogenetic analysis placed SDRV1 in the genus Alphanemrhavirus, which includes two recognized species that were identified in nematodes. Finally, a highly divergent rhabdovirus, Sodak rhabdovirus 2 (SDRV2), was identified in two of 23 (8.7%) big brown bats. Phylogenetic analysis placed SDRV2 as ancestral to the dimarhabdovirus supergroup and Lyssavirus. Intracranial inoculation of mouse pups with rhabdovirus-positive tissue homogenates failed to elicit clinical disease. Further research is needed to determine the zoonotic potential of these non-rabies rhabdoviruses.
Asunto(s)
Quirópteros/virología , Filogenia , Rhabdoviridae/clasificación , Animales , COVID-19/virología , Femenino , Genotipo , Humanos , Metagenómica , Ratones , Virus de la Rabia , Rhabdoviridae/aislamiento & purificación , South Dakota , Zoonosis Virales/transmisiónRESUMEN
: Among more than twenty species belonging to the class Mollecutes, Mycoplasma bovis is the most common cause of bovine mycoplasmosis in North America and Europe. Bovine mycoplasmosis causes significant economic loss in the cattle industry. The number of M. bovis positive herds recently has increased in North America and Europe. Since antibiotic treatment is ineffective and no efficient vaccine is available, M. bovis induced mycoplasmosis is primarily controlled by herd management measures such as the restriction of moving infected animals out of the herds and culling of infected or shedders of M. bovis. To better understand the population structure and genomic factors that may contribute to its transmission, we sequenced 147 M. bovis strains isolated from four different countries viz. USA (n = 121), Canada (n = 22), Israel (n = 3) and Lithuania (n = 1). All except two of the isolates (KRB1 and KRB8) were isolated from two host types i.e., bovine (n = 75) and bison (n = 70). We performed a large-scale comparative analysis of M. bovis genomes by integrating 103 publicly available genomes and our dataset (250 total genomes). Whole genome single nucleotide polymorphism (SNP) based phylogeny using M. agalactiae as an outgroup revealed that M. bovis population structure is composed of five different clades. USA isolates showed a high degree of genomic divergence in comparison to the Australian isolates. Based on host of origin, all the isolates in clade IV was of bovine origin, whereas majority of the isolates in clades III and V was of bison origin. Our comparative genome analysis also revealed that M. bovis has an open pangenome with a large breadth of unexplored diversity of genes. The function based analysis of autogenous vaccine candidates (n = 10) included in this study revealed that their functional diversity does not span the genomic diversity observed in all five clades identified in this study. Our study also found that M. bovis genome harbors a large number of IS elements and their number increases significantly (p = 7.8x10-6) as the genome size increases. Collectively, the genome data and the whole genome-based population analysis in this study may help to develop better understanding of M. bovis induced mycoplasmosis in cattle.
RESUMEN
Bats are the reservoir for a large number of zoonotic viruses, including members of Coronaviridae (severe acute respiratory syndrome coronavirus [SARS-CoV] and SARS-CoV-2), Paramyxoviridae (Hendra and Nipah viruses), Rhabdoviridae (rabies virus), and Filoviridae (Ebola virus) as exemplars. Many retroviruses, such as human immunodeficiency virus, are similarly zoonotic; however, only infectious exogenous gammaretroviruses have recently been identified in bats. Here, viral metagenomic sequencing of samples from bats submitted for rabies virus testing, largely due to human exposure, identified a novel, highly divergent exogenous Deltaretrovirus from a big brown bat (Eptesicus fuscus) in South Dakota. The virus sequence, corresponding to Eptesicus fuscus deltaretrovirus (EfDRV), comprised a nearly complete coding region comprised of canonical 5'-gag-pro-pol-env-3' genes with 37% to 51% identity to human T-lymphotropic virus (HTLV), an infectious retrovirus that causes T-cell lymphoma. A putative tax gene with 27% identity to HTLV was located downstream of the pol gene along with a gene harbored in an alternative reading frame which possessed a conserved domain for an Epstein-Barr virus nuclear antigen involved in gene transactivation, suggesting a regulatory function similar to that of the deltaretrovirus rex gene. A TaqMan reverse transcriptase PCR (RT-PCR) targeting the pol gene identified 4/60 (6.7%) bats as positive for EfDRV, which, combined with a search of the E. fuscus genome that failed to identify sequences with homology to EfDRV, suggests that EfDRV is an infectious exogenous virus. As all known members of Deltaretrovirus can cause malignancies and E. fuscus is widely distributed in the Americas, often with a colonial roosting behavior in human dwellings, further studies are needed to investigate potential zoonosis.IMPORTANCE Bats host a large numbers of viruses, many of which are zoonotic. In the United States, the big brown bat (Eptesicus fuscus) is widely distributed and lives in small colonies that roost in cavities, often in human dwellings, leading to frequent human interaction. Viral metagenomic sequencing of samples from an E. fuscus bat submitted for rabies testing identified the first exogenous bat Deltaretrovirus The E. fuscus deltaretrovirus (EfDRV) genome consists of the typical deltaretrovial 5'-gag-pro-pol-env-3' genes along with genes encoding two putative transcriptional transactivator proteins distantly related to the Tax protein of human T-cell lymphotrophic virus and nuclear antigen 3B of Epstein-Barr virus. Searches of the E. fuscus genome sequence failed to identify endogenous EfDRV. RT-PCR targeting the EfDRV pol gene identified 4/60 (6.7%) bats with positive results. Together, these results suggest that EfDRV is exogenous. As all members of Deltaretrovirus are associated with T- and B-cell malignancies or neurologic disease, further studies on possible zoonosis are warranted.
Asunto(s)
Quirópteros/virología , Deltaretrovirus/genética , Deltaretrovirus/aislamiento & purificación , Genoma Viral/genética , Animales , Productos del Gen tax/genética , Humanos , ARN Viral/genética , South Dakota , Estados Unidos , Zoonosis/virologíaRESUMEN
This consensus document presents the suggested guidelines developed by the Laboratory Technology Committee (LTC) of the American Association of Veterinary Laboratory Diagnosticians (AAVLD) for development, validation, and modification (methods comparability) of real-time PCR (rtPCR) assays. These suggested guidelines are presented with reference to the World Organisation for Animal Health (OIE) guidelines for validation of nucleic acid detection assays used in veterinary diagnostic laboratories. Additionally, our proposed practices are compared to the guidelines from the Foods Program Regulatory Subdivision of the U.S. Food and Drug Administration (FDA) and from the American Society for Veterinary Clinical Pathology (ASVCP). The LTC suggestions are closely aligned with those from the OIE and comply with version 2021-01 of the AAVLD Requirements for an Accredited Veterinary Medical Diagnostic Laboratory, although some LTC recommendations are more stringent and extend beyond the AAVLD requirements. LTC suggested guidelines are substantially different than the guidelines recently published by the U.S. FDA for validation and modification of regulated tests used for detection of pathogens in pet food and animal-derived products, such as dairy. Veterinary diagnostic laboratories that perform assays from the FDA Bacteriological Analytical Method (BAM) manual must be aware of the different standard.
Asunto(s)
Adhesión a Directriz/normas , Laboratorios/normas , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Animales , Guías como Asunto/normas , Patología Clínica/normas , Control de Calidad , Reproducibilidad de los Resultados , Estados UnidosRESUMEN
Influenza remains a global health risk and challenge. Currently, neuraminidase (NA) inhibitors are extensively used to treat influenza, but their efficacy is compromised by the emergence of drug-resistant variants. Neutralizing antibodies targeting influenza A virus surface glycoproteins are critical components of influenza therapeutic agents and may provide alternative strategies to the existing countermeasures. However, the major hurdle for the extensive application of antibody therapies lies in the difficulty of generating nonimmunogenic antibodies in large quantities rapidly. Here, we report that one human monoclonal antibody (MAb), 53C10, isolated from transchromosomic (Tc) cattle exhibits potent neutralization and hemagglutination inhibition titers against different clades of H1N1 subtype influenza A viruses. In vitro selection of antibody escape mutants revealed that 53C10 recognizes a novel noncontinuous epitope in the hemagglutinin (HA) head domain involving three amino acid residues, glycine (G), serine (S), and glutamic acid (E) at positions 172, 207, and 212, respectively. The results of our experiments supported a critical role for substitution of arginine at position 207 (S207R) in mediating resistance to 53C10, while substitutions at either G172E or E212A did not alter antibody recognition and neutralization. The E212A mutation may provide structural stability for the epitope, while the substitution G172E probably compensates for loss of fitness introduced by S207R. Our results offer novel insights into the mechanism of action of MAb 53C10 and indicate its potential role in therapeutic treatment of H1 influenza virus infection in humans.IMPORTANCE Respiratory diseases caused by influenza viruses still pose a serious concern to global health, and neutralizing antibodies constitute a promising area of antiviral therapeutics. However, the potential application of antibodies is often hampered by the challenge in generating nonimmunogenic antibodies in large scale. In the present study, transchromosomic (Tc) cattle were used for the generation of nonimmunogenic monoclonal antibodies (MAbs), and characterization of such MAbs revealed one monoclonal antibody, 53C10, exhibiting a potent neutralization activity against H1N1 influenza viruses. Further characterization of the neutralization escape mutant generated using this MAb showed that three amino acid substitutions in the HA head domain contributed to the resistance. These findings emphasize the importance of Tc cattle in the production of nonimmunogenic MAbs and highlight the potential of MAb 53C10 in the therapeutic application against H1 influenza virus infection in humans.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Bovinos , Línea Celular , Humanos , Evasión Inmune , Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A/genética , Modelos Moleculares , Mutación , Pruebas de Neutralización , Análisis de Secuencia de ProteínaRESUMEN
We developed a model to predict the cyclic pattern of porcine reproductive and respiratory syndrome virus (PRRSV) RNA detection by reverse-transcription real-time PCR (RT-rtPCR) from 4 major swine-centric veterinary diagnostic laboratories (VDLs) in the United States and to use historical data to forecast the upcoming year's weekly percentage of positive submissions and issue outbreak signals when the pattern of detection was not as expected. Standardized submission data and test results were used. Historical data (2015-2017) composed of the weekly percentage of PCR-positive submissions were used to fit a cyclic robust regression model. The findings were used to forecast the expected weekly percentage of PCR-positive submissions, with a 95% confidence interval (CI), for 2018. During 2018, the proportion of PRRSV-positive submissions crossed 95% CI boundaries at week 2, 14-25, and 48. The relatively higher detection on week 2 and 48 were mostly from submissions containing samples from wean-to-market pigs, and for week 14-25 originated mostly from samples from adult/sow farms. There was a recurring yearly pattern of detection, wherein an increased proportion of PRRSV RNA detection in submissions originating from wean-to-finish farms was followed by increased detection in samples from adult/sow farms. Results from the model described herein confirm the seasonal cyclic pattern of PRRSV detection using test results consolidated from 4 VDLs. Wave crests occurred consistently during winter, and wave troughs occurred consistently during the summer months. Our model was able to correctly identify statistically significant outbreak signals in PRRSV RNA detection at 3 instances during 2018.
Asunto(s)
Brotes de Enfermedades/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/epidemiología , Virus del Síndrome Respiratorio y Reproductivo Porcino/fisiología , Animales , Reacción en Cadena de la Polimerasa/veterinaria , Síndrome Respiratorio y de la Reproducción Porcina/virología , ARN Viral/análisis , Estaciones del Año , Porcinos , Estados Unidos/epidemiologíaRESUMEN
Bacterial communities resident in the hindgut of pigs, have profound impacts on health and disease. Investigations into the pig microbiome have utilized either culture-dependent, or far more commonly, culture-independent techniques using next generation sequencing. We contend that a combination of both approaches generates a more coherent view of microbiome composition. In this study, we surveyed the microbiome of Tamworth breed and feral pigs through the integration high throughput culturing and shotgun metagenomics. A single culture medium was used for culturing. Selective screens were added to the media to increase culture diversity. In total, 46 distinct bacterial species were isolated from the Tamworth and feral samples. Selective screens successfully shifted the diversity of bacteria on agar plates. Tamworth pigs are highly dominated by Bacteroidetes primarily composed of the genus Prevotella whereas feral samples were more diverse with almost equal proportions of Firmicutes and Bacteroidetes. The combination of metagenomics and culture techniques facilitated a greater retrieval of annotated genes than either method alone. The single medium based pig microbiota library we report is a resource to better understand pig gut microbial ecology and function. It allows for assemblage of defined bacterial communities for studies in bioreactors or germfree animal models.