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It has been proposed that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that spread through human populations as a pandemic originated in Asian bats. There is concern that infected humans could transmit the virus to native North American bats; therefore, the susceptibility of several North American bat species to the pandemic virus has been experimentally assessed. Big brown bats (Eptesicus fuscus) were shown to be resistant to infection by SARS-CoV-2, whereas Mexican free-tailed bats (Tadarida brasiliensis) became infected and orally excreted moderate amounts of virus for up to 18 d postinoculation. Little brown bats (Myotis lucifugus) frequently contact humans, and their populations are threatened over much of their range due to white-nose syndrome, a fungal disease that is continuing to spread across North America. We experimentally challenged little brown bats with SARS-CoV-2 to determine their susceptibility and host potential and whether the virus presents an additional risk to this species. We found that this species was resistant to infection by SARS-CoV-2. These findings provide reassurance to wildlife rehabilitators, biologists, conservation scientists, and the public at large who are concerned with possible transmission of this virus to threatened bat populations.
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Monkeypox virus (MPXV) is a re-emerging zoonotic poxvirus responsible for producing skin lesions in humans. Endemic in sub-Saharan Africa, the 2022 outbreak with a clade IIb strain has resulted in ongoing sustained transmission of the virus worldwide. MPXV has a relatively wide host range, with infections reported in rodent and non-human primate species. However, the susceptibility of many domestic livestock species remains unknown. Here, we report on a susceptibility/transmission study in domestic pigs that were experimentally inoculated with a 2022 MPXV clade IIb isolate or served as sentinel contact control animals. Several principal-infected and sentinel contact control pigs developed minor lesions near the lips and nose starting at 12 through 18 days post-challenge (DPC). No virus was isolated and no viral DNA was detected from the lesions; however, MPXV antigen was detected by IHC in tissue from a pustule of a principal infected pig. Viral DNA and infectious virus were detected in nasal and oral swabs up to 14 DPC, with peak titers observed at 7 DPC. Viral DNA was also detected in nasal tissues or skin collected from two principal-infected animals at 7 DPC post-mortem. Furthermore, all principal-infected and sentinel control animals enrolled in the study seroconverted. In conclusion, we provide the first evidence that domestic pigs are susceptible to experimental MPXV infection and can transmit the virus to contact animals.
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Monkeypox virus , Mpox , Doenças dos Suínos , Animais , Monkeypox virus/fisiologia , Monkeypox virus/patogenicidade , Monkeypox virus/genética , Suínos , Mpox/transmissão , Mpox/virologia , Mpox/veterinária , Doenças dos Suínos/virologia , Doenças dos Suínos/transmissão , DNA Viral/genética , Anticorpos Antivirais/sangue , Humanos , Pele/virologia , Nariz/virologiaRESUMO
ABSTRACTRapid evolution of highly pathogenic avian influenza viruses (HPAIVs) is driven by antigenic drift but also by reassortment, which might result in robust replication in and transmission to mammals. Recently, spillover of clade 2.3.4.4b HPAIV to mammals including humans, and their transmission between mammalian species has been reported. This study aimed to evaluate the pathogenicity and transmissibility of a mink-derived clade 2.3.4.4b H5N1 HPAIV isolate from Spain in pigs. Experimental infection caused interstitial pneumonia with necrotizing bronchiolitis with high titers of virus present in the lower respiratory tract and 100% seroconversion. Infected pigs shed limited amount of virus, and importantly, there was no transmission to contact pigs. Notably, critical mammalian-like adaptations such as PB2-E627 K and HA-Q222L emerged at low frequencies in principal-infected pigs. It is concluded that pigs are highly susceptible to infection with the mink-derived clade 2.3.4.4b H5N1 HPAIV and provide a favorable environment for HPAIV to acquire mammalian-like adaptations.
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Virus da Influenza A Subtipo H5N1 , Vison , Infecções por Orthomyxoviridae , Doenças dos Suínos , Animais , Vison/virologia , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/veterinária , Suínos , Virus da Influenza A Subtipo H5N1/patogenicidade , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Doenças dos Suínos/virologia , Doenças dos Suínos/transmissão , Espanha , Proteínas Virais/genética , Proteínas Virais/metabolismo , Eliminação de Partículas ViraisRESUMO
Feline respiratory disease complex (FRDC) is caused by a wide range of viral and bacterial pathogens. Both Influenza A virus (IAV) and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) also induce respiratory diseases in cats. Two one-step multiplex qPCR/RT-qPCR assays were developed and validated: FRA_1 (Feline respiratory assay 1) for the detection of four viral targets and FRA_2 for the detection of three bacteria associated with FRDC. Both multiplex assays demonstrated high specificity, efficiency (93.51%-107.8%), linearity (> 0.998), analytical sensitivity (≤ 15 genome copies/µl), repeatability (coefficient of variation [CV] < 5%), and reproducibility (CV < 6%). Among the 63 clinical specimens collected from FRDC-suspected cats, 92.1% were positive for at least one pathogen and co-infection was detected in 57.1% of samples. Mycoplasma felis (61.9%) was the most found pathogen, followed by feline herpesvirus-1 (30.2%), Chlamydia felis (28.7%) and feline calicivirus (27.0%). SARS-CoV-2 was detected in two specimens. In summary, this new panel of qPCR/RT-qPCR assays constitutes a useful and reliable tool for the rapid detection of SARS-CoV-2 and viral and bacterial pathogens associated with FRDC in cats.
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COVID-19 , Doenças Respiratórias , Gatos , Animais , SARS-CoV-2/genética , Reprodutibilidade dos Testes , COVID-19/diagnóstico , Bactérias/genética , Sensibilidade e EspecificidadeRESUMO
(1) Background: equid alphaherpesvirus-1 (EHV-1) is a highly contagious viral pathogen prevalent in most horse populations worldwide. Genome-editing technologies such as CRISPR/Cas9 have become powerful tools for precise RNA-guided genome modifications; (2) Methods: we designed single guide RNAs (sgRNA) to target three essential (ORF30, ORF31, and ORF7) and one non-essential (ORF74) EHV-1 genes and determine their effect on viral replication dynamics in vitro; (3) Results: we demonstrated that sgRNAs targeting essential lytic genes reduced EHV-1 replication, whereas those targeting ORF74 had a negligible effect. The sgRNAs targeting ORF30 showed the strongest effect on the suppression of EHV-1 replication, with a reduction in viral genomic copy numbers and infectious progeny virus output. Next-generation sequencing identified variants with deletions in the specific cleavage site of selective sgRNAs. Moreover, we evaluated the combination between different sgRNAs and found that the dual combination of sgRNAs targeting ORF30 and ORF7 significantly suppressed viral replication to lower levels compared to the use of a single sgRNA, suggesting a synergic effect; (4) Conclusion: data demonstrate that sgRNA-guided CRISPR/Cas9 can be used to inhibit EHV-1 replication in vitro, indicating that this programmable technique can be used to develop a novel, safe, and efficacious therapeutic and prophylactic approach against EHV-1.
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Edição de Genes , Herpesvirus Equídeo 1 , Animais , Cavalos , Edição de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas , Herpesvirus Equídeo 1/genética , Genoma ViralRESUMO
A wide range of animal species show variable susceptibility to SARS-CoV-2; however, host factors associated with varied susceptibility remain to be defined. Here, we examined whether susceptibility to SARS-CoV-2 and virus tropism in different animal species are dependent on the expression and distribution of the virus receptor angiotensin-converting enzyme 2 (ACE2) and the host cell factor transmembrane serine protease 2 (TMPRSS2). We cataloged the upper and lower respiratory tract of multiple animal species and humans in a tissue-specific manner and quantitatively evaluated the distribution and abundance of ACE2 and TMPRSS2 mRNA in situ. Our results show that: (i) ACE2 and TMPRSS2 mRNA are abundant in the conduction portion of the respiratory tract, (ii) ACE2 mRNA occurs at a lower abundance compared to TMPRSS2 mRNA, (iii) co-expression of ACE2-TMPRSS2 mRNAs is highest in those species with the highest susceptibility to SARS-CoV-2 infection (i.e., cats, Syrian hamsters, and white-tailed deer), and (iv) expression of ACE2 and TMPRSS2 mRNA was not altered following SARS-CoV-2 infection. Our results demonstrate that while specific regions of the respiratory tract are enriched in ACE2 and TMPRSS2 mRNAs in different animal species, this is only a partial determinant of susceptibility to SARS-CoV-2 infection.IMPORTANCESARS-CoV-2 infects a wide array of domestic and wild animals, raising concerns regarding its evolutionary dynamics in animals and potential for spillback transmission of emerging variants to humans. Hence, SARS-CoV-2 infection in animals has significant public health relevance. Host factors determining animal susceptibility to SARS-CoV-2 are vastly unknown, and their characterization is critical to further understand susceptibility and viral dynamics in animal populations and anticipate potential spillback transmission. Here, we quantitatively assessed the distribution and abundance of the two most important host factors, angiotensin-converting enzyme 2 and transmembrane serine protease 2, in the respiratory tract of various animal species and humans. Our results demonstrate that while specific regions of the respiratory tract are enriched in these two host factors, they are only partial determinants of susceptibility. Detailed analysis of additional host factors is critical for our understanding of the underlying mechanisms governing viral susceptibility and reservoir hosts.
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COVID-19 , Cervos , Humanos , Animais , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Sistema Respiratório , RNA Mensageiro , Tropismo , Serina EndopeptidasesRESUMO
Group A rotaviruses are a well-known cause of viral gastroenteritis in infants and children, as well as in many mammalian species and birds, affecting them at a young age. This group of viruses has a double-stranded, segmented RNA genome with high genetic diversity linked to point mutations, recombination, and, importantly, reassortment. While initial molecular investigations undertaken in the 1900s suggested host range restriction among group A rotaviruses based on the fact that different gene segments were distributed among different animal species, recent molecular surveillance and genome constellation genotyping studies conducted by the Rotavirus Classification Working Group (RCWG) have shown that animal rotaviruses serve as a source of diversification of human rotavirus A, highlighting their zoonotic potential. Rotaviruses occurring in various animal species have been linked with contributing genetic material to human rotaviruses, including horses, with the most recent identification of equine-like G3 rotavirus A infecting children. The goal of this article is to review relevant information related to rotavirus structure/genomic organization, epidemiology (with a focus on human and equine rotavirus A), evolution, inter-species transmission, and the potential zoonotic role of equine and other animal rotaviruses. Diagnostics, surveillance and the current status of human and livestock vaccines against RVA are also reviewed.
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Infecções por Enterovirus , Saúde Única , Rotavirus , Criança , Lactente , Cavalos , Animais , Humanos , Rotavirus/genética , Saúde Pública , Gado , MamíferosRESUMO
Canine pneumovirus was detected by RT-qPCR in 2022 from nasal swabs collected from two dogs with upper respiratory disease in a shelter in Louisiana, United States. The genomes from the designated strains CPnV USA/LA/2022/124423 and USA/LA/2022/123696 were sequenced and show the closest similarity to the pneumonia virus of mice J3666.
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Since emerging in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly crossed the species barrier with natural infections reported in various domestic and wild animal species. The emergence and global spread of SARS-CoV-2 variants of concern (VOCs) has expanded the range of susceptible host species. Previous experimental infection studies in cattle using Wuhan-like SARS-CoV-2 isolates suggested that cattle were not likely amplifying hosts for SARS-CoV-2. However, SARS-CoV-2 sero- and RNA-positive cattle have since been identified in Europe, India, and Africa. Here, we investigated the susceptibility and transmission of the Delta and Omicron SARS-CoV-2 VOCs in cattle. Eight Holstein calves were co-infected orally and intranasally with a mixed inoculum of SARS-CoV-2 VOCs Delta and Omicron BA.2. Twenty-four hours post-challenge, two sentinel calves were introduced to evaluate virus transmission. The co-infection resulted in a high proportion of calves shedding SARS-CoV-2 RNA at 1- and 2-days post-challenge (DPC). Extensive tissue distribution of SARS-CoV-2 RNA was observed at 3 and 7 DPC and infectious virus was recovered from two calves at 3 DPC. Next-generation sequencing revealed that only the SARS-CoV-2 Delta variant was detected in clinical samples and tissues. Similar to previous experimental infection studies in cattle, we observed only limited seroconversion and no clear evidence of transmission to sentinel calves. Together, our findings suggest that cattle are more permissive to infection with SARS-CoV-2 Delta than Omicron BA.2 and Wuhan-like isolates but, in the absence of horizontal transmission, are not likely to be reservoir hosts for currently circulating SARS-CoV-2 variants.
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COVID-19 , Coinfecção , Animais , Bovinos , COVID-19/veterinária , Coinfecção/veterinária , RNA Viral/genética , SARS-CoV-2/genéticaRESUMO
BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is a global viral pathogen of domestic equids which causes reproductive, respiratory and neurological disease. Few isolates acquired from naturally infected USA-based hosts have been fully sequenced and analyzed to date. An ORF 30 (DNA polymerase) variant (A2254G) has previously been associated with neurological disease in host animals. The purpose of this study was to perform phylogenomic analysis of EHV-1 isolates acquired from USA-based hosts and compare these isolates to previously sequenced global isolates. METHODS: EHV-1 was isolated from 23 naturally infected USA-based equids (6 different states, 15 disease outbreaks) with reproductive (22/23) or neurological disease (1/23). Following virus isolation, EHV-1 DNA was extracted for sequencing using Illumina MiSeq. Following reference-based assembly, whole viral genomes were annotated and assessed. Previously sequenced EHV-1 isolates (n = 114) obtained from global host equids were included in phylogenomic analyses. RESULTS: The overall average genomic distance was 0.0828% (SE 0.004%) for the 23 newly sequenced USA isolates and 0.0705% (SE 0.003%) when all 137 isolates were included. Clade structure was predominantly based on geographic origin. Numerous nucleotide substitutions (mean [range], 179 [114-297] synonymous and 81 [38-120] non-synonymous substitutions per isolate) were identified throughout the genome of the newly sequenced USA isolates. The previously described ORF 30 A2254G substitution (associated with neurological disease) was found in only one isolate obtained from a host with non-neurological clinical signs (reproductive disease), six additional, unique, non-synonymous ORF 30 substitutions were detected in 22/23 USA isolates. Evidence of recombination was present in most (22/23) of the newly sequenced USA isolates. CONCLUSIONS: Overall, the genomes of the 23 newly sequenced EHV-1 isolates obtained from USA-based hosts were broadly similar to global isolates. The previously described ORF 30 A2254G neurological substitution was infrequently detected in the newly sequenced USA isolates, most of which were obtained from host animals with reproductive disease. Recombination was likely to be partially responsible for genomic diversity in the newly sequenced USA isolates.
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Infecções por Herpesviridae , Herpesvirus Equídeo 1 , Doenças dos Cavalos , Doenças do Sistema Nervoso , Animais , Cavalos , Filogenia , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/genética , Genoma Viral , Sequência de Bases , Doenças dos Cavalos/epidemiologiaRESUMO
Canine infectious respiratory disease complex (CIRDC) is the primary cause of respiratory disease in the canine population and is caused by a wide array of viruses and bacterial pathogens with coinfections being common. Since its recognition in late 2019, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been reported to cause respiratory disease in dogs. Therefore, the rapid detection and differentiation of SARS-CoV-2 from other common viral and bacterial agents is critical from a public health standpoint. Here, we developed and validated a panel of four one-step multiplex qPCR/RT-qPCR assays for the detection and identification of twelve pathogens associated with CIRDC (canine adenovirus-2, canine distemper virus, canine herpesvirus-1, canine influenza A virus, canine parainfluenza virus, canine pneumovirus, canine respiratory coronavirus, SARS-CoV-2, Bordetella bronchiseptica, Streptococcus equi subsp. zooepidemicus, Mycoplasma cynos, and M. canis), as well as the identification of three main CIV subtypes (i.e., H3N2, H3N8, and H1N1). All developed assays demonstrated high specificity and analytical sensitivity. This panel was used to test clinical specimens (n = 76) from CIRDC-suspected dogs. M. canis, M. cynos, and CRCoV were the most frequently identified pathogens (30.3%, 25.0%, and 19.7% of samples, respectively). The newly emerging pathogens CPnV and SARS-CoV-2 were detected in 5.3% of samples and coinfections were identified in 30.3%. This new multiplex qPCR/RT-qPCR panel is the most comprehensive panel developed thus far for identifying CIRDC pathogens, along with SARS-CoV-2.
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COVID-19 , Canidae , Coinfecção , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N8 , Infecções Respiratórias , Cães , Animais , SARS-CoV-2/genética , Coinfecção/diagnóstico , Coinfecção/veterinária , Vírus da Influenza A Subtipo H3N2 , COVID-19/diagnóstico , COVID-19/veterinária , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/veterináriaRESUMO
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was transmitted from humans to dogs and cats (reverse zoonosis) during the COVID-19 pandemic. SARS-CoV-2 has been detected in fecal samples of infected dogs and cats, indicating potential fecal-oral transmission, environmental contamination, and zoonotic transmission (i.e., spillback). Additionally, gastrointestinal viral infections are prevalent in dogs and cats. In this study, we developed and validated a panel of multiplex one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays for the simultaneous detection of SARS-CoV-2 and common canine enteric viruses: Canine Enteric Assay_1 (CEA_1) for the detection of canine adenovirus-1, canine enteric coronavirus, canine distemper virus, and canine parvovirus, and CEA_2 for the detection of rotavirus A (RVA), and SARS-CoV-2); or common feline enteric viruses (Feline Enteric Assay_1 (FEA_1) for the detection of feline enteric coronavirus, feline panleukopenia virus, RVA, and SARS-CoV-2). All assays demonstrated high analytical sensitivity, detecting as few as 5-35 genome copies/µL in multiplex format. The repeatability and reproducibility of the multiplex assays were excellent, with coefficient of variation <4%. Among the 58 clinical samples tested, 34.5% were positive for at least one of these viruses, and SARS-CoV-2 was detected in two samples collected from one dog and one cat, respectively. In conclusion, these newly developed one-step multiplex RT-qPCR assays allow for rapid diagnosis of enteric viral infections, including SARS-CoV-2, in dogs and cats.
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COVID-19 , Doenças do Gato , Doenças do Cão , Infecções por Enterovirus , Enterovirus , Rotavirus , Cães , Gatos , Animais , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , COVID-19/veterinária , Pandemias , Doenças do Gato/diagnóstico , Reprodutibilidade dos Testes , Doenças do Cão/diagnósticoRESUMO
Equine rotavirus A (ERVA) is the leading cause of diarrhea in foals, with G3P[12] and G14P[12] genotypes being the most prevalent. Recently, equine G3-like RVA was recognized as an emerging infection in children, and a group B equine rotavirus (ERVB) was identified as an emergent cause of foal diarrhea in the US. Thus, there is a need to adapt molecular diagnostic tools for improved detection and surveillance to identify emerging strains, understand their molecular epidemiology, and inform future vaccine development. We developed a quadruplex TaqMan® RT-qPCR assay for differentiation of ERVA and ERVB and simultaneous G-typing of ERVA strains, evaluated its analytical and clinical performance, and compared it to (1) a previously established ERVA triplex RT-qPCR assay and (2) standard RT-PCR assay and Sanger sequencing of PCR products. This quadruplex RT-qPCR assay demonstrated high sensitivity (>90%)/specificity (100%) for every target and high overall agreement (>96%). Comparison between the triplex and quadruplex assays revealed only a slightly higher sensitivity for the ERVA NSP3 target using the triplex format (p-value 0.008) while no significant differences were detected for other targets. This quadruplex RT-qPCR assay will significantly enhance rapid surveillance of both ERVA and ERVB circulating and emerging strains with potential for interspecies transmission.
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Doenças dos Cavalos , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Rotavirus , Rotavirus , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Rotavirus/isolamento & purificação , Animais , Cavalos , Doenças dos Cavalos/virologia , Infecções por Rotavirus/veterinária , Fezes/virologia , Sensibilidade e EspecificidadeRESUMO
The current global COVID-19 pandemic once again highlighted the urgent need for a simple, cost-effective, and sensitive diagnostic platform that can be rapidly developed for distribution and easy access in resource-limited areas. Here, we present a simple and low-cost plasmonic photothermal (PPT)-reverse transcription-colorimetric polymerase chain reaction (RTcPCR) for molecular diagnosis of dengue virus (DENV) infection. The assay can be completed within 54 min with an estimated detection limit of 1.6 copies/µL of viral nucleic acid. The analytical sensitivity and specificity of PPT-RTcPCR were comparable to that of the reference RT-qPCR assay. Moreover, the clinical performance of PPT-RTcPCR was evaluated and validated using 158 plasma samples collected from patients suspected of dengue infection. The results showed a diagnostic agreement of 97.5% compared to the reference RT-qPCR and demonstrated a clinical sensitivity and specificity of 97.0% and 100%, respectively. The simplicity and reliability of our PPT-RTcPCR strategy suggest it can provide a foundation for developing a field-deployable diagnostic assay for dengue and other infectious diseases.
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COVID-19 , Vírus da Dengue , Dengue , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus da Dengue/genética , Reprodutibilidade dos Testes , Colorimetria , Pandemias , Sensibilidade e Especificidade , Testes Diagnósticos de Rotina , RNA Viral/genética , Teste para COVID-19RESUMO
The liver is an early systemic target of Ebola virus (EBOV), but characterization beyond routine histopathology and viral antigen distribution is limited. We hypothesized Ebola virus disease (EVD) systemic proinflammatory responses would be reflected in temporally altered liver myeloid phenotypes. We utilized multiplex fluorescent immunohistochemistry (mfIHC), multispectral whole slide imaging, and image analysis to quantify molecular phenotypes of myeloid cells in the liver of rhesus macaques (Macaca mulatta; n = 21) infected with EBOV Kikwit. Liver samples included uninfected controls (n = 3), 3 days postinoculation (DPI; n = 3), 4 DPI (n = 3), 5 DPI (n = 3), 6 DPI (n = 3), and terminal disease (6-8 DPI; n = 6). Alterations in hepatic macrophages occurred at ≥ 5 DPI characterized by a 1.4-fold increase in CD68+ immunoreactivity and a transition from primarily CD14-CD16+ to CD14+CD16- macrophages, with a 2.1-fold decrease in CD163 expression in terminal animals compared with uninfected controls. An increase in the neutrophil chemoattractant and alarmin S100A9 occurred within hepatic myeloid cells at 5 DPI, followed by rapid neutrophil influx at ≥ 6 DPI. An acute rise in the antiviral myxovirus resistance protein 1 (MxA) occurred at ≥ 4 DPI, with a predilection for enhanced expression in uninfected cells. Distinctive expression of major histocompatibility complex (MHC) class II was observed in hepatocytes during terminal disease. Results illustrate that EBOV causes macrophage phenotype alterations as well as neutrophil influx and prominent activation of interferon host responses in the liver. Results offer insight into potential therapeutic strategies to prevent and/or modulate the host proinflammatory response to normalize hepatic myeloid functionality.
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Ebolavirus , Doença pelo Vírus Ebola , Animais , Doença pelo Vírus Ebola/veterinária , Doença pelo Vírus Ebola/patologia , Ebolavirus/fisiologia , Macaca mulatta , Fígado/patologia , FenótipoRESUMO
Introduction: Accurate quantitative analysis of equine insulin in blood samples is critical for assessing hyperinsulinemia in horses. Although there are various laboratory methods for evaluating equine serum insulin, different immunoassays show significant discrepancies between the determined insulin concentrations and are often not comparable. The aim of this study was to evaluate the Immulite® 1000 chemiluminescent immunoassay (CLIA) to establish independent laboratory and assay-specific cut values to provide an accurate diagnosis of hyperinsulinemia in horses. Thus, the analytical and clinical performance of Immulite® 1000 CLIA in terms of precision (intra- and inter-assay coefficient of variance, CV) and recovery upon dilution were evaluated and compared with radioimmunoassay (RIA), which has been previously validated for use in horses. Material and methods: Archived serum samples (n = 106) from six Quarter horse mares enrolled in the glucose phase of a Frequently Sampled Insulin and Glucose Test (FSIGT) study were used to measure blood insulin. Results: The Immulite® 1000 CLIA had good precision with acceptable intra- and inter-assay CVs, adequate recovery on dilution, and a strong correlation with the RIA (r = 0.974, P < 0.0001), with constant bias resulting in consistently lower values. Discussion: On this basis, the Immulite® 1000 Insulin Assay is valid for measuring equine serum insulin for diagnostic and monitoring purposes when cut values are appropriately adjusted.
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Several models were developed to study the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the in vivo efficacy of vaccines and therapeutics. Since wild-type mice are naturally resistant to infection by ancestral SARS-CoV-2 strains, several transgenic mouse models expressing human angiotensin-converting enzyme 2 (hACE2) were developed. An alternative approach has been to develop mouse-adapted SARS-CoV-2 strains. Here, we compared the clinical progression, viral replication kinetics and dissemination, pulmonary tropism, and host innate immune response dynamics between the mouse-adapted MA10 strain and its parental strain (USA-WA1/2020) following intranasal inoculation of K18-hACE2 mice, a widely used model. Compared to its parental counterpart, the MA10 strain induced earlier clinical decline with significantly higher viral replication and earlier neurodissemination. Importantly, the MA10 strain also showed a wider tropism, with infection of bronchiolar epithelia. While both SARS-CoV-2 strains induced comparable pulmonary cytokine/chemokine responses, many proinflammatory and monocyte-recruitment chemokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IP-10/CXCL10, and MCP-1/CCL2, showed an earlier peak in MA10-infected mice. Furthermore, both strains induced a similar downregulation of murine Ace2, with only a transient downregulation of Tmprss2 and no alterations in hACE2 expression. Overall, these data demonstrate that in K18-hACE2 mice, the MA10 strain has a pulmonary tropism that more closely resembles SARS-CoV-2 tropism in humans (airways and pneumocytes) than its parental strain. Its rapid replication and neurodissemination and early host pulmonary responses can have a significant impact on the clinical outcomes of infection and are, therefore, critical features to consider for study designs using these strains and mouse model. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, is still significantly impacting health care systems around the globe. Refined animal models are needed to study SARS-CoV-2 pathogenicity as well as efficacy of vaccines and therapeutics. In line with this, thorough evaluation of animal models and virus strains/variants are paramount for standardization and meaningful comparisons. Here, we demonstrated differences in replication dynamics between the Wuhan-like USA-WA1/2020 strain and the derivative mouse-adapted MA10 strain in K18-hACE2 mice. The MA10 strain showed accelerated viral replication and neurodissemination, differential pulmonary tropism, and earlier pulmonary innate immune responses. The observed differences allow us to better refine experimental designs when considering the use of the MA10 strain in the widely utilized K18-hACE2 murine model.
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COVID-19 , SARS-CoV-2 , Camundongos , Humanos , Animais , COVID-19/patologia , Enzima de Conversão de Angiotensina 2/genética , Pandemias , Pulmão/patologia , Replicação Viral , Camundongos Transgênicos , TropismoRESUMO
Actinobacillus equuli subsp. equuli is the etiological agent of sleepy foal disease, an acute form of fatal septicemia in newborn foals. A. equuli is commonly found in the mucous membranes of healthy horses' respiratory and alimentary tracts and rarely causes disease in adult horses. In this study, we report a case of a 22-year-old American Paint gelding presenting clinical signs associated with an atypical pattern of pleuropneumonia subjected to necropsy. The gross and histopathological examinations revealed a unilateral fibrinosuppurative and hemorrhagic pleuropneumonia with an infrequent parenchymal distribution and heavy isolation of A. equuli. The whole genome sequence analysis indicated that the isolate shared 95.9% homology with the only other complete genome of A. equuli subsp. equuli available in GenBank. Seven virulence-associated genes specific to the isolate were identified and categorized as iron acquisition proteins, lipopolysaccharides (LPS), and capsule polysaccharides. Moreover, four genes (glf, wbaP, glycosyltransferase family 2 protein, and apxIB) shared higher amino acid similarity with the invasive Actinobacillus spp. than the reference A. equuli subsp. equuli genome. Availability of the whole genome sequence will allow a better characterization of virulence determinants of A. equuli subsp. equuli, which remain largely elusive.
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This paper reports a presumptive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a cat. A cat with respiratory disease living with three individuals with coronavirus disease 2019 showed bilateral ground-glass opacities in the lung on X-ray and computed tomography. The clinical swabs were negative for SARS-CoV-2 RNA, but the serum was positive for SARS-CoV-2 antibodies. Interstitial pneumonia and prominent type 2 pneumocyte hyperplasia were noted on histopathology. Respiratory tissues were negative for SARS-CoV-2 RNA or antigen, but the cat was positive for feline parvovirus DNA. In conclusion, the respiratory disease and associated pathology in this cat could have been due to exposure to SARS-CoV-2.
Assuntos
COVID-19 , Doenças do Gato , Animais , Anticorpos Antivirais , COVID-19/veterinária , Doenças do Gato/diagnóstico por imagem , Gatos , RNA Viral , SARS-CoV-2 , Tomografia Computadorizada por Raios X/veterináriaRESUMO
Equine arteritis virus (EAV) is the causative agent of equine viral arteritis (EVA), a respiratory, systemic, and reproductive disease of equids. Following natural infection, up to 70% of the infected stallions can remain persistently infected over 1 year (long-term persistent infection [LTPI]) and shed EAV in their semen. Thus, the LTP-infected stallions play a pivotal role in maintaining and perpetuating EAV in the equine population. Previous studies identified equine C-X-C motif chemokine ligand 16 (CXCL16) as a critical host cell factor determining LTPI in the stallion's reproductive tract. Two alleles (CXCL16 S and CXCL16 r ) were identified in the equine population and correlated with the susceptibility or resistance of a CD3+ T cell subpopulation in peripheral blood to in vitro EAV infection, respectively. Interestingly, CXCL16 S has been linked to the establishment of LTPI in stallions, and thus, genotyping stallions based on CXCL16 S/r would allow identification of those at the highest risk of establishing LTPI. Thus, we developed a TaqMan® allelic discrimination qPCR assay for the genotyping of the equine CXCL16 gene based on the identification of a single nucleotide polymorphism in position 1,073 based on NCBI gene ID: 100061442 (or position 527 based on Ensembl: ENSECAG00000018406.2) located in exon 2. One hundred and sixty horses from four breeds were screened for the CD3+ T cell susceptibility phenotype to EAV infection by flow cytometry and subsequently sequenced to determine CXCL16 allelic composition. Genotyping by Sanger sequencing determined that all horses with the resistant CD3+ T cell phenotype were homozygous for CXCL16 r while horses with the susceptible CD3+ T cell phenotype carried at least one CXCL16 S allele or homozygous for CXCL16 S . In addition, genotypification with the TaqMan® allelic discrimination qPCR assay showed perfect agreement with Sanger sequencing and flow cytometric analysis. In conclusion, the new TaqMan® allelic discrimination genotyping qPCR assay can be used to screen prepubertal colts for the presence of the CXCL16 genotype. It is highly recommended that colts that carry the susceptible genotype (CXCL16 S/S or CXCL16 S/r ) are vaccinated against EAV after 6 months of age to prevent the establishment of LTPI carriers following possible natural infection with EAV.