RESUMO
Strains of Rocahepevirus ratti, an emerging hepatitis E virus (HEV), have recently been found to be infectious to humans. Rats are a primary reservoir of the virus; thus, it is referred to as "rat HEV". Rats are often found on swine farms in close contact with pigs. Our goal was to determine whether swine may serve as a transmission host for zoonotic rat HEV by characterizing an infectious cDNA clone of a zoonotic rat HEV, strain LCK-3110, in vitro and in vivo. RNA transcripts of LCK-3110 were constructed and assessed for their replicative capacity in cell culture and in gnotobiotic pigs. Fecal suspension from rat HEV-positive gnotobiotic pigs was inoculated into conventional pigs co-housed with naïve pigs. Our results demonstrated that capped RNA transcripts of LCK-3110 rat HEV replicated in vitro and successfully infected conventional pigs that transmit the virus to co-housed animals. The infectious clone of rat HEV may afford an opportunity to study the genetic mechanisms of rat HEV cross-species infection and tissue tropism.
RESUMO
Porcine epidemic diarrhea virus (PEDV) causes a highly contagious enteric disease with major economic losses to swine production worldwide. Due to the immaturity of the neonatal piglet immune system and given the high virulence of PEDV, improving passive lactogenic immunity is the best approach to protect suckling piglets against the lethal infection. We tested whether oral vitamin A (VA) supplementation and PEDV exposure of gestating and lactating VA-deficient (VAD) sows would enhance their primary immune responses and boost passive lactogenic protection against the PEDV challenge of their piglets. We demonstrated that PEDV inoculation of pregnant VAD sows in the third trimester provided higher levels of lactogenic protection of piglets as demonstrated by >87% survival rates of their litters compared with <10% in mock litters and that VA supplementation to VAD sows further improved the piglets' survival rates to >98%. We observed significantly elevated PEDV IgA and IgG antibody (Ab) titers and Ab-secreting cells (ASCs) in VA-sufficient (VAS)+PEDV and VAD+VA+PEDV sows, with the latter maintaining higher Ab titers in blood prior to parturition and in blood and milk throughout lactation. The litters of VAD+VA+PEDV sows also had the highest serum PEDV-neutralizing Ab titers at piglet post-challenge days (PCD) 0 and 7, coinciding with higher PEDV IgA ASCs and Ab titers in the blood and milk of their sows, suggesting an immunomodulatory role of VA in sows. Thus, sows that delivered sufficient lactogenic immunity to their piglets provided the highest passive protection against the PEDV challenge. Maternal immunization during pregnancy (± VA) and VA sufficiency enhanced the sow primary immune responses, expression of gut-mammary gland trafficking molecules, and passive protection of their offspring. Our findings are relevant to understanding the role of VA in the Ab responses to oral attenuated vaccines that are critical for successful maternal vaccination programs against enteric infections in infants and young animals.
Assuntos
Imunidade Adaptativa , Anticorpos Antivirais , Infecções por Coronavirus , Imunidade Materno-Adquirida , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Vitamina A , Animais , Vírus da Diarreia Epidêmica Suína/imunologia , Feminino , Suínos , Gravidez , Vitamina A/administração & dosagem , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Anticorpos Antivirais/sangue , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Animais Recém-Nascidos , Lactação/imunologia , Suplementos Nutricionais , Deficiência de Vitamina A/imunologia , ImunizaçãoRESUMO
The role of gut microbiota [especially, histo-blood group antigen (HBGA)-expressing bacteria] in influencing human norovirus (HuNoV) infections is unclear. We investigated if infectivity of GII.12 HuNoV in gnotobiotic (Gn) pigs is altered by intestinal colonization with Escherichia fergusonii known to express HBGA A and H on their cell surface. Fifteen piglets were randomly grouped: (1) E. fergusonii + HuNoV (n = 6), (2) HuNoV alone (n = 6), and (3) Mock-inoculated (n = 3). Pigs (8-11-day-old) were inoculated orally with GII.12 HuNoV strain HS206 (9.5 log10 genomic equivalents/pig) or mock. For 2 days prior to viral inoculation, pigs were inoculated orally with E. fergusonii [8 log10 colony forming units/pig/day]. Daily fecal consistency, fecal viral RNA or E. fergusonii shedding, and histopathology (at euthanasia) were evaluated. Unlike the reduced infectivity of GII.4 HuNoV observed previously in Gn pigs colonized with Enterobacter cloacae known to express HBGA A, B, and H on the surface, E. fergusonii + HuNoV pigs exhibited significantly higher cumulative fecal HuNoV RNA shedding at PIDs 6-14 and 1-21 compared with HuNoV alone pigs. Mean days of fecal HuNoV RNA shedding were also significantly greater in E. fergusonii + HuNoV pigs (11.8 ± 1.6 days) compared with HuNoV alone pigs (7.0 ± 1.0 days). By immunofluorescent staining, HuNoV antigen-positive bacteria were detected on the surface of the intestinal epithelium, possibly enhancing attachment of HuNoV to enterocytes, suggesting a potential mechanism by which intestinal colonization with E. fergusonii promoted infectivity of GII.12 HuNoV in Gn pigs.
Assuntos
Antígenos de Grupos Sanguíneos , Norovirus , Suínos , Humanos , Animais , Norovirus/genética , Intestinos , Antígenos de Grupos Sanguíneos/metabolismo , Vida Livre de Germes , RNARESUMO
Rotavirus A (RVA) causes ~200,000 diarrheal deaths annually in children <5yrs, mostly in low- and middle-income countries. Risk factors include nutritional status, social factors, breastfeeding status, and immunodeficiency. We evaluated the effects of vitamin A (VA) deficiency/VA supplementation and RVA exposure (anamnestic) on innate and T cell immune responses in RVA seropositive pregnant and lactating sows and passive protection of their piglets post-RVA challenge. Sows were fed VA deficient (VAD) or sufficient (VAS) diets starting at gestation day (GD)30. A subset of VAD sows received VA supplementation from GD|76 (30,000IU/day, VAD+VA). Sows (6 groups) were inoculated with porcine RVA G5P[7] (OSU strain) or Minimal Essential Medium (mock) at GD~90: VAD+RVA; VAS+RVA; VAD+VA+RVA; VAD-mock; VAS-mock; and VAD+VA-mock. Blood, milk, and gut-associated tissues were collected from sows at several time points to examine innate [natural killer (NK), dendritic (DC) cells], T cell responses and changes in genes involved in the gut-mammary gland (MG)-immunological axis trafficking. Clinical signs of RVA were evaluated post inoculation of sows and post-challenge of piglets. We observed decreased frequencies of NK cells, total and MHCII+ plasmacytoid DCs, conventional DCs, CD103+ DCs and CD4+/CD8+ and T regulatory cells (Tregs) and NK cell activity in VAD+RVA sows. Polymeric Ig receptor and retinoic acid receptor alpha (RARα) genes were downregulated in mesenteric lymph nodes and ileum of VAD+RVA sows. Interestingly, RVA-specific IFN-γ producing CD4+/CD8+ T cells were increased in VAD-Mock sows, coinciding with increased IL-22 suggesting inflammation in these sows. VA supplementation to VAD+RVA sows restored frequencies of NK cells and pDCs, and NK activity, but not tissue cDCs and blood Tregs. In conclusion, similar to our recent observations of decreased B cell responses in VAD sows that led to decreased passive immune protection of their piglets, VAD impaired innate and T cell responses in sows, while VA supplementation to VAD sows restored some, but not all responses. Our data reiterate the importance of maintaining adequate VA levels and RVA immunization in pregnant and lactating mothers to achieve optimal immune responses, efficient function of the gut-MG-immune cell-axis and to improve passive protection of their piglets.
Assuntos
Infecções por Rotavirus , Rotavirus , Deficiência de Vitamina A , Gravidez , Suínos , Animais , Feminino , Vitamina A/farmacologia , Linfócitos T CD8-Positivos/metabolismo , Lactação , Suplementos Nutricionais , ImunidadeRESUMO
Porcine deltacoronavirus (PDCoV) was first identified approximately a decade ago, but much is still obscure in terms of its pathogenesis. We aimed to further characterize PDCoV infection by investigating the presence of virus in respiratory and biliary tissues or fluids; T cell population frequencies in blood; and altered serum cholesterol levels. Twelve, 6-day-old, gnotobiotic piglets were inoculated oronasally with PDCoV OH-FD22 (2.6 × 107 FFU/pig). Six control piglets were not inoculated. Rectal swab (RS), nasal swab (NS), nasal wash (NW), bronchoalveolar lavage (BAL), and biliary fluid (BF) samples were collected at 2, 4, and 7 days post-inoculation (DPI) and tested for PDCoV RNA by RT-qPCR. Blood T cell populations and serum cholesterol levels were determined by flow cytometry and a colorimetric assay, respectively. Moderate to high, and low to moderate titers of PDCoV RNA were detected in RS and in NS, NW, BAL, and BF samples, respectively, of inoculated piglets. There were trends toward decreased CD4+CD8-, CD4-CD8+, and CD4+CD8+ blood T cell frequencies in inoculated piglets. Furthermore, serum cholesterol levels were increased in inoculated piglets. Overall, we found that PDCoV infection does not exclusively involve the intestine, since the respiratory and biliary systems and cholesterol metabolism also can be affected.
RESUMO
The aim of this study was to determine the impact of vitamin A deficiency (VAD)/supplementation (±VA) and group A RV (RVA) maternal immunization of RVA seropositive multiparous pregnant sows, on their immune responses (anamnestic response) and on passive protection of their piglets against RVA challenge. Our results showed that VAD- mock sows had increased RVA RNA shedding at 1-5 days post piglet RVA challenge, and their litters had increased RVA shedding and diarrhea frequency throughout the experiment. VAD decreased memory B cell frequencies while VA supplementation increased RVA specific IgA/IgG antibody (Ab) secreting cell (ASC) numbers in blood, milk, and tissues of RVA inoculated VAD sows. The increased numbers of RVA specific IgA/IgG ASCs in blood, milk/colostrum, intestinal contents, and tissues in VA supplemented VAD sows, suggest a role of VA in B cell immunity and trafficking to tissues. We also observed that RVA inoculated sows had the highest viral neutralizing Ab titers in serum and milk while VA supplementation of VAD sows and RVA inoculation increased IgA+ B cell frequencies in sow colostrum. In summary, we demonstrated that daily oral VA-supplementation (2nd trimester-throughout lactation) to RVA inoculated VAD sows improved the function of their gut-mammary-IgA immunological axis, reducing viral RNA shedding, diarrhea, and increasing weight gain in suckling piglets.
Assuntos
Rotavirus , Gravidez , Suínos , Animais , Feminino , Vitamina A , Imunidade Adaptativa , Leite , Imunoglobulina A , Suplementos Nutricionais , Diarreia/prevenção & controleRESUMO
Campylobacter jejuni is the most common cause of bacterial foodborne gastroenteritis and holds significant public health importance. The continuing increase of antibiotic-resistant Campylobacter necessitates the development of antibiotic-alternative approaches to control infections in poultry and in humans. Here, we assessed the ability of E. coli Nissle 1917 (EcN; free and chitosan-alginate microencapsulated) to reduce C. jejuni colonization in chickens and measured the effect of EcN on the immune responses, intestinal morphology, and gut microbes of chickens. Our results showed that the supplementation of 3-week-old chickens daily with free EcN in drinking water resulted in a 2.0 log reduction of C. jejuni colonization in the cecum, whereas supplementing EcN orally three times a week, either free or microencapsulated, resulted in 2.0 and 2.5 log reductions of C. jejuni colonization, respectively. Gavaged free and microencapsulated EcN did not have an impact on the evenness or the richness of the cecal microbiota, but it did increase the villous height (VH), crypt depth (CD), and VH:CD ratio in the jejunum and ileum of chickens. Further, the supplementation of EcN (all types) increased C. jejuni-specific and total IgA and IgY antibodies in chicken's serum. Microencapsulated EcN induced the expression of several cytokines and chemokines (1.6 to 4.3-fold), which activate the Th1, Th2, and Th17 pathways. Overall, microencapsulated EcN displayed promising effects as a potential nonantibiotic strategy to control C. jejuni colonization in chickens. Future studies on testing microencapsulated EcN in the feed and water of chickens raised on built-up floor litter would facilitate the development of EcN for industrial applications to control Campylobacter infections in poultry.
Assuntos
Infecções por Campylobacter , Campylobacter jejuni , Quitosana , Água Potável , Microbioma Gastrointestinal , Doenças das Aves Domésticas , Probióticos , Animais , Humanos , Alginatos/farmacologia , Antibacterianos/farmacologia , Infecções por Campylobacter/microbiologia , Ceco/microbiologia , Quimiocinas , Galinhas/microbiologia , Quitosana/farmacologia , Citocinas , Escherichia coli , Imunidade , Imunoglobulina A , Doenças das Aves Domésticas/microbiologia , Probióticos/farmacologia , Probióticos/uso terapêuticoRESUMO
Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus of swine that causes acute diarrhoea, vomiting, dehydration and mortality in seronegative neonatal piglets. PDCoV was first reported in Hong Kong in 2012 and its etiological features were first characterized in the United States in 2014. Currently, PDCoV is a concern due to its broad host range, including humans. Chickens, turkey poults, and gnotobiotic calves can be experimentally infected by PDCoV. Therefore, as discussed in this review, a comprehensive understanding of the origin, evolution, cross-species transmission and zoonotic potential of epidemic PDCoV strains is urgently needed.
RESUMO
Live attenuated vaccines (LAVs) replicate in the respiratory/oral mucosa, mimic natural infection, and can induce mucosal and systemic immune responses to the full repertoire of SARS-CoV-2 structural/nonstructural proteins. Generally, LAVs produce broader and more durable protection than current COVID-19 vaccines. We generated a temperature-sensitive (TS) SARS-CoV-2 mutant TS11 via cold-adaptation of the WA1 strain in Vero E6 cells. TS11 replicated at >4 Log10-higher titers at 32 °C than at 39 °C. TS11 has multiple mutations, including those in nsp3, a 12-amino acid-deletion spanning the furin cleavage site of the S protein and a 371-nucleotide-deletion spanning the ORF7b-ORF8 genes. We tested the pathogenicity and protective efficacy of TS11 against challenge with a heterologous virulent SARS-CoV-2 D614G strain 14B in Syrian hamsters. Hamsters were randomly assigned to mock immunization-challenge (Mock-C) and TS11 immunization-challenge (TS11-C) groups. Like the mock group, TS11-vaccinated hamsters did not show any clinical signs and continuously gained body weight. TS11 replicated well in the nasal cavity but poorly in the lungs and caused only mild lesions in the lungs. After challenge, hamsters in the Mock-C group lost weight. In contrast, the animals in the TS11-C group continued gaining weight. The virus titers in the nasal turbinates and lungs of the TS11-C group were significantly lower than those in the Mock-C group, confirming the protective effects of TS11 immunization of hamsters. Histopathological examination demonstrated that animals in the Mock-C group had severe pulmonary lesions and large amounts of viral antigens in the lungs post-challenge; however, the TS11-C group had minimal pathological changes and few viral antigen-positive cells. In summary, the TS11 mutant was attenuated and induced protection against disease after a heterologous SARS-CoV-2 challenge in Syrian hamsters.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Cricetinae , Anticorpos Neutralizantes , Anticorpos Antivirais , Antígenos Virais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Mesocricetus , SARS-CoV-2/genética , Temperatura , Vacinas Atenuadas/genéticaRESUMO
Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) cause acute diarrhea/vomiting in neonatal pigs and share similar tissue or cellular tropisms in the gastrointestinal tract. We investigated if or how these two swine enteric coronaviruses interact with each other in gnotobiotic (Gn) piglets. Seventeen 9-10-day-old Gn piglets were randomly assigned to 5 groups and inoculated with PEDV strain PC21A [9.3 log10 genomic equivalents (GE)/pig] and/or PDCoV strain OH-FD22 (8.6 log10 GE/pig) as follows: dually with PEDV and PDCoV [16 h later (n = 4) or simultaneously (n = 3)] or singly with PEDV (n = 4), PDCoV (n = 4), or mock (n = 3). No enhanced clinical disease or fecal PEDV shedding were observed in dually inoculated pigs compared with PEDV or PDCoV singly inoculated pigs, coinciding with no significant differences in jejunal VH:CD ratios and PEDV antigen-positive scores at post-inoculation days (PIDs) 3-4 among the groups. These observations indicate no increased severity of PEDV infectivity by PDCoV co-infection. Notably, compared with PDCoV singly inoculated pigs, low to moderate fecal PDCoV RNA titers were detected only at PID 1 in both dually inoculated pig groups. At PIDs 2-4, however, there was no detectable PDCoV RNA in the feces, coinciding with no or few PDCoV antigen-positive cells in the small and large intestine of the dually inoculated pigs at PIDs 3-4. These observations indicate a possible interference or inhibition of PDCoV replication in the gastrointestinal tract of pigs co-infected with PEDV and may influence PDCoV infection in PEDV co-infected pigs.
Assuntos
Coinfecção/veterinária , Deltacoronavirus/fisiologia , Trato Gastrointestinal/virologia , Interações Microbianas/fisiologia , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/virologia , Replicação Viral , Animais , Animais Recém-Nascidos , Coinfecção/virologia , Distribuição Aleatória , SuínosRESUMO
Porcine deltacoronavirus (PDCoV) strain OH-FD22 infects poultry and shares high nucleotide identity with sparrow-origin deltacoronaviruses (SpDCoV) ISU73347 and HKU17 strains. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from these SpDCoVs would alter the host and tissue tropism of PDCoV. First, an infectious cDNA clone of PDCoV OH-FD22 strain (icPDCoV) was generated and used to construct chimeric icPDCoVs harboring the S protein of HKU17 (icPDCoV-SHKU17) or the RBD of ISU73347 (icPDCoV-RBDISU). To evaluate their pathogenesis, neonatal gnotobiotic pigs were inoculated orally/oronasally with the recombinant viruses or PDCoV OH-FD22. All pigs inoculated with icPDCoV or OH-FD22 developed severe diarrhea and shed viral RNA at moderate-high levels (7.62-10.56 log10 copies/mL) in feces, and low-moderate levels in nasal swabs (4.92-8.48 log10 copies/mL). No pigs in the icPDCoV-SHKU17 and icPDCoV-RBDISU groups showed clinical signs. Interestingly, low-moderate levels (5.07-7.06 log10 copies/mL) of nasal but not fecal viral RNA shedding were detected transiently at 1-4 days post-inoculation in 40% (2/5) of icPDCoV-SHKU17- and 50% (1/2) of icPDCoV-RBDISU-inoculated pigs. These results confirm that PDCoV infected both the upper respiratory and intestinal tracts of pigs. The chimeric viruses displayed an attenuated phenotype with the loss of tropism for the pig intestine. The SpDCoV S protein and RBD reduced viral replication in pigs, suggesting limited potential for cross-species spillover upon initial passage.
Assuntos
Infecções por Coronavirus/patologia , Deltacoronavirus/genética , Intestinos/patologia , Sistema Respiratório/patologia , Glicoproteína da Espícula de Coronavírus/genética , Tropismo Viral/genética , Motivos de Aminoácidos , Animais , Doenças das Aves/virologia , Linhagem Celular , Deltacoronavirus/patogenicidade , Intestinos/virologia , Proteínas Recombinantes/genética , Sistema Respiratório/virologia , Pardais , Suínos , Doenças dos Suínos/virologia , Virulência/genéticaRESUMO
Coronaviruses (CoVs) comprise a large group of positive stranded RNA viruses that infect a diverse host range including birds and mammals. Infection with CoVs typically presents as mild to severe respiratory or enteric disease, but CoVs have the potential to cause significant morbidity or mortality in highly susceptible age groups. CoVs have exhibited a penchant for jumping species barriers throughout history with devastating effects. The emergence of highly pathogenic or infectious CoVs in humans over the past 20 years, including severe acute respiratory syndrome CoV (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and most recently severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), underscores the significant threat that CoV spillovers pose to humans. Similar to the emergence of SARS-CoV-2, CoVs have been devastating to commercial animal production over the past century, including infectious bronchitis virus in poultry and bovine CoV, as well as the emergence and reemergence of multiple CoVs in swine including transmissible gastroenteritis virus, porcine epidemic diarrhea virus, and porcine deltacoronavirus. These naturally occurring animal CoV infections provide important examples for understanding CoV disease as many animal CoVs have complex pathogenesis similar to SARS-CoV-2 and can shed light on the ongoing SARS-CoV-2 outbreak. We provide an overview and update regarding selected existing animal CoVs and their primary host species, diseases caused by CoVs, how CoVs jump species, whether these CoVs pose an outbreak risk or risk to humans, and how we can mitigate these risks.
Assuntos
Infecções por Coronavirus/virologia , Coronavirus/fisiologia , Animais , Aves , Coronavirus/classificação , Coronavirus/patogenicidade , Infecções por Coronavirus/transmissão , Modelos Animais de Doenças , Humanos , Mamíferos , FilogeniaRESUMO
Mycoplasma gallisepticum (MG) causes chronic respiratory disease in chickens, leading to severe economic losses to the poultry industry. Currently the disease is managed with antimicrobials and vaccination; however, emergence of multi-drug resistant Mycoplasma and the limited effect of vaccines necessitate development of novel approaches. A library of 4,182 small molecules (SMs) was screened for identification of narrow spectrum anti-MG compounds using high throughput screening. A total of 584 SMs were identified. Ten SMs possessed low MICs (0.78-100 µM) with efficacy against multiple MG strains and MG biofilm. These 10 SMs did not affect commensal/probiotic bacteria and other avian and foodborne pathogens. They displayed no or little toxicity on the avian macrophage HD-11 cells, human epithelial Caco-2 cells, and chicken red blood cells (RBCs); but, they were effective in reducing MG in chicken RBCs. Six SMs (SM1, SM3-5, and SM9-10) were tested in three-week-old chickens infected with MG (nasal spray; 109 CFU/bird). SM4 and SM9 reduced airsacculitis by 77.2 % and 82.9 %, MG load in the trachea by 0.9 log (p < 0.05) and 2.7 log (p < 0.0001), and tracheal mucosal thickness by 23 % and 61 %, respectively with no impact on the richness and evenness of the cecal (P = 0.6; H = 1.0) and tracheal (P = 0.8; H = 0.8) microbiota compared to the MG-infected controls. Both SM4 and SM9 treatments resulted in a significant alteration in the cell membrane conformation of MG. In conclusion; we identified two novel growth inhibitors of MG that are effective in chickens. These findings will facilitate development of novel antibacterials to control mycoplasmosis in poultry.
Assuntos
Antibacterianos/farmacologia , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/efeitos dos fármacos , Doenças das Aves Domésticas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Antibacterianos/administração & dosagem , Membrana Externa Bacteriana/efeitos dos fármacos , Células CACO-2 , Galinhas/microbiologia , Farmacorresistência Bacteriana , Células Epiteliais/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Infecções por Mycoplasma/tratamento farmacológico , Doenças das Aves Domésticas/microbiologia , Infecções Respiratórias , Organismos Livres de Patógenos EspecíficosRESUMO
Porcine deltacoronavirus (PDCoV) has emerged as a novel, contagious swine enteric coronavirus that causes watery diarrhea and/or vomiting and intestinal villous atrophy in nursing piglets. PDCoV-related diarrhea first occurred in the USA in 2014 and was subsequently reported in South Korea, China, Thailand, Vietnam, and Lao People's Democratic Republic, leading to massive economic losses and posing a threat to the swine industry worldwide. Currently, no treatments or vaccines for PDCoV are available. The critical step in the development of potential vaccines against PDCoV infection is the isolation and propagation of PDCoV in cell culture. This chapter provides a detailed protocol for isolation and propagation of PDCoV in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures supplemented with pancreatin and trypsin, respectively. Filtered clinical samples (swine intestinal contents or feces) applied to ST or LLC-PK cells produce cytopathic effects characterized by rounding, clumping, and detachment of cells. PDCoV replication in cells can be quantifiably monitored by qRT-PCR, immunofluorescence assays, and immune-electron microscopy. Infectious viral titers can be evaluated by using plaque assays or 50% tissue culture infectious dose (TCID50) assays. The ST or LLC-PK cells efficiently supported serial passage and propagation of PDCoV. After serial passage of PDCoV in either ST or LLC-PK cells, the virus can be purified further in ST cells by plaque assays.
Assuntos
Coronavirus/isolamento & purificação , Doenças dos Suínos/virologia , Técnicas de Cultura de Tecidos/métodos , Animais , Células Cultivadas , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Fezes/virologia , Inoculações Seriadas , SuínosRESUMO
Discovery of bats with severe acute respiratory syndrome (SARS)-related coronaviruses (CoVs) raised the specter of potential future outbreaks of zoonotic SARS-CoV-like disease in humans, which largely went unheeded. Nevertheless, the novel SARS-CoV-2 of bat ancestral origin emerged to infect humans in Wuhan, China, in late 2019 and then became a global pandemic. Less than 5 months after its emergence, millions of people worldwide have been infected asymptomatically or symptomatically and at least 360,000 have died. Coronavirus disease 2019 (COVID-19) in severely affected patients includes atypical pneumonia characterized by a dry cough, persistent fever, and progressive dyspnea and hypoxia, sometimes accompanied by diarrhea and often followed by multiple organ failure, especially of the respiratory and cardiovascular systems. In this minireview, we focus on two endemic respiratory CoV infections of livestock: bovine coronavirus (BCoV) and porcine respiratory coronavirus (PRCV). Both animal respiratory CoVs share some common features with SARS-CoV and SARS-CoV-2. BCoV has a broad host range including wild ruminants and a zoonotic potential. BCoV also has a dual tropism for the respiratory and gastrointestinal tracts. These aspects, their interspecies transmission, and certain factors that impact disease severity in cattle parallel related facets of SARS-CoV or SARS-CoV-2 in humans. PRCV has a tissue tropism for the upper and lower respiratory tracts and a cellular tropism for type 1 and 2 pneumocytes in lung but is generally a mild infection unless complicated by other exacerbating factors, such as bacterial or viral coinfections and immunosuppression (corticosteroids).
Assuntos
Betacoronavirus/crescimento & desenvolvimento , Doenças dos Bovinos/fisiopatologia , Infecções por Coronavirus/veterinária , Coronavirus Bovino/crescimento & desenvolvimento , Pneumonia Viral/fisiopatologia , Infecções Respiratórias/veterinária , Doenças dos Suínos/fisiopatologia , Animais , Betacoronavirus/patogenicidade , COVID-19 , Bovinos , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Coronavirus Bovino/patogenicidade , Especificidade de Hospedeiro , Humanos , Pandemias , Pneumonia Viral/patologia , Coronavirus Respiratório Porcino/crescimento & desenvolvimento , Coronavirus Respiratório Porcino/patogenicidade , Infecções Respiratórias/patologia , Infecções Respiratórias/fisiopatologia , SARS-CoV-2 , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Tropismo ViralRESUMO
Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus in the family Coronaviridae, causes acute diarrhea and/or vomiting, dehydration and high mortality in neonatal piglets. Two different genogroups of PEDV, S INDEL [PEDV variant containing multiple deletions and insertions in the S1 subunit of the spike (S) protein, G1b] and non-S INDEL (G2b) strains were detected during the diarrheal disease outbreak in US swine in 2013-2014. Similar viruses are also circulating globally. Continuous improvement and update of biosecurity and vaccine strains and protocols are still needed to control and prevent PEDV infections worldwide. Although the non-S INDEL PEDV was highly virulent and the S INDEL PEDV caused milder disease, the latter has the capacity to cause illness in a high number of piglets on farms with low biosecurity and herd immunity. The main PEDV transmission route is fecal-oral, but airborne transmission via the fecal-nasal route may play a role in pig-to-pig and farm-to-farm spread. PEDV infection of neonatal pigs causes fecal virus shedding (alongside frequent detection of PEDV RNA in the nasal cavity), acute viremia, severe atrophic enteritis (mainly jejunum and ileum), and increased pro-inflammatory and innate immune responses. PEDV-specific IgA effector and memory B cells in orally primed sows play a critical role in sow lactogenic immunity and passive protection of piglets. This review focuses on the etiology, transmission, pathogenesis, and prevention and control of PEDV infection.
Assuntos
Infecções por Coronavirus/patologia , Infecções por Coronavirus/transmissão , Mucosa Intestinal/virologia , Vírus da Diarreia Epidêmica Suína/patogenicidade , Doenças dos Suínos/transmissão , Aerossóis , Animais , Infecções por Coronavirus/prevenção & controle , Progressão da Doença , Imunidade Humoral/imunologia , Mucosa Intestinal/patologia , Vírus da Diarreia Epidêmica Suína/genética , Vírus da Diarreia Epidêmica Suína/imunologia , Receptores Virais/metabolismo , Suínos , Doenças dos Suínos/patologia , Doenças dos Suínos/virologia , Tropismo Viral/fisiologia , Viremia/sangueRESUMO
Unlike porcine epidemic diarrhea virus (PEDV) that infects only pigs, porcine deltacoronavirus (PDCoV) has the capacity to infect different animal species. In vivo gnotobiotic calves were previously confirmed to be susceptible to infection with PDCoV, but not with PEDV. We next investigated in vitro whether primary bovine cells are susceptible to PDCoV or PEDV infection. We conducted quantification of viral RNA in cell culture supernatants and immunofluorescent staining for the detection of PDCoV or PEDV antigen in two primary bovine cell types inoculated with the PDCoV strain OH-FD22 or PEDV strain PC22-P40 grown in LLC-PK or Vero cells, respectively, and supplemented with 1.25â¼5 µg/mL of trypsin in the cell culture medium. The primary cells were isolated from the kidney or heart of a gnotobiotic calf, and both cell types were vimentin-positive, but E-Cadherin-negative, resembling mesenchymal cells. Similar to the previous in vivo observation, cytopathic effects (CPE) that consisted of enlarged and rounded cells, followed by cell shrinkage and detachment, were identified in the two primary cell types inoculated with PDCoV. Unexpectedly, similar CPE was also identified in the two cell types inoculated with PEDV. High PDCoV or PEDV RNA titers and PDCoV or PEDV antigens were detected in the cell culture supernatants and CPE-positive cells, respectively. Our study revealed that primary bovine mesenchymal cells are susceptible to infection with PDCoV and PEDV. The in vitro observation partially coincided with the corresponding in vivo data from gnotobiotic calves.
Assuntos
Deltacoronavirus/fisiologia , Vírus da Diarreia Epidêmica Suína/fisiologia , Replicação Viral , Animais , Bovinos , Células Cultivadas , Chlorocebus aethiops , Efeito Citopatogênico Viral , Vida Livre de Germes , Miocárdio/citologia , Cultura Primária de Células , Suínos , Células VeroRESUMO
Deltacoronavirus (DCoV)-the only coronavirus that can infect multiple species of mammals and birds-was initially identified in several avian and mammalian species, including pigs, in China in 2009-2011. Porcine DCoV has since spread worldwide and is associated with multiple outbreaks of diarrheal disease of variable severity in farmed pigs. In contrast, avian DCoV is being reported in wild birds in different countries without any evidence of disease. The DCoV transboundary nature and the recent discovery of its remarkably broad reactivity with its cellular receptor-aminopeptidase N (APN)-from different species emphasize its epidemiological relevance and necessitate additional research. Further, the ability of porcine DCoV to infect and cause disease in chicks and turkey poults and gnotobiotic calves is suggestive of its increased potential for interspecies transmission or of its avian origin. Whether, porcine DCoVs were initially acquired by one or several mammalian species from birds and whether avian and porcine DCoVs continue co-evolving with frequent spillover events remain to be major unanswered questions. In this review, we will discuss the current information on the prevalence, genetic diversity, and pathogenic potential of porcine and avian DCoVs. We will also analyze the existing evidence of the ongoing interspecies transmission of DCoVs that may provide novel insights into their complex evolution.
RESUMO
Human noroviruses (HuNoVs) are a leading cause of acute gastroenteritis worldwide. It is unclear which arm of the immune system regulates resistance to HuNoV infection. Thus, we studied the pathogenesis of human norovirus (HuNoV) in T-B-NK+ Severe Combined Immunodeficiency (SCID) gnotobiotic pigs to investigate the role of innate (especially, natural killer (NK) cells) immunity in HuNoV infection. Forty SCID and non-SCID pigs were randomly grouped: 1) SCID+HuNoV (n = 12); 2) non-SCID+HuNoV (n = 14); 3) SCID mock-inoculated (n = 6); and 4) non-SCID mock-inoculated (n = 8). Pigs (8-14-day-old) were inoculated orally with GII.4 HuNoV strain HS292 (mean 9.1 log10 genomic equivalents/pig) or mock. Daily fecal consistency and fecal viral RNA shedding, and histopathology (at euthanasia) were evaluated. Frequencies of blood and ileal T, B, and NK cells were analyzed by flow cytometry, and a NK cell cytotoxicity assay was performed at post-inoculation day (PID) 8. Unlike the increased infectivity of HuNoV observed previously in T-B-NK- SCID pigs (Lei et al., 2016. Sci. Rep. 6, 25,222), there was no significant difference in frequency of pigs with diarrhea and diarrhea days between T-B-NK+ SCID+HuNoV and non-SCID+HuNoV groups. Cumulative fecal HuNoV RNA shedding at PIDs 1-8, PIDs 9-27, and PIDs 1-27 also did not differ statistically. These observations coincided with the presence of NK cells and NK cell cytotoxicity in the ileum and blood of the SCID pigs. Based on our observations, innate immunity, including NK cell activity, may be critical to mediate or reduce HuNoV infection in T-B-NK+ SCID pigs, and potentially in immunocompetent patients.