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1.
Theriogenology ; 132: 22-26, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30981942

RESUMO

Adenovirus is a kind of non-enveloped,double-stranded DNA virus. As a member of the mammalian adenoviruses of the Adenoviridae family, porcine adenovirus causes gastrointestinal disease in piglets. In this study, the modified adenovirus was manipulated to carry a green fluorescence EGFP marker. The modified adenovirus was added to medium199 for co-incubation or microinjected into the cytoplasm of porcine oocytes. The effect of adenovirus on the first polar body extrusion was not significant during porcine oocyte maturation. Our data demonstrated the zona pellucida plays a vital role in porcine oocytes being resistant to modified adenovirus. Additionally, the results suggested that oocytes protect themselves from nonself substances.


Assuntos
Adenoviridae/fisiologia , Fertilização In Vitro/veterinária , Oócitos/fisiologia , Suínos , Zona Pelúcida/fisiologia , Animais , Oócitos/virologia
2.
Res Vet Sci ; 123: 135-140, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30641473

RESUMO

Bovine herpesvirus 1 (BoHV-1) disseminates easily, is difficult to control, and is widely spread in cattle herds worldwide. BoHV-1 causes a broad range of losses to the cattle industry, mainly concerning reproduction. Previous studies involving experimental infection of BoHV-1 in an in vitro embryo production system have reported impairment of embryonic development by BoHV-1. In this study, we evaluated the interference of BoHV-1 in the in vitro maturation system of cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) cultured with a cumulus cell suspension. Blood samples and ovaries were collected from slaughterhouse cows unvaccinated against BoHV-1. Using virus neutralization assays, the seropositive animals were classified according to their antibody titers. The oocytes were recovered by follicular aspiration and divided into two groups, COCs and DOs, which were evaluated for their nuclear maturation capacity using immunofluorescence assays by laser scanning confocal microscopy. Two experiments were carried out: (I) in vitro maturation of COCs and DOs after artificial infection of seronegative animals and (II) in vitro maturation of COCs and DOs of seropositive animals. In experiment I, a difference (P < 0.01) was observed between the maturation rates of the control group COCs (78.2%) and the infected COCs (43.6%). In experiment II, there was a difference (P < 0.01) in the maturation rate between animals with antibody titers ≥16 (56.9%) and the control group (79.4%). Immunofluorescence assays identified BoHV-1 in the COCs and DOs. Therefore, it was concluded that BoHV-1 affects the in vitro maturation process in both in vitro and natural infections.


Assuntos
Bovinos , Herpesvirus Bovino 1/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/virologia , Animais , Feminino , Gravidez
3.
Theriogenology ; 126: 75-80, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30537656

RESUMO

In vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) are important breeding techniques for livestock. High-quality MII oocytes produced from in vitro maturation (IVM) are required for the two techniques listed above. The ovaries used for IVM operations are primarily acquired from commercial abattoirs, and the pathogen status of slaughtered animals becomes an unavoidable issue. Our previous monitoring data showed that porcine circovirus type 2 (PCV-2) is the main pathogen present in ovaries from abattoirs. However, the characteristics and effects of PCV-2 infection in oocyte maturation and in vitro production (IVP) of embryos are unclear, and currently there are no relevant studies. Therefore, the aim of this study was to determine the PCV-2 infection pattern and determine whether it affects oocyte in vitro maturation and IVP embryo development. More than five hundred ovaries and five thousand oocytes were utilized in the present study. Polymerase chain reaction (PCR) was used to detect PCV-2 DNA in ovaries, follicular fluid (FF), oocytes, cumulus cells and IVP embryos. The effects of viral infections on the rate of oocyte maturation and IVP embryo development were evaluated. We also analyzed the number of copies of the virus in the IVM and IVP process by absolute quantitative fluorescence PCR. Our study showed that the prevalent virus subgenotype in ovaries was PCV-2a. PCV-2a infection did not significantly affect ovarian/oocyte morphology and maturation. Moreover, virus infection did not have a significant effect on the development of the IVP embryos except for a reduction in IVF blastocyst cell numbers. Further tests showed that the viral copy numbers fluctuated at different stages between the IVP embryos and culture medium. For the first time, this study identified the infection pattern of naturally sourced PCV-2 in the course of oocyte maturation and embryo development.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus , Fertilização In Vitro/veterinária , Oócitos/virologia , Doenças dos Suínos/embriologia , Suínos/embriologia , Animais , Infecções por Circoviridae/embriologia , Meios de Cultura , DNA Viral/isolamento & purificação , Desenvolvimento Embrionário , Fertilização In Vitro/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/crescimento & desenvolvimento , Suínos/virologia
4.
Pesqui. vet. bras ; 38(12): 2207-2212, dez. 2018. tab, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-976420

RESUMO

Bovine herpesvirus 1 (BoHV-1) is an important bovine pathogen that is responsible for causing respiratory diseases and reproductive failures. The presence of BoHV-1 in an in vitro embryo production system affects fertilization, maturation, and embryonic development. The objective of this study was to evaluate the developmental capacity of oocytes from naturally infected cows with no reproductive history. Moreover, this study investigated the presence of viral DNA in cumulus oophorus complexes (COCs). Experimental groups were differentiated by titrating the antibodies detected through seroneutralization assays, establishing three groups: seronegative animals (titer lower than 2), low titer (2 to 8), and animals with a titer above or equal to 16. COCs were obtained from 15 donors during 22 sessions of ultrasound-guided follicular aspiration. DNA was extracted from a pool of COCs obtained from all aspirations from the same donor as well as from whole blood and nested PCR reactions were performed. Only COCs with a compact layer of cumulus cells, an intact zona pellucida, and homogeneous cytoplasm were selected for in vitro culture and evaluation of nuclear maturation rate. After culturing for 24 hours, the oocytes were fixed and stained to evaluate the meiotic cell cycle stage. Oocytes that showed a chromosomal configuration in metaphase II were considered to have reached nuclear maturation. Compared with the other groups, the oocyte nuclear maturation rate in animals with a titer greater than or equal to 16 (50%) was compromised (P<0.05). However, the viral titer did not influence the maturation rate of bovine oocytes in animals exhibiting low titration (62.2%) when compared with the control group (76.7%). Viral DNA was not observed in the blood samples but was detected in the COC pool from three seropositive donors. In view of the results obtained, we conclude that natural infections by the BoHV-1 virus can compromise the nuclear maturation rate in cows, depending on the titration levels of antibodies against the virus. Moreover, viral DNA could be present in COCs, contradicting the hypothesis that seropositive animals with no history of clinical symptomatology pose a negligible risk of transmitting BoHV-1 by COCs.(AU)


Herpesvírus bovino 1 (BoHV-1) é um importante patógeno bovino, responsável por causar doenças respiratórias e falhas reprodutivas. A presença do BoHV-1 em sistema de produção in vitro de embriões afeta a fertilização, a maturação e o desenvolvimento embrionário. O objetivo deste estudo foi avaliar a capacidade de desenvolvimento de ovócitos oriundos de vacas infectadas naturalmente sem histórico reprodutivo. Além disso, este estudo investigou a presença do DNA viral em Complexos Cumulus Ooforus (COCs). Os tratamentos foram definidos a partir do título de anticorpos detectados pelos ensaios de soroneutralização, sendo estabelecidos três grupos: animais soronegativos (título menor do que 2), título baixo (2 a 8) e animais com título maior ou igual a 16. Os COCs foram obtidos de 15 doadoras durante 22 sessões de aspiração folicular guiada por ultrassom. A extração do DNA foi realizada em um pool de COCs de todas as aspirações de uma mesma doadora e no sangue total para a realização das reações de Nested-PCR. Para avaliação da taxa de maturação nuclear, foram selecionados para o cultivo in vitro somente os COCs com camada compacta de células do cumulus, zona pelúcida íntegra e citoplasma homogêneo. Após 24 horas de cultivo, os ovócitos foram fixados e corados em lâmina para a avaliação do estádio do ciclo celular meiótico. Os ovócitos que apresentaram configuração cromossômica em metáfase II foram considerados como tendo alcançado a maturação nuclear. Verificou-se comprometimento na taxa de maturação nuclear ovocitária (P<0.05) nos animais de título maior ou igual a 16 (50%). No entanto, não houve influência do título viral na taxa de maturação de ovócitos bovinos em animais que apresentaram titulação baixa (62,2%) quando comparados com o grupo controle (76,7%). O DNA viral não foi identificado nas amostras de sangue, mas foi detectado no pool de COCs de três doadoras soropositivas. Diante dos resultados encontrados conclui-se que vacas infectadas naturalmente pelo vírus BoHV-1 apresentam comprometimento na taxa de maturação nuclear, dependendo do grau de titulação de anticorpos contra o vírus. Ademais, o DNA viral pode estar presente em COCs contrariando a hipótese de que animais sorologicamente positivos e sem histórico de sintomatologia clínica oferecem risco negligível de transmissão do BoHV-1 por COCs.(AU)


Assuntos
Animais , Feminino , Bovinos , Oócitos/patologia , Oócitos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Bovino 1 , Rinotraqueíte Infecciosa Bovina , Técnicas de Maturação in Vitro de Oócitos/veterinária
5.
Res Vet Sci ; 120: 54-56, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30218851

RESUMO

Bovine herpesvirus 1 (BoHV-1) is the causative agent of infectious bovine rhinotracheitis (IBR) and is also associated with reproductive failure. This study investigated the presence of BoHV-1 in cumulus-oocyte complexes (COCs) of naturally-infected cows without clinical signs of IBR. The presence of BoHV-1 in COCs was evaluated by immunofluorescence using confocal laser scanning microscopy. Blood samples and ovaries from 82 cows that had not been vaccinated against BoHV-1 were collected for serological analysis. COCs were divided into two pools: COCs derivate from seropositive cows and from seronegative cows. Then, the samples were processed for confocal microscopy analysis. The results indicated that 61% (50/82) of cows were seropositive for BoHV-1. A total of 719 COCs were obtained from the cows and processed. None of 276 COCs from the 32 seronegative cows presented BoHV-1. However, BoHV-1 was present in the cytoplasm of cumulus cells from 158 out of 443 COCs aspirated from the seropositive cows. The detection of BoHV-1 in the COCs of seropositive cows suggests that the COCs of naturally-infected, asymptomatic cows may be infected with BoHV-1.


Assuntos
Bovinos , Células do Cúmulo/virologia , Herpesvirus Bovino 1/isolamento & purificação , Oócitos/virologia , Animais , Feminino , Herpesvirus Bovino 1/imunologia
6.
Sci Rep ; 8(1): 1152, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29348472

RESUMO

Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin ß revealing previously unknown dynamics of the entry pathway.


Assuntos
Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Núcleo Celular/virologia , Citosol/virologia , Parvovirus Canino/metabolismo , Vírion/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Capsídeo/ultraestrutura , Proteínas do Capsídeo/ultraestrutura , Gatos , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Citosol/metabolismo , Citosol/ultraestrutura , Células Epiteliais , Corantes Fluorescentes/química , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Processamento de Imagem Assistida por Computador , Microscopia de Força Atômica , Microscopia Confocal/métodos , Oócitos/metabolismo , Oócitos/ultraestrutura , Oócitos/virologia , Compostos Orgânicos/química , Parvovirus Canino/ultraestrutura , Espectrometria de Fluorescência/métodos , Vírion/ultraestrutura , Xenopus laevis , beta Carioferinas/genética , beta Carioferinas/metabolismo
7.
Biomed Res Int ; 2017: 1840417, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28890894

RESUMO

The introduction and widespread application of vitrification are one of the most important achievements in human assisted reproduction techniques (ART) of the past decade despite controversy and unclarified issues, mostly related to concerns about disease transmission. Guidance documents published by US Food and Drug Administration, which focused on the safety of tissue/organ donations during Zika virus spread in 2016, as well as some reports of virus, bacteria, and fungi survival to cryogenic temperatures, highlighted the need for a review of the way how potentially infectious material is handled and stored in ART-related procedures. It was experimentally demonstrated that cross-contamination between liquid nitrogen (LN2) and embryos may occur when infectious agents are present in LN2 and oocytes/embryos are not protected by a hermetically sealed device. Thus, this review summarizes pertinent data and opinions regarding the potential hazard of infectious transmission through cryopreserved and banked reproductive cells and tissues in LN2. Special attention is given to the survival of pathogens in LN2, the risk of cross-contamination, vitrification methods, sterility of LN2, and the risks associated with the use of straws, cryovials, and storage dewars.


Assuntos
Criopreservação , Embrião de Mamíferos/virologia , Células Germinativas/virologia , Infecção por Zika virus/virologia , Células Germinativas/crescimento & desenvolvimento , Humanos , Oócitos/virologia , Técnicas de Reprodução Assistida , Obtenção de Tecidos e Órgãos , Estados Unidos , United States Food and Drug Administration , Vitrificação , Zika virus/patogenicidade , Infecção por Zika virus/transmissão
8.
Theriogenology ; 97: 67-72, 2017 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-28583610

RESUMO

As production of in vitro (IVP) bovine embryos steadily increases, the sanitary risk associated with IVP embryos remains a concern. One of the greatest concerns is how BVDV may be transmitted through IVP embryos. The objective of this study was to evaluate the effects caused by BVDV-1, BVDV-2 and Hobi-like virus exposure during in vitro maturation on embryo development and viral infection. Abittior-derived oocytes were randomly assigned for in vitro maturation with serial concentrations of BVDV-1 (3.12 × 102 - 2.50 × 103 TCID50/100 µL), BVDV-2 (6.25 × 101 - 5.20 × 102 TCID50/100 µL) or Hobi-like virus (1.90 × 102 - 1.58 × 103 TCID50/100 µL) for 22-24 h. After maturation, oocytes were fertilized and embryo cultured following standard in vitro procedures. Embryo development was evaluated and percentage of respective, positive BVDV degenerated and viable embryos were evaluated by RT-qPCR. No concentration of BVDV-1 altered embryo development as measured by cleavage and blastocyst rates, compared to negative control group. However 100% of degenerated embryos and 50-100% of viable embryos tested positive for BVDV-1, depending on the viral concentration. BVDV-2 exposed oocytes had higher cleavage rates than the negative control group (60.2-64.1% vs 49.8%; P = 0.003-0.032). However, no difference was detected for blastocyst rates. In aadition, 100% of degenerated embryos and 20-50% of viable embryos tested positive for BVDV-2. Hobi-like virus treated oocytes had reduced cleavage rates for the three highest viral concentrations (33.3-38.0% vs 49.8% for negative controls; P ≤ 0.001-0.014). Blastocyst rates were only reduced in the 7.9 × 102 Hobi-like virus concentration (6.9 ± 0.9% vs 15.1 ± 1.6%; P = 0.009), when calculated by oocyte number. 50-80% of degenerated embryos tested positive for Hobi-like virus. No viable embryos from the Hobi-like virus treated oocytes tested positive. These results suggest that IVP embryos from BVDV-1 and -2 infected oocytes develop normally, but carry the virus. However, Hobi-like virus infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus.


Assuntos
Bovinos , Desenvolvimento Embrionário/fisiologia , Oócitos/fisiologia , Oócitos/virologia , Infecções por Pestivirus/embriologia , Pestivirus/fisiologia , Animais , Vírus da Diarreia Viral Bovina Tipo 1/fisiologia , Vírus da Diarreia Viral Bovina Tipo 2/fisiologia , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro/veterinária
9.
Pol J Vet Sci ; 20(1): 25-29, 2017 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-28525333

RESUMO

Porcine circovirus type 2 (PCV2) has been known as a causative agent of reproductive failure in the sow. In the present study mouse model was used to investigate PCV2 infection. In order to investigate whether PCV2 can induce lesions of spermatocytes and oocytes, 6 male and 6 female mice were each inoculated intraperitoneally with PCV2b, and 3 male and 3 female mice mock-infected with cell culture supernatant served as controls. Samples of testes and ovaries from PCV2b-inoculated and mock-infected mice were investigated using PCR, histopathological, ultrastructural and immunofluorescent histochemical methods at 14 and 21 day post infection (dpi). The study revealed that in the virus-inoculated mice leydig cells in testes and granulosa cells in ovaries were degenerated, and a small number of spermatocytes and oocytes showed apoptosis. Positive PCV2b antigen signals were also observed in these apoptotic cells. It can be suggested that PCV2 can cause lesions of spermatocyte and oocyte prior to zygotes formation in its host.


Assuntos
Infecções por Circoviridae/patologia , Circovirus/classificação , Oócitos/virologia , Espermatozoides/virologia , Animais , DNA Viral , Feminino , Masculino , Camundongos , Oócitos/ultraestrutura , Espermatozoides/ultraestrutura
10.
J Gen Virol ; 98(4): 652-661, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28141490

RESUMO

Viral covert mortality disease (VCMD) has caused serious losses to shrimp aquaculture in China in recent years and the ridgetail white prawn Exopalaemon carinicauda has been suspected to be one important factor in perpetuating the high prevalence of covert mortality nodavirus (CMNV) infections due to its perennial presence in shrimp farming ponds and water from natural habitats. Experiments were carried out to determine the possibility of vertical transmission of CMNV in E. carinicauda in this study. CMNV infection in gonads, fertilized eggs and larvae was investigated by using the methods of reverse transcription nested PCR (nRT-PCR), in situ hybrization (ISH) and transmission electron microscopy (TEM). The ovarian tissue and testis tissue of artificially infected parental E. carinicauda were proved to be CMNV-positive by nRT-PCR. Fertilized eggs were also found to be CMNV-positive by nRT-PCR whether the fertilized eggs originated from the CMNV-positive female broodstock mated with the CMNV-negative male broodstock, or they originated from the CMNV-negative female broodstock mated with the CMNV-positive males. The results of ISH indicated that the positive signals were evident in the oocytes within ovarian tissue and nauplii. By TEM analysis, CMNV virions were observed in oogonia, oocytes, spermatocytes, fertilized eggs and nauplii. The presence of CMNV in fertilized eggs and larvae indicates that CMNV can transmit vertically via sperm and oocytes in E. carinicauda, which highlights the high probability of vertical transmission of CMNV in the main species of cultured shrimp and prawns.


Assuntos
Transmissão Vertical de Doença Infecciosa , Nodaviridae/crescimento & desenvolvimento , Palaemonidae/virologia , Animais , China , Gônadas/virologia , Hibridização In Situ , Larva/virologia , Masculino , Microscopia Eletrônica de Transmissão , Oócitos/virologia , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatócitos/virologia , Zigoto/virologia
11.
Theriogenology ; 86(8): 1999-2003, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27481815

RESUMO

Structural changes in the zona pellucida (ZP) of bovine oocytes seem to modulate their interaction with various viral agents, facilitating the viral infection in in vitro production systems. To evaluate the susceptibility of bovine oocytes to noncytopathogenic bovine viral diarrhea virus (ncp-BVDV), cumulus-oocyte complexes were exposed to 10(7) ​tissue culture-infective doses (TCID50)/mL of an ncp-BVDV strain during IVM (in vitro maturation). After that, cumulus cells and the ZP were removed by hyaluronidase and pronase treatment, respectively, and the percentages of oocytes with polar body were analyzed as a sign of nuclear maturation. After passage through cell culture, the virus was isolated from granulosa cells, ZP-free mature oocytes, and ZP-intact mature oocytes. These results were confirmed by reverse transcription-polymerase chain reaction. After consecutive washes, the virus remained associated with ZP-free oocytes, maintaining its replication and infectivity in permissive cells. Based on these findings, it is concluded that the classical viral isolation procedure has a predictive value to detect BVDV associated with ZP-free oocytes and that it was novelty demonstrated that both washing and trypsin treatment of oocytes were ineffective to remove BVDV infection.


Assuntos
Bovinos , Células do Cúmulo/virologia , Vírus da Diarreia Viral Bovina/isolamento & purificação , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/virologia , Animais , Membrana Celular , Corpos Polares , Zona Pelúcida
12.
Fertil Steril ; 105(4): 1010-3, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26730499

RESUMO

OBJECTIVE: To investigate the vertical transmission of hepatitis B virus (HBV) via embryos to children and whether HBV in embryos has an effect on the development of the fetus and pregnancy outcomes of in vitro fertilization and embryo transfer (IVF-ET). DESIGN: Long-term follow-up study. SETTING: Research laboratory. PATIENT(S): Thirty-one couples with a hepatitis B surface antigen (HBsAg)-negative woman and HBsAg-positive man, and 41 couples with a HBsAg-positive woman and HBsAg-negative man, whose unfertilized oocytes and nonviable embryos were tested for HBV DNA, RNA, or HBsAg. INTERVENTION(S): HBV DNA, RNA, or HBsAg analyses in unfertilized oocytes and nonviable embryos. MAIN OUTCOME MEASURE(S): HBV serologic markers analyses. RESULT(S): We obtained follow-up data for 71 couples. A total of 24 babies were born, and no newborns exhibited defects at birth. Twelve babies were born to couples with HBV-positive oocytes and/or embryos. The pregnancy outcomes were not associated with the presence of HBV in oocytes and embryos. Three patterns of HBV serologic markers were screened. Twenty babies were anti-HBs-positive. Three babies were negative for HBsAg, antibody to hepatitis B surface antigen (anti-HBs), antibody to hepatitis B core antigen (anti-HBc), HBeAg, and antibody to hepatitis B e antigen (anti-HBe). One baby was seropositive for anti-HBs, anti-HBc, and anti-HBe at 6 months of age but seroconverted from anti-HBe-positive to anti-HBe-negative at 9 months of age. CONCLUSION(S): The presence of HBsAg in oocytes and embryos may not result in the vertical transmission of HBV in the offspring of HBV carriers.


Assuntos
Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B/metabolismo , Transmissão Vertical de Doença Infecciosa , Oócitos/metabolismo , Complicações Infecciosas na Gravidez/metabolismo , Desenvolvimento Embrionário/fisiologia , Feminino , Seguimentos , Hepatite B/diagnóstico , Hepatite B/transmissão , Antígenos de Superfície da Hepatite B/análise , Humanos , Lactente , Recém-Nascido , Masculino , Oócitos/virologia , Gravidez , Complicações Infecciosas na Gravidez/diagnóstico
13.
Biochem Biophys Res Commun ; 468(1-2): 179-84, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26522226

RESUMO

BACKGROUND: Clinical disorders caused by parvovirus B19 (B19V) infection include endothelial dysfunction with cardiac ischemia. The virus is effective in part by lysophosphatidylcholine-producing phospholipase A2 (PLA2) activity of B19V capsid protein VP1. Mechanisms compromising endothelial function include up-regulation of amiloride sensitive epithelial Na(+)-channel ENaC leading to endothelial cell stiffness. Regulators of ENaC include ubiquitin-ligase Nedd4-2. The present study explored whether VP1 modifies ENaC-activity. METHODS: cRNA encoding ENaC was injected into Xenopus oocytes without or with cRNA encoding VP1. Experiments were made with or without coexpression of Nedd4-2. ENaC activity was estimated from amiloride (50 µM) sensitive current. RESULTS: Injection of cRNA encoding ENaC into Xenopus oocytes was followed by appearance of amiloride sensitive current, which was significantly enhanced by additional injection of cRNA encoding VP1, but not by additional injection of cRNA encoding PLA2-negative VP1 mutant (H153A). The effect of VP1 on ENaC was mimicked by treatment of ENaC expressing oocytes with lysophosphatidylcholine (1 µg/ml). The effect of VP1 and lysophosphatidylcholine was not additive. ENaC activity was downregulated by Nedd4-2, an effect not reversed by VP1. CONCLUSIONS: The B19V capsid protein VP1 up-regulates ENaC, an effect at least partially due to phospholipase A2 (PLA) dependent formation of lysophosphatidylcholine.


Assuntos
Proteínas do Capsídeo/metabolismo , Canais Epiteliais de Sódio/metabolismo , Interações Hospedeiro-Patógeno , Infecções por Parvoviridae/metabolismo , Parvovirus B19 Humano/fisiologia , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Lisofosfatidilcolinas/metabolismo , Ubiquitina-Proteína Ligases Nedd4 , Oócitos/virologia , Infecções por Parvoviridae/virologia , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima , Proteínas de Xenopus , Xenopus laevis
14.
PLoS One ; 10(7): e0132212, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26177382

RESUMO

Bovine herpesvirus 4 (BoHV-4) is increasingly considered as responsible for various problems of the reproductive tract. The virus infects mainly blood mononuclear cells and displays specific tropism for vascular endothelia, reproductive and fetal tissues. Epidemiological studies suggest its impact on reproductive performance, and its presence in various sites in the reproductive tract highlights its potential transmission in transfer-stage embryos. This work describes the biological and genetic characterization of BoHV-4 strains isolated from an in vitro bovine embryo production system. BoHV-4 strains were isolated in 2011 and 2013 from granulosa cells and bovine oocytes from ovary batches collected at a local abattoir, used as "starting material" for in vitro production of bovine embryos. Compatible BoHV-4-CPE was observed in the co-culture of granulosa cells and oocytes with MDBK cells. The identity of the isolates was confirmed by PCR assays targeting three ORFs of the viral genome. The phylogenetic analyses of the strains suggest that they were evolutionary unlinked. Therefore it is possible that BoHV-4 ovary infections occurred regularly along the evolution of the virus, at least in Argentina, which can have implications in the systems of in vitro embryo production. Thus, although BoHV-4 does not appear to be a frequent risk factor for in vitro embryo production, data are still limited. This study reveals the potential of BoHV-4 transmission via embryo transfer. Moreover, the high variability among the BoHV-4 strains isolated from aborted cows in Argentina highlights the importance of further research on the role of this virus as an agent with the potential to cause reproductive disease in cattle. The genetic characterization of the isolated strains provides data to better understand the pathogenesis of BoHV-4 infections. Furthermore, it will lead to fundamental insights into the molecular aspects of the virus and the means by which these strains circulate in the herds.


Assuntos
Embrião de Mamíferos/virologia , Células da Granulosa/virologia , Herpesvirus Bovino 4/genética , Oócitos/virologia , Animais , Argentina , Teorema de Bayes , Bovinos , Células Cultivadas , Técnicas de Cocultura , DNA Viral/análise , Cães , Feminino , Células da Granulosa/citologia , Herpesvirus Bovino 4/classificação , Herpesvirus Bovino 4/isolamento & purificação , Células Madin Darby de Rim Canino , Oócitos/citologia , Fases de Leitura Aberta/genética , Filogenia , Análise de Sequência de DNA
15.
J Infect Dis ; 212 Suppl 1: S47-51, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26116732

RESUMO

Complete genomic reprogramming happens twice during the life of a genome, once during the formation of gametes in their parents and once after their union at fertilization. For that matter complete genomic reprogramming happens twice in the same parental cell, the oocyte, when it is forming and after it matures and receives the paternal gamete. Control of these processes in this unique single cell is epigenetic, and our understanding of it is based on information gleaned from imprinting, X chromosome inactivation, and activation and silencing of endogenous retroviruses (ERV). Activation of ERVs is attributed to demethylation of chromatin and DNA, whereas silencing requires methylation, attributed to the nuage proteins, which engage the Piwi-interacting RNA pathway and other posttranscriptional mechanisms. This reprogramming process has evolved throughout speciation because in mammals, but not fish, flies and worms, nuage-component muations affect male and female gametogenesis differentially. Transcription of ERVs is derepressed in both sexes in nuage-mutant mice, but whereas males are sterile, females are fertile. Using a proteomic approach we now report molecular interactions between nuage proteins and components of the oocyte cytoplasmic lattice and speculate how this interaction could preserve ERV/host chimeric gene products affecting female fertility.


Assuntos
Reprogramação Celular/genética , Retrovirus Endógenos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Oócitos/virologia , RNA Interferente Pequeno/genética , Animais , Retrovirus Endógenos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oogênese/genética , RNA Interferente Pequeno/metabolismo
16.
mBio ; 5(5): e01529-14, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25182324

RESUMO

UNLABELLED: The endosymbiotic bacteria of the genus Wolbachia are present in most insects and are maternally transmitted through the germline. Moreover, these intracellular bacteria exert antiviral activity against insect RNA viruses, as in Drosophila melanogaster, which could explain the prevalence of Wolbachia bacteria in natural populations. Wolbachia is maternally transmitted in D. melanogaster through a mechanism that involves distribution at the posterior pole of mature oocytes and then incorporation into the pole cells of the embryos. In parallel, maternal transmission of several endogenous retroviruses is well documented in D. melanogaster. Notably, gypsy retrovirus is expressed in permissive follicle cells and transferred to the oocyte and then to the offspring by integrating into their genomes. Here, we show that the presence of Wolbachia wMel reduces the rate of gypsy insertion into the ovo gene. However, the presence of Wolbachia does not modify the expression levels of gypsy RNA and envelope glycoprotein from either permissive or restrictive ovaries. Moreover, Wolbachia affects the pattern of distribution of the retroviral particles and the gypsy envelope protein in permissive follicle cells. Altogether, our results enlarge the knowledge of the antiviral activity of Wolbachia to include reducing the maternal transmission of endogenous retroviruses in D. melanogaster. IMPORTANCE: Animals have established complex relationships with bacteria and viruses that spread horizontally among individuals or are vertically transmitted, i.e., from parents to offspring. It is well established that members of the genus Wolbachia, maternally inherited symbiotic bacteria present mainly in arthropods, reduce the replication of several RNA viruses transmitted horizontally. Here, we demonstrate for the first time that Wolbachia diminishes the maternal transmission of gypsy, an endogenous retrovirus in Drosophila melanogaster. We hypothesize that gypsy cannot efficiently integrate into the germ cells of offspring during embryonic development in the presence of Wolbachia because both are competitors for localization to the posterior pole of the egg. More generally, it would be of interest to analyze the influence of Wolbachia on vertically transmitted exogenous viruses, such as some arboviruses.


Assuntos
Drosophila melanogaster/microbiologia , Drosophila melanogaster/virologia , Retrovirus Endógenos/fisiologia , Proteínas do Envelope Viral/metabolismo , Wolbachia/fisiologia , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Feminino , Masculino , Oócitos/microbiologia , Oócitos/virologia , Retroelementos , Simbiose , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas do Envelope Viral/genética , Replicação Viral
17.
Vopr Virusol ; 59(1): 42-6, 2014.
Artigo em Russo | MEDLINE | ID: mdl-25065146

RESUMO

The goal of this work was to study the capacity of the herpes simplex virus (HSV) of infecting ovary with disease in case of the intravaginal experimental animals. The results of the study demonstrated that the ascending HSV infection in mice lead to modification of all the cells of the ovary, including follicular cells synthesizing estrogen and progesterone. The two hormones influence the development of the disease. Estrogens provide the protective effects against the virus. Progesterone does not modify the body sensitivity to HSV, but reduces the effectiveness of the antiviral immunity, resulting in increased mortality of animals. We demonstrated that infection of oocytes in ovarian follicles of female mice during infection with HSV modified the process in vitro and for the first time demonstrated the detection of viral antigens in mature oocytes in patient with infertility. During the intracytoplasmic sperm injection into the infected oocytes (ICSI), the failure of fertilization was observed. These results are of interest, because there is no available literature on whether HSV infection of oocytes can have a direct negative impact on the process of fertilization in humans.


Assuntos
Herpes Simples/metabolismo , Herpesvirus Humano 1/metabolismo , Oócitos/virologia , Folículo Ovariano/virologia , Adulto , Animais , Estrogênios/farmacologia , Feminino , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos DBA , Oócitos/metabolismo , Oócitos/ultraestrutura , Folículo Ovariano/metabolismo , Folículo Ovariano/ultraestrutura , Progesterona/farmacologia , Progestinas/farmacologia
18.
Theriogenology ; 82(6): 790-9, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25062959

RESUMO

The present aimed to study if porcine circovirus type 2 (PCV2), which adhered to zona pellucida (ZP), was able to enter mature porcine oocytes with intact and damaged ZP. Four groups, including uninfected ZP-intact oocytes (UOZI), uninfected ZP-damaged oocytes (UOZD), PCV2-infected ZP-intact oocytes (POZI), and PCV2-infected ZP-damaged oocytes (POZD) were studied. The oocytes were incubated with 1 mL minimum essential medium, containing 3.1 × 10(8) copies of PCV2 DNA for 1 hour. Mechanical procedure of the insertion by microneedle induced injuries to the ZP of porcine oocytes. At the blastocyst stage, the percentage of PCV2-infected embryos and the ratio of viral antigen-positive cells per embryo were determined by indirect immunofluorescence. To assess the effect of ZP injury on the developmental competence and quality of porcine PCV2-infected oocytes after parthenogenetic activation, blastocyst formation rates and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining were analyzed. Moreover, real-time polymerase chain reaction was used to evaluate the expression of genes related to apoptosis and pluripotency at different developmental stages. The results of indirect immunofluorescence showed that only POZD group presented PCV2-infected embryos and viral-positive cells. The blastocyst rate of POZD group dropped down to approximately half of POZI group's (7.1 ± 1.5 vs. 14.5 ± 3.3). At the blastocyst stage, ZP injury increased apoptotic index of PCV2-infected embryos. The relative expression levels of Caspase 3 were higher in POZD group than the ones in POZI group at the two- and four-cell stages (not statistically significant). Compared with the one in POZI group, the ratio of antiapoptotic Bcl-xl gene to proapoptotic Bax gene, an indicator of the ability to resist apoptosis, was lower in POZD group at the one-cell stage, but higher at the two- and four-cell stages. Expression levels of Oct4 and Nanog associated with pluripotency were lower in POZD group than the ones in POZI group at the morula stage (not statistically significant). Noteworthily, the expression of Nanog was significantly lower in POZD group versus POZI group (P < 0.05), whereas relative expression of Oct4 was significantly higher in the former at the blastocyst stage (P < 0.01). In conclusion, PCV2, which attached to ZP, was able to enter mature porcine oocytes with damaged ZP and subsequently reduced the developmental competence and quality of the oocytes after parthenogenetic activation.


Assuntos
Circovirus/fisiologia , Oócitos/virologia , Partenogênese/fisiologia , Suínos/fisiologia , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Apoptose , Embrião de Mamíferos/virologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Zona Pelúcida
19.
Mol Cell Probes ; 28(4): 113-7, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24334246

RESUMO

In vitro-produced bovine embryos become infected after exposure to bovine Herpesvirus type 5 (BoHV-5), yet no changes in developmental rates, mitochondrial activity and inhibition of apoptosis are detected in comparison to unexposed embryos. Thus, the aim of the present study was to assess the transcription of mitochondria-mediated apoptosis genes using TaqMan real-time polymerase chain reaction. Transcripts of mcl-1, caspase-2, -3, Apaf-1 and Bax genes were measured after exposure to BoHV-5 in vitro. Mitochondrial dehydrogenase activity was evaluated by MTT test and compared between groups of exposed and unexposed embryos, at day 7 of development. The rate of oocyte maturation was assessed by the extrusion of the first polar body. In summary, BoHV-5 exposed embryos retained their viability, mitochondrial dehydrogenase activity and displayed up-regulation of transcription of survival mcl-1 gene and down-regulation of Bax transcription in relation to mitochondria-mediated pathway which might improve embryo viability. These findings demonstrate that BoHV-5 exposed embryos maintain their viability and mitochondrial dehydrogenase activity with no compromise of embryos produced in vitro.


Assuntos
Embrião de Mamíferos/citologia , Embrião de Mamíferos/virologia , Genes Mitocondriais , Infecções por Herpesviridae/patologia , Herpesvirus Bovino 5/fisiologia , Animais , Apoptose , Bovinos , Doenças dos Bovinos/embriologia , Doenças dos Bovinos/virologia , Regulação da Expressão Gênica no Desenvolvimento , Infecções por Herpesviridae/embriologia , Infecções por Herpesviridae/veterinária , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Oócitos/fisiologia , Oócitos/virologia , Reação em Cadeia da Polimerase em Tempo Real
20.
Vet Microbiol ; 165(3-4): 326-32, 2013 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-23726223

RESUMO

The aim of this study was to study the effect of Bovine Viral Diarrhea Virus on the reproductive female tract by means of analyzing the ovarian follicular population of persistently infected (PI) heifers, and evaluating the performance of oocytes procured form those heifers in in vitro fertilization procedures. Seven BVDV PI Aberdeen Angus and British crossbred heifers ranging from 18 to 36 months of age were spayed and their ovaries used for viral isolation, microscopic examination, and in vitro fertilization procedures. Bovine Viral Diarrhea Virus was detected from the follicular fluid and sera of all PI heifers. Microscopic examination of the ovaries from PI heifers showed a significant drop in the number of follicles cortical regions, compared with controls. A comparative analysis of the stages of follicular development showed a significant decrease in the number of primordial and tertiary follicles in the cortical regions of ovaries from PI heifers. Viral antigen was detected by immunohistochemistry, and was widely distributed throughout the ovarian tissues. There were differences in the rate of cleavage and embryo development between oocytes obtained from the ovaries of control animals and PI heifers. Furthermore, two developed embryos obtained from oocytes from one of the PI heifers were positive to BVDV, as well as two media from in vitro fertilization (IVF) procedures. The results of this study demonstrate that BVDV PI heifers exhibit alterations in follicular population through of the early interaction between the virus and germ cell line affecting directly the mechanisms involved in the ontogenesis of the ovary.


Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Vírus da Diarreia Viral Bovina/fisiologia , Ovário/patologia , Ovário/virologia , Animais , Anticorpos Neutralizantes/imunologia , Antígenos Virais/metabolismo , Doença das Mucosas por Vírus da Diarreia Viral Bovina/virologia , Bovinos , Vírus da Diarreia Viral Bovina/isolamento & purificação , Feminino , Fertilização In Vitro/veterinária , Oócitos/virologia , Reprodução/fisiologia
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