Investigation of apoptosis in cultured cells infected with equine herpesvirus 1.

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin-Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris.

The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation. The G-protein expression was evaluated by SDS-PAGE, ELISA, and western blot assays. Like native G-protein, the recombinant G-protein was found reactive when it was challenged against specific antibodies. The data indicate that the recombinant G-protein can be easily expressed and isolated, and may be useful as a safe source in the production of diagnostic kits and subunit vaccines to prevent rabies.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1.

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.

Protective effects of intranasal immunization with recombinant glycoprotein d in pregnant BALB/c mice challenged with different strains of equine herpesvirus 1.

Equine herpesvirus (EHV)-1 induces respiratory infection, neurological disorders and abortion in horses. Most of the currently available attenuated or inactivated vaccines against this infection are administered intramuscularly and only provide partial protection against the respiratory disease. The present study examines the effect of intranasal immunization with purified EHV-1 recombinant glycoprotein D (gD) in BALB/c mice followed by challenge with three different EHV-1 strains during early to mid-pregnancy. The induced viral infection was evaluated by virus isolation, DNA detection by polymerase chain reaction, histopathology and immunohistochemical localization of antigen in the lung, placenta and uterus. Non-immunized mice showed clinical signs of infection, positive virus isolation from lungs and uteri, and abortion induced by one of the virus strains. Endometrial lesions developed in some of these animals that have been described previously only in horses. Immunized mice and their offspring had no viral infection or typical lesions. Intranasally administered gD therefore induced partial or complete protection against three different EHV-1 strains in BALB/c mice.

First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America / Primeira detecção molecular de co-infecção de vírus de abelhas em Bombus atratus assintomática na América do Sul

Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees.

A polinização é essencial para a produção de alimentos e tem como particularidade a conexão entre os ecossistemas naturais com sistemas de produção agrícola. Recentemente, as perdas de espécies de bumblebee em todo o mundo têm sido relatadas. Neste trabalho, amostras de uma exploração comercial de bumblebee da Argentina, com recente história de mortes foram estudadas utilizando uma Multiplex PCR para a detecção de vírus de abelha mais frequentemente detectados na América do Sul. Todas as amostras analisadas foram positivas para as co-infecções com Deformed wing virus, Black queen cell viruses e Sacbrood virus. Este trabalho descreve o primeiro relato de infecção de Bombus atratus com vírus de abelhas. Uma melhor compreensão das infecções virais em bumblebee e da epidemiologia dos vírus poderia ser de grande importância, uma vez que tais abelhas podem servir como reservatório viral, com possível repercussão tanto na produtividade de abelhas melíferas como afetando-as diretamente.

Systemic infection induced by intranasal inoculation of Bovine herpesvirus 1.1 in pregnant and non-pregnant rabbits.

Bovine herpesvirus (BoHV) type 1.1 (BoHV-1.1) causes repeated outbreaks of upper respiratory disease and abortion in cattle. The systemic effects of BoHV-1.1 in rabbits, using intranasal inoculation are reported. Female rabbits were divided into four groups and inoculated with the virus 10 days before mating, and at 15 or 22 days of pregnancy. Studies of the clinical signs, antibody production, virus isolation, and DNA detection as well as histological and immunohistochemical studies were carried out on lungs, kidneys, spleen, placentas, uteri and foetal tissues. All virus-inoculated animals developed respiratory clinical signs and a humoral response. BoHV-1.1 was isolated from nasal swabs and plasma rich in leukocytes, and viral DNA was detected in blood, dead foetuses and placentas. Histopathological lesions were found in the respiratory tract and some placentas and foetuses were immunohistochemically positive. Intranasal inoculation might be useful to study the systemic effects of BoHV-1.1 infection in the rabbit model.

Effects of different anesthetics in the murine model of EHV-1 infection.

Mice are commonly used as an experimental model to investigate the Equid herpesvirus 1 (EHV-1) infection. This model easily reproduces the disease, and the clinical signs are more or less similar to those observed in the horse, the natural host. During natural infection, the acute course of respiratory infection is mandatory for the development of adaptive immune response. Since interactions between EHV-1 and anesthetics are possible, the study investigated whether the early events of murine pulmonary immune response could be affected by different anesthetics. Therefore, mice were experimentally infected with a unique EHV-1 strain under the effects of ether, ketamine/xylazine, or isoflurane. Clinical signs and histopathological lesions in the lungs were described, and the cell death and proliferation rates of sham-inoculated or infected animals were quantified using immunohistochemistry. Clinical signs were more severe in animals anesthetized with ether. Qualitative differences in the recruited inflammatory cells were observed following application of anesthesia. The level of infection between the infected groups was not statistically significant. However, lungs from ketamine/xylazine-anesthetized animals showed the highest cell death rates, whereas those from isoflurane-anesthetized animals showed the highest proliferation rates. It has been emphasized that anesthetics alone or their interactions with EHV-1 modify the response against the infection. An appropriate selection of the anesthetic during experimental studies is relevant to minimize wrong conclusions.

First molecular detection of co-infection of honey bee viruses in asymptomatic Bombus atratus in South America.

Pollination is critical for food production and has the particularity of linking natural ecosystems with agricultural production systems. Recently, losses of bumblebee species have been reported worldwide. In this study, samples from a commercial exploitation of bumblebees of Argentina with a recent history of deaths were studied using a multiplex PCR for the detection of the honey bee viruses most frequently detected in South America. All samples analysed were positive for co-infections with Deformed wing virus, Black queen cell virus and Sacbrood virus. This is the first report of infection of Bombus atratus with honey bee viruses. A better understanding of viral infections in bumblebees and of the epidemiology of viruses could be of great importance as bumblebees can serve as possible viral reservoirs, resulting in pathogen spillover towards honey bees and native bumblebees.

An atypical clinical presentation for the first isolation of canid herpesvirus 1 in Argentina / Uma apresentação clínica atípica para o primeiro isolamento do herpesvirus canino 1 na Argentina

Relatam-se o primeiro isolamento de herpesvirus canino 1 (CaHV-1) e a localização atípica das lesões vesiculares associadas a este vírus na Argentina. A amostra foi recuperada de lesões vesiculares, localizadas na parte interna da coxa direita, em uma fêmea de raça Labrador. A cadela tinha quatro anos de idade e era de propriedade privada. O primeiro diagnóstico foi realizado pela reação em cadeia da polimerase e, posteriormente, o vírus foi isolado e sua identificação confirmada por imunofluorescência indireta e pelo teste de neutralização viral.

Prevalence of antibodies against Kilham virus in experimental rat colonies of Argentina.

The Kilham rat virus (KRV) is a parvovirus originally isolated from a rat sarcoma in the late 1950s. The clinical signs associated with a natural KRV infection include foetal resorption in dams, runting, ataxia, cerebellar hypoplasia and jaundice in suckling rats, and sudden death, scrotal cyanosis, abdominal swelling and dehydration in juvenile rats. The ability of this virus to produce persistent infections has resulted in a high frequency of contamination of cell cultures and transplantable-tumor system. In addition, the virus may interfere with research in other ways. The remarkable resistance to environmental conditions determines the importance of the detection and control of this agent, especially in the laboratory animal production. This study determines the seroprevalence of Kilham antibodies from sera of adult rats from conventional facilities, using the haemagglutination inhibition test. The seroprevalence varied between 27.8% and 75%. This result confirms that the virus is circulating in Argentinean conventional facilities and might be interfering with research. The recognized Kilham virus may be prevented from supply sources by implementing a health monitoring schedule including a regular serological surveillance, and by keeping the animals under barrier systems.

Complete genome amplification of equine influenza virus subtype 2.

This work reports a method for rapid amplification of the complete genome of equine influenza virus subtype 2 (H3N8). A ThermoScript reverse transcriptase instead of the avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase was used. This enzyme has demonstrated higher thermal stability and is described as suitable to make long cDNA with a complex secondary structure. The product obtained by this method can be cloned, used in later sequencing reactions or nested-PCR with the purpose of achieving a rapid diagnosis and characterization of the equine influenza virus type A. This detection assay might be a valuable tool for diagnosis and screening of field samples as well as for conducting molecular studies.

Genomic and phylogenetic analysis of Argentinian Equid Herpesvirus 1 strains.

Equid Herpesvirus 1 (EHV-1) has long been causally implicated in the occurrence of abortion, neonatal death, respiratory disease, and neurological disorders in horses. This study analyzed for the first time the characteristics of the genomic section of Argentinian EHV-1 strains and reconstructed the phylogeny in order to establish their origin. The phylogenetic dataset included 22 Argentinian strains and four additional reference strains isolated in other countries. The intergenic region between ORF 62 and ORF 63 was amplified by PCR and sequenced. The phylogenetic analysis carried out by parsimony algorithms showed that six of the Argentinian strains had the same origin as British and Japanese strains. The mapping of symptoms caused by EHV-1 suggested that neonatal disease developed through convergent evolution, which would constitute an adaptation mechanism of the virus. This study constitutes the first analysis carried out in South-American strains that establishes the phylogenetic relationship between Argentinian strains and rebuilds the evolutionary history of symptoms. This study focuses on a very important aspect of evolution of Herpesviridae infecting perissodactyls and attempts to shed light on the evolution of symptoms, an issue of high clinical interest.

Immunohistochemical and molecular detection of equine herpesvirus 1 in Uruguay.

Equine herpesvirus 1 (EHV-1) is a major cause of epidemic abortion, neonatal mortality, respiratory disease and neurological disorders in horses. In South America, the virus has been isolated in Brazil, Argentina and Colombia. In Chile pathological findings from one aborted foetus have been reported, and in Uruguay only serological data about EHV-1 activity have been found. Some pathological findings were reported in Uruguay several years ago, but these data have never been officially confirmed. The present work describes the relevant findings of a study of EHV-1 infections in the Uruguayan equine population using polymerase chain reaction (PCR) and histological and immunohistochemical analysis techniques. The sequence analysis of a portion of the glycoprotein C gene amplified by PCR confirmed EHV-1 activity. The real-time PCR revealed the association of the virus with the non-neuropathogenic genotype. This study describes for the first time the immunohistochemical and molecular detection of EHV-1 in Uruguay.

A simple method of infecting rabbits with Bovine herpesvirus 1 and 5.

This report describes an alternative technique to inoculate rabbits and to reproduce infection by Bovine herpesvirus 1 and 5. First, the nostrils are anaesthetized by aspersion with local anaesthetic. A few seconds later, and after proving the insensitivity of the zone, the rabbits are put on their back legs with their nostrils upwards and the inoculum is introduced slowly into each nostril by using disposable droppers. Clinical signs, viral isolation from nasal swabs, histological lesions found, positive polymerase chain reaction and antibodies production confirm the infection. This very simple and bloodless technique, where the animals are exposed to minor distress, may be useful for evaluating the virulence of BoHV-1 and BoHV-5 strains, to study the establishment of latent virus infection and to test the potential of experimental vaccines or properties of antiviral drugs. It may be also suitable for experimental infection with other respiratory viruses in this animal model.

Equine herpesvirus 1: characterisation of the first strain isolated in Colombia.

This paper describes the isolation and characterisation of equine herpesvirus 1 (EHV-1) in Colombia. The virus was isolated from a nasal swab and an aborted foetus of a pregnant mare imported from Argentina, with clinical signs of rhinopneumonitis. The new strain was characterised through culture and morphological, serological and immunocytochemical studies. Polymerase chain reaction and DNA restriction maps revealed an EHV-1 1P genome. This is the first report on the isolation and characterisation of EHV-1 in Colombia.

An Argentine equine herpesvirus strain with special restriction patterns protect mice challenged with a pathogenic strain.

Equine herpesvirus 1 (EHV-1) was first isolated in Argentina in 1979. This strain SPv has special restriction patterns, but a previous study demonstrated that SPv did not modify its growth in cell culture. In addition, it showed low virulence in the mouse respiratory model consistently with results found in female BALB/C at different state of gestation. This study evaluates in a mouse respiratory model, if primary infection with SPv strain protects animals from subsequent challenge with a pathogenic strain. Body weight loss was not observed in mice intranasally inoculated with SPv strain and challenged with HH1 Japanese strain. The SPv primary infection does not completely prevent clinical presentation by HH1 infection but the SPv inoculated animals recovered more quickly, with less intense and less persistent histological lesions. The challenge infection caused a rapid and prolonged increase in anti-EHV-1 antibodies in the mice previously infected with SPv, along with a more rapid reduction of viral titres in lungs. In this work it was demonstrated that this EHV-1 strain constitute a good immunogen. These results show that this SPv strain could be considered to produce an EHV-1 vaccine.

Detection of equine herpesvirus 1 genome 1B in Argentina.

To determine the genomic variation of equine herpesviruses (EHVs) isolated in Argentina between 1979 and the first half of 2004, DNA sequences from all 69 strains isolated were analysed. Sixty strains were recovered from aborted fetuses, one from leucocyte-rich plasma from a horse with respiratory signs and eight from cases of neonatal disease. The DNA was extracted from rabbit kidney epithelial (RK13) cells infected with each strain and digested with three restriction endonucleases (BamHI, Bg/II and KpnI). Two strains could be differentiated using BamHI restriction and were assigned to the EHV-1 1B prototype group. Only one of these two strains was typed EHV-1 1B with Bg/II. DNA digestion with KpnI was ineffective. The results obtained in this study demonstrate that the EHV-1 1B genome has been present in Argentina since at least 1996. The finding of two strains with this electropherotype suggests that there is genomic heterogeneity among Argentinian isolates.

Argentine strain of equine herpesvirus 1 isolated from an aborted foetus shows low virulence in mouse respiratory and abortion models.

The equine herpesvirus 1 (EHV-1) was isolated in Argentina from an aborted equine foetus in 1979. This virus (SPv) has special restriction patterns (RP) in comparison with other Argentine isolates. In addition, SPv could be distinguished on the basis of its pathogenicity in baby mice inoculated intracerebrally. We studied the growth properties of the SPv in cell culture and its effects in a mouse respiratory and abortion model. We observed that SPv did not modify its capacity to grow in cell culture with respect to reference HH1 strain. Nevertheless, we found significant differences between the titres of the two strains at 8-14 h post-infection (PI). In this work we demonstrated that SPv showed low virulence in female at different stages of gestation, consistently, with results found in the mouse respiratory model. We considered that this low virulence of SPv could be related to its RP because the RP of HH1 strain are similar to those of the HVS25A strain and both showed effect on pregnant mice. More specific studies about genomic alterations to the SPv are necessary for identifying, more clearly, if the intra-strain variations have relation with the low virulence in the mouse respiratory and abortion model.

[Development of an antigen for the diagnosis of Kilham rat parvovirus by hemagglutination inhibition test]. / Desarrollo de un antígeno para diagnóstico del parvovirus de las ratas (virus Kilham) por la técnica de inhibición de la hemaglutinación.

An antigen of rat parvovirus (Kilham virus) was developed for the diagnosis of viral infection in rat colonies by using hemagglutination inhibition (HAI) test. Primary cell cultures from rat embryos were infected with Kilham rat virus. Infected cells obtained at different time post infection were scraped, centrifuged, concentrated one hundred times, sonicated and centrifuged again. The supernatants obtained were titrated by hemagglutination. The specificity was confirmed with positive and negative reference sera. Ninety eight serum samples were studied by using HAI test. The results coincided with those obtained in a reference laboratory. Kilham rat parvovirus antigen obtained from 5 days-infected-cells was specific, sensitive, easy to prepare, with a high yield and it is useful to detect this virus in experimental and production rat colonies.

Desarrollo de un antígeno para diagnóstico del parvovirus de las ratas (virus Kilham) por la técnica de inhibición de la hemaglutinación / [Development of an antigen for the diagnosis of Kilham rat parvovirus by hemagglutination inhibition test]

An antigen of rat parvovirus (Kilham virus) was developed for the diagnosis of viral infection in rat colonies by using hemagglutination inhibition (HAI) test. Primary cell cultures from rat embryos were infected with Kilham rat virus. Infected cells obtained at different time post infection were scraped, centrifuged, concentrated one hundred times, sonicated and centrifuged again. The supernatants obtained were titrated by hemagglutination. The specificity was confirmed with positive and negative reference sera. Ninety eight serum samples were studied by using HAI test. The results coincided with those obtained in a reference laboratory. Kilham rat parvovirus antigen obtained from 5 days-infected-cells was specific, sensitive, easy to prepare, with a high yield and it is useful to detect this virus in experimental and production rat colonies.