Characterization of Cetacean Morbillivirus in Humpback Whales, Brazil.

Cetacean morbillivirus is an etiologic agent associated with strandings of live and dead cetacean species occurring sporadically or as epizootics worldwide. We report 2 cases of cetacean morbillivirus in humpback whales (Megaptera novaeangliae) in Brazil and describe the anatomopathological, immunohistochemical, and molecular characterization findings in the specimens.

Molecular Analysis of Copy-Back Defective Interfering RNAs of Morbilliviruses.

Copy-back defective interfering RNAs are major contaminants of viral stock preparations of morbilliviruses and other negative strand RNA viruses. They are hybrid molecules of positive sense antigenome and negative sense genome. They possess perfectly complementary ends allowing the formation of extremely stable double-stranded RNA panhandle structures. The presence of the 3'-terminal promoter allows replication of these molecules by the viral polymerase. They thereby negatively interfere with replication of standard genomes. In addition, the double-stranded RNA stem structures are highly immunostimulatory and activate antiviral cell-intrinsic innate immune responses. Thus, copy-back defective interfering RNAs severely affect the virulence and pathogenesis of morbillivirus stocks. We describe two biochemical methods to analyze copy-back defective interfering RNAs in virus-infected samples, or purified viral RNA. First, we present our Northern blotting protocol that allows accurate size determination of defective interfering RNA molecules and estimation of the relative contamination level of virus preparations. Second, we describe a PCR approach to amplify defective interfering RNAs specifically, which allows detailed sequence analysis.

In Vitro Modeling of Feline Morbillivirus Infections Using Primary Feline Kidney Cells.

Domestic cats are the natural host of feline morbilliviruses (FeMV). Although other species can also be infected (such as dogs and opossums), no laboratory animal infection model is established so far. In vitro models for studying the molecular pathogenesis are therefore needed. For this purpose, propagation and titration of FeMV are key techniques. Unlike other morbilliviruses, such as canine distemper virus (CDV) or measles virus (MV), FeMV is a slow growing virus in cell culture and is difficult to titrate using classical plaque techniques. Here we describe methods for the efficient isolation of FeMV from natural sources (e.g., urine), the propagation of viral stocks, and their titration. In addition, we establish the generation of a three-dimensional infection model mimicking the feline tubular epithelium.

Generation of Defective Interfering Particles of Morbilliviruses Using Reverse Genetics.

RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource competition or by interferon stimulation are known as defective interfering (DI) genomes. DI genomes can be successfully packaged into virus-like-particles referred to as defective interfering particles (DIPs). Such DIPs can sustainably coexist with the full-length virus particles and have been shown to negatively impact virus replication in vitro and in vivo. Here, we describe a method to generate a clonal DI genome population by reverse genetics. This method is applicable to other RNA viruses and will enable assessment of DIPs for their antiviral properties.

Rational attenuation of canine distemper virus (CDV) to develop a morbillivirus animal model that mimics measles in humans.

Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimental MeV infections in non-human primates are restricted by ethical and practical constraints, and animal morbillivirus infections in natural host species have been considered as alternatives. Inoculation of ferrets with wild-type canine distemper virus (CDV) has been used for this purpose, but in most cases, the virus overwhelms the immune system and causes highly lethal disease. Introduction of an additional transcription unit and an additional attenuating point mutation in the polymerase yielded a candidate virus that caused self-limiting disease with transient viremia and virus shedding. This rationally attenuated CDV strain can be used for experimental morbillivirus infections in ferrets that reflect measles in humans.

Comparison between Sampling Techniques for Virological Molecular Analyses: Dolphin Morbillivirus and Herpesvirus Detection from FTA® Card and Frozen Tissue.

Stranded animals offer valuable information on marine mammal physiology and pathology; however, the decomposition state of the carcasses and lack of a rigorous cold chain for sample preservation can sometimes discourage diagnostic analyses based on nucleic acid detection. The present paper aims at evaluating the reliability of FTA® card tissue imprints as an alternative matrix to frozen tissues for virological analyses based on biomolecular methods. Given the contribution of Cetacean morbillivirus (CeMV) to strandings and the increase of herpesvirus detection in cetaceans, these two pathogens were selected as representative of RNA and DNA viruses. Dolphin morbillivirus (DMV) and herpesvirus presence was investigated in parallel on tissue imprints on FTA® cards and frozen tissues collected during necropsy of dolphins stranded in Italy. Samples were analysed by nested RT-PCR for DMV and nested-PCR for herpesvirus. Only one animal was positive for herpesvirus, hampering further considerations on this virus. DMV was detected in all animals, both in FTA® card imprints and tissue samples, with differences possibly related to the decomposition condition category of the carcasses. Tissue sampling on FTA® cards seems a promising alternative to frozen tissues for biomolecular analyses, especially when ensuring adequate storage and shipment conditions for frozen tissues is difficult.

Global genetic diversity of measles virus (Paramyxoviridae: Morbillivirus: Morbillivirus hominis): historical aspects and current state.

Monitoring the circulation of the measles virus and studying its genetic diversity is an important component of the measles elimination program. A methodological approach to molecular genetic studies and their interpretation in the measles surveillance was developed in the early 2000s. During its development, clear areas of circulation of each genotype of the virus were identified, therefore, the determination of viruses' genotypes was proposed to monitor circulation and identify transmission pathways. However, in the future, due to a significant decrease in the number of active genotypes, an approach based on sub-genotyping was proposed: determining not only the genotype of the virus, but also its genetic lineage/genetic variant. The Global Measles and Rubella Laboratory Network (GMRLN) systematically monitors the circulation of the measles virus at the sub-genotypic level, depositing the results in a specialized database MeaNS2. It is this database that is the most complete and reliable source of information about the genetic characteristic of measles viruses. This review presents both historical information and the latest data on the global genetic diversity of the measles virus.

4-hydroxy-2-nonenal as a marker of the oxidative stress in brains of dogs with canine distemper / 4-hidroxi-2-nonenal como marcador de estresse oxidativo no cérebro de cães com cinomose

Canine Distemper is a disease caused by Canine morbillivirus (CM), a pantropic virus that can affect the central nervous system (CNS), causing demyelination. However, the pathogenesis of this lesion remains to be clarified. Brain samples of 14 naturally infected dogs by CM were analyzed to evaluate the presence of oxidative stress and demyelination. RT-PCR assay was performed to confirm a diagnosis of canine distemper in the brain, immunohistochemistry anti-CM was used to localize the viral proteins in the tissue, and anti-4-hydroxy-2-nonenal (4-HNE) was a marker of a product of lipid peroxidation. The results showed the presence of viral proteins in the demyelinated area with the presence of 4-HNE. Our results suggest that the CM virus infection causes oxidative stress leading to lipid peroxidation, which causes tissue damage and demyelination. In conclusion, oxidative stress plays a significant role in canine distemper pathogenesis in the CNS.(AU)

A cinomose canina é uma doença causada pelo Morbilivírus canino (CM), um vírus pantrópico que pode afetar o sistema nervoso central (SNC), causando desmielinização. No entanto, a patogênese dessa lesão não está totalmente esclarecida. RT-PCR e imuno-histoquímica foram realizadas para confirmação do diagnóstico de cinomose em amostras de encéfalo de 14 cães naturalmente infectados. Após confirmação, foi realizada uma avaliação do estresse oxidativo por imuno-histoquímica com uso de anti-4-hidroxi-nonenal (4HNE) como marcador de produtos resultantes da peroxidação lipídica. Os resultados sugerem que a infecção pelo CM causa estresse oxidativo no tecido, levando a peroxidação lipídica, a qual causa danos ao tecido, culminando com desmielinização. Conclui-se que o estresse oxidativo tem papel importante na patogênese da cinomose canina no sistema nervoso central.(AU)

Sarampión: la enfermedad, epidemiología, historia y los programas de vacunación en Chile / Measles: the disease, epidemiology, history and vaccination programs in Chile

Measles, one of most important inmuno-preventable diseases, remains as a worldwide concern issue with an important morbidity and mortality. Particularly in the America region declared free of measles in 2010 by WHO, they still appear imported cases that origin outbreaks of variable magnitude in susceptible subjects usually none vaccinated which is the current situation in Santiago, the capital city of Chile. In this review we present characteristics of the etiological agent, the disease, epidemiological aspects with national historical focus, impact of immunization programs and outbreaks in Chile, in order to contribute to knowledge and management of this always present public health problem.

Sarampión una de las más importantes enfermedades inmuno-prevenibles, se mantiene como foco de preocupación en el mundo, con elevada carga de morbilidad y mortalidad. Particularmente en América, región declarada libre de esta enfermedad en 2010 por la OMS, se siguen identificando casos importados que originan brotes de diferente magnitud en personas susceptibles, habitualmente no vacunados, situación recientemente diagnosticada en Santiago, ciudad capital de Chile. En esta revisión, se presenta características del agente etiológico, de la enfermedad, aspectos epidemiológicos con foco en datos históricos nacionales, el impacto de los programas de vacunación, la ocurrencia de brotes en Chile, para contribuir al conocimiento y enfrentamiento de este siempre presente problema de salud pública.

Análise microscópica do miocárdio ventricular esquerdo em cães soropositivos para cinomose / Microscopic analysis of the left ventricular myocardium in positive serum dogs to distemper disease

Classificado no gênero Morbillivirus da família Paramixoviridae, o vírus da cinomose possui RNA de fita simples de polaridade negativa, é causador de doença multissistêmica, altamente contagiosa e grave dos cães e carnívoros selvagens, e com elevado índice de mortalidade em animais não vacinados ou com falhas vacinais. Com o objetivo de avaliar as alterações histopatológicas no coração, particularmente na região do miocárdio ventricular esquerdo, de cães naturalmente infectados com o vírus da cinomose, foram estudados 35 animais, de ambos os sexos e com idades variadas. Das 35 amostras enviadas ao Laboratório de Medicina Veterinária Preventiva do Hospital Veterinário de Uberaba, 100 por cento (35/35) mostrou-se soropositivas para a cinomose (técnica de imunoensaio em fase sólida) e tiveram no miocárdio ventricular esquerdo as seguintes alterações histopatológicas: miocardite, degeneração hialina, hiperemia e hemorragia, com 42,8 por cento (15/35), 31,4 por cento (11/35), 14,3 por cento (5/35) e 11,4 por cento (4/35), respectivamente. Tendo utilizado o teste Qui-Quadrado com nível de significância de 0,05, conclui-se que existe alta correlação (p=0,02) entre os animais infectados com o vírus da cinomose e as alterações histopatológicas observadas no miocárdio ventricular esquerdo.

Classified pertaining to the genus Morbillivirus of the Paramyxoviridae family, the canine distemper virus is a RNA single-stranded virus with negative polarity and causes a multisystemic disease, serious and highly contagious for dogs and wild carnivores, with a high mortality rate in non-vaccinated animals or with vaccine fails. With the objective to evaluate heart histopathological alterations, particularly in the left ventricular myocardium, in dogs naturally infected with canine distemper virus, 35 dogs, males and females of different ages, were studied. All the 35 samples sent to the Veterinary Hospital of Uberaba were serum-positive for distemper (immunoassay technique in solid phase) and had in the left ventricular myocardium the following histopathologic alterations: myocarditis, hyalin degeneration, hyperemia and hemorrhage, in 42.8 percent (15/35), 31.4 percent (11/35), 14.3 percent (5/35) and 11.4 percent (4/35), respectively. Having carried out the Qui-Quadrado test with a significancy level of 0.05, it can be concluded that there is a high correlation (p=0.02) between the infected animals with canine distemper virus and histopathological alterations found in the left ventricular myocardium.

Freqüência de sarampo, rubéola, dengue e eritema infeccioso entre casos suspeitos de sarampo e rubéola no estado de Pernambuco, no período de 2001 a 2004 / Frequency of measles, rubella, dengue and erythema infectiosum among suspected cases of measles and rubella in the State of Pernambuco between 2001 and 2004

O estudo teve como objetivo verificar a freqüência de sarampo, rubéola, dengue e eritema infeccioso entre casos suspeitos de sarampo e rubéola, no estado de Pernambuco, Brasil. Foram testadas 1.161 amostras de soro coletadas no período de 2001 a 2004, para as quatro viroses, utilizando-se ensaios imunoenzimáticos para detecção de anticorpos IgM. Desse total, 276 (23,8 por cento) amostras foram positivas para uma das quatro viroses analisadas. Foram detectados 196 (16,9 por cento) casos positivos para dengue, 38 (3,3 por cento) para eritema infeccioso (parvovírus B19), 32 (2,8 por cento) para rubéola e 10 (0,9 por cento) para sarampo. Entre os casos suspeitos de sarampo e rubéola, a infecção pelo vírus dengue foi a mais freqüente, seguida pelo parvovírus B19. A semelhança de manifestações clínicas entre as doenças exantemáticas contribui para dificultar o diagnóstico de sarampo, rubéola, dengue e eritema infeccioso, quando observados apenas os critérios clínicos. Deve-se salientar que os quatro testes utilizados foram insuficientes para diagnosticar 76,2 por cento das doenças febris exantemáticas notificadas. Este é o primeiro estudo que evidencia a circulação de parvovírus B19 humano em Pernambuco.

This study had the aim of investigating the frequency of measles, rubella, dengue and erythema infectiosum among suspected cases of measles and/or rubella in the state of Pernambuco, Brazil. A total of 1,161 serum samples collected between 2001 and 2004 were tested for these four viral diseases, using enzyme immunoassays to detect IgM antibodies. Out of this total, 276 (23.8 percent) samples were positive for one of the four viral diseases analyzed. There were 196 positive cases (16.9 percent) for dengue, 38 (3.3 percent) for erythema infectiosum (parvovirus B19), 32 (2.8 percent) for rubella and 10 (0.9 percent) for measles. Among the suspected cases of measles and rubella, dengue infection was the most frequent, followed by parvovirus B19. The similarity of the clinical manifestations among rash diseases contributes towards making it difficult to diagnose measles, rubella, dengue and erythema infectiosum on clinical grounds alone. It must be emphasized that the four tests used were insufficient for diagnosing 76.2 percent of the febrile and rash diseases notified. This is the first study bringing evidence of human parvovirus B19 circulation in Pernambuco.

Serological surveillance of measles in blood donors in Rio de Janeiro, Brazil / Vigilancia serológica de sarampión en donantes de sangre de Río de Janeiro, Brasil

OBJETIVOS: Determinar la proporción de personas seronegativas a anticuerpos contra el sarampión entre los donantes de un banco de sangre de la ciudad de Río de Janeiro, Brasil, y describir sus características sociales y demográficas, con el fin de explorar el uso eventual de los bancos de plasma para complementar la vigilancia serológica con datos que permitan apoyar el programa de erradicación del sarampión en la ciudad de Río de Janeiro y en otras localidades. MÉTODOS: Se estudió la presencia de inmunoglobulinas G contra el sarampión en muestras de plasma de 1 101 donaciones de sangre consecutivas realizadas en noviembre de 2000 en HEMORIO, el mayor banco de sangre del estado de Río de Janeiro, mediante un ensayo inmunoenzimático comercial y una prueba de neutralización por reducción de placas. Se calculó la proporción (y el intervalo de confianza (IC) de 95 por ciento) de las muestras negativas a anticuerpos contra el sarampión en la totalidad de las donaciones y en subgrupos conformados según la edad, el sexo, el vecindario de residencia, la educación y la ocupación laboral. La significación estadística de las diferencias entre las proporciones y entre sus tendencias lineales se determinó mediante la prueba de ji al cuadrado. RESULTADOS: Del total de donantes de sangre, el 6,9 por ciento (IC95 por ciento: 5,4 a 8,4 por ciento) resultó seronegativo al sarampión. Las mujeres tuvieron una mayor proporción de resultados seronegativos (10,1 por ciento; IC95 por ciento: 6,8 a 13,4 por ciento) que los hombres (5,6 por ciento; IC95 por ciento: 4,0 a 7,2 por ciento). En relación con la edad, 86,8 por ciento de las personas seronegativas nacieron entre 1971 y 1982. La seronegatividad fue inversamente proporcional a la edad (ji al cuadrado = 58,0; P < 0,0001). En cuanto a la ocupación laboral, los estudiantes presentaron la mayor proporción de seronegativos (17,8 por ciento), mientras que en lo referente a la educación, la mayoría de las personas susceptibles a la enfermedad se encontraban en la categoría de "estudios universitarios incompletos" o "estudios secundarios incompletos". La proporción de seronegativos entre las diferentes áreas de residencia estuvo entre 2,1 por ciento y 11,4 por ciento. CONCLUSIONES: El plasma de los bancos de sangre puede ser una fuente útil y conveniente de datos complementarios para la vigilancia serológica en adultos, tanto de sarampión como de otras enfermedades infecciosas para las que se hayan implementado...

Objetivos. Determinar la proporción de personas seronegativas a anticuerpos contra el sarampión entre los donantes de un banco de sangre de la ciudad de Río de Janeiro, Brasil, y describir sus características sociales y demográficas, con el fin de explorar el uso eventual de los bancos de plasma para complementar la vigilancia serológica con datos que permitan apoyar el programa de erradicación del sarampión en la ciudad de Río de Janeiro y en otras localidades. Métodos. Se estudió la presencia de inmunoglobulinas G contra el sarampión en muestras de plasma de 1 101 donaciones de sangre consecutivas realizadas en noviembre de 2000 en HEMORIO, el mayor banco de sangre del estado de Río de Janeiro, mediante un ensayo inmunoenzimático comercial y una prueba de neutralización por reducción de placas. Se calculó la proporción (y el intervalo de confianza (IC) de 95%) de las muestras negativas a anticuerpos contra el sarampión en la totalidad de las donaciones y en subgrupos conformados según la edad, el sexo, el vecindario de residencia, la educación y la ocupación laboral. La significación estadística de las diferencias entre las proporciones y entre sus tendencias lineales se determinó mediante la prueba de ji al cuadrado. Resultados. Del total de donantes de sangre, el 6,9% (IC95%: 5,4 a 8,4%) resultó seronegativo al sarampión. Las mujeres tuvieron una mayor proporción de resultados seronegativos (10,1%; IC95%: 6,8 a 13,4%) que los hombres (5,6%; IC95%: 4,0 a 7,2%). En relación con la edad, 86,8% de las personas seronegativas nacieron entre 1971 y 1982. La seronegatividad fue inversamente proporcional a la edad (ji al cuadrado = 58,0; P < 0,0001). En cuanto a la ocupación laboral, los estudiantes presentaron la mayor proporción de seronegativos (17,8%), mientras que en lo referente a la educación, la mayoría de las personas susceptibles a la enfermedad se encontraban en la categoría de "estudios universitarios incompletos" o "estudios secundarios incompletos". La proporción de seronegativos entre las diferentes áreas de residencia estuvo entre 2,1% y 11,4%. Conclusiones. El plasma de los bancos de sangre puede ser una fuente útil y conveniente de datos complementarios para la vigilancia serológica en adultos, tanto de sarampión como de otras enfermedades infecciosas para las que se hayan implementado actividades de inmunización y vigilancia. Este enfoque puede ser beneficioso en otras regiones de Brasil y en otros países en los que se pueda contar para la vigilancia con el plasma de los donantes de sangre. El empleo del remanente de los sueros de los pacientes y del plasma de los donantes de sangre representa una solución de compromiso entre la representatividad y la oportunidad, y el buen uso de los recursos