Mumps virus genotypes identified during disease outbreaks in the state of São Paulo, Brazil: 2011-2016 / Genótipos dos vírus da caxumba identificados durantesurtos da doença no estado de São Paulo, Brasil: 2011 - 2016

In São Paulo the mumps virus (MuV) outbreaks have been increasing from In São Paulo the mumps virus (MuV) outbreaks have been increasing from 2011 to nowadays. MuV epidemiological surveillance has been improving by using the polymerase chain reaction in real time (rRT-PCR) in addition to the specific IgM antibody (IgM-Ab) detection; in some cases,genome sequencing studies were performed. Increased virus transmission and recent outbreakshave raised interest on MuV genotyping, as a means to understand the transmission pathways andto identify the vaccine-associated cases. From January 2011 to August 2016, MuV infection was analyzed at Institute Adolfo Lutz. A total of 232 (77.33 %) throat wash samples showed positivity to mumps genome, and 68 (22.66 %) were negative when analyzed by rRT-PCR. Among 15 samples for molecular analysis, 10 serum samples from respective patients were also available for detecting anti-MuV IgM-Ab; and from these, four (40%) samples were seropositive. Vaccination statuswas available only for patients from Cedral and Araraquara. Phylogenetic analysis revealed the circulation of the following mumps virus genotypes in the investigated periods: 2011(M), 2012, and 2013 (K); 2014 (N); 2015 (G, K, and N); 2016 (G). Knowledge on MuV molecular epidemiology in São Paulo-Brazil could contribute to the surveillance and epidemiological program in Brazil, and globally as well.

No estado de São Paulo têm ocorrido surtos de caxumba desde 2011. O diagnóstico laboratorialtem sido realizado no Instituto Adolfo Lutz utilizando-se a técnica de identificação de material genético viral por meio de reação de cadeia de polimerase-em tempo real (rRT-PCR) e peladetecção de anticorpos IgM (Ac-IgM) específicos circulantes. Os recentes surtos de caxumbatêm aumentado o interesse em investigar os genótipos dos vírus prevalentes para identificaros casos associadas à vacina. De janeiro de 2011 a agosto de 2016, 300 amostras de lavadosda orofaringe coletadas de pacientes suspeitos de infecção foram analisadas. O material genéticoviral específico foi detectado em 232 (77,33 %) amostras e 68 (22,66 %) foram negativas.Das 10 amostras analisadas pelo teste sorológico, quatro (40 %) demonstraram positividadepara Ac-IgM específicos anti-vírus da caxumba e seis foram negativas. Somente os municípios Cedral e Araraquara forneceram os dados referentes à vacinação. Análise filogenética mostroua circulação dos seguintes genótipos do vírus da caxumba no período investigado: 2011 (M),2012 e 2013 (K); 2014 (N); 2015 (GKN); 2016 (G). A vigilância virológica é mundialmente imprescindível, para identificar a diversidade e a distribuição dos diferentes genótipos, com vistasà composição de vacinas específicas.

Mumps virus genotypes identified during disease outbreaks in the state of São Paulo, Brazil: 2011 – 2016 / Genótipos dos vírus da caxumba identificados durante surtos da doença no estado de São Paulo, Brasil: 2011 - 2016

In São Paulo the mumps virus (MuV) outbreaks have been increasing from 2011 to nowadays. MuV epidemiological surveillance has been improving by using the polymerase chain reaction in real time (rRT-PCR) in addition to the specific IgM antibody (IgM-Ab) detection; in some cases, genome sequencing studies were performed. Increased virus transmission and recent outbreaks have raised interest on MuV genotyping, as a means to understand the transmission pathways and to identify the vaccine-associated cases. From January 2011 to August 2016, MuV infection was analyzed at Institute Adolfo Lutz. A total of 232 (77.33 %) throat wash samples showed positivity to mumps genome, and 68 (22.66 %) were negative when analyzed by rRT-PCR. Among 15 samples for molecular analysis, 10 serum samples from respective patients were also available for detecting anti-MuV IgM-Ab; and from these, four (40%) samples were seropositive. Vaccination status was available only for patients from Cedral and Araraquara. Phylogenetic analysis revealed the circulation of the following mumps virus genotypes in the investigated periods: 2011(M), 2012, and 2013 (K); 2014 (N); 2015 (G, K, and N); 2016 (G). Knowledge on MuV molecular epidemiology in São Paulo-Brazil could contribute to the surveillance and epidemiological program in Brazil, and globally as well.

No estado de São Paulo têm ocorrido surtos de caxumba desde 2011. O diagnóstico laboratorial tem sido realizado no Instituto Adolfo Lutz utilizando-se a técnica de identificação de material genético viral por meio de reação de cadeia de polimerase-em tempo real (rRT-PCR) e pela detecção de anticorpos IgM (Ac-IgM) específicos circulantes. Os recentes surtos de caxumba têm aumentado o interesse em investigar os genótipos dos vírus prevalentes para identificar os casos associadas à vacina. De janeiro de 2011 a agosto de 2016, 300 amostras de lavados da orofaringe coletadas de pacientes suspeitos de infecção foram analisadas. O material genético viral específico foi detectado em 232 (77,33 %) amostras e 68 (22,66 %) foram negativas. Das 10 amostras analisadas pelo teste sorológico, quatro (40 %) demonstraram positividade para Ac-IgM específicos anti-vírus da caxumba e seis foram negativas. Somente os municípios Cedral e Araraquara forneceram os dados referentes à vacinação. Análise filogenética mostrou a circulação dos seguintes genótipos do vírus da caxumba no período investigado: 2011 (M), 2012 e 2013 (K); 2014 (N); 2015 (GKN); 2016 (G). A vigilância virológica é mundialmente imprescindível, para identificar a diversidade e a distribuição dos diferentes genótipos, com vistas à composição de vacinas específicas.

Mumps virus genotypes identified during disease outbreaks in the state of São Paulo, Brazil: 2011 2016 / Genótipos dos vírus da caxumba identificados durante surtos da doença no estado de São Paulo, Brasil: 2011 - 2016

In São Paulo the mumps virus (MuV) outbreaks have been increasing from 2011 to nowadays. MuV epidemiological surveillance has been improving by using the polymerase chain reaction in real time (rRT-PCR) in addition to the specific IgM antibody (IgM-Ab) detection; in some cases, genome sequencing studies were performed. Increased virus transmission and recent outbreaks have raised interest on MuV genotyping, as a means to understand the transmission pathways and to identify the vaccine-associated cases. From January 2011 to August 2016, MuV infection was analyzed at Institute Adolfo Lutz. A total of 232 (77.33 %) throat wash samples showed positivity to mumps genome, and 68 (22.66 %) were negative when analyzed by rRT-PCR. Among 15 samples for molecular analysis, 10 serum samples from respective patients were also available for detecting anti-MuV IgM-Ab; and from these, four (40%) samples were seropositive. Vaccination status was available only for patients from Cedral and Araraquara. Phylogenetic analysis revealed the circulation of the following mumps virus genotypes in the investigated periods: 2011(M), 2012, and 2013 (K); 2014 (N); 2015 (G, K, and N); 2016 (G). Knowledge on MuV molecular epidemiology in São Paulo-Brazil could contribute to the surveillance and epidemiological program in Brazil, and globally as well.(AU)

No estado de São Paulo têm ocorrido surtos de caxumba desde 2011. O diagnóstico laboratorial tem sido realizado no Instituto Adolfo Lutz utilizando-se a técnica de identificação de material genético viral por meio de reação de cadeia de polimerase-em tempo real (rRT-PCR) e pela detecção de anticorpos IgM (Ac-IgM) específicos circulantes. Os recentes surtos de caxumba têm aumentado o interesse em investigar os genótipos dos vírus prevalentes para identificar os casos associadas à vacina. De janeiro de 2011 a agosto de 2016, 300 amostras de lavados da orofaringe coletadas de pacientes suspeitos de infecção foram analisadas. O material genético viral específico foi detectado em 232 (77,33 %) amostras e 68 (22,66 %) foram negativas. Das 10 amostras analisadas pelo teste sorológico, quatro (40 %) demonstraram positividade para Ac-IgM específicos anti-vírus da caxumba e seis foram negativas. Somente os municípios Cedral e Araraquara forneceram os dados referentes à vacinação. Análise filogenética mostrou a circulação dos seguintes genótipos do vírus da caxumba no período investigado: 2011 (M), 2012 e 2013 (K); 2014 (N); 2015 (GKN); 2016 (G). A vigilância virológica é mundialmente imprescindível, para identificar a diversidade e a distribuição dos diferentes genótipos, com vistas à composição de vacinas específicas.(AU)

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

Diversidad antigénica de las vacunas antiparotiditicas / Antigenic diversity of vaccines againts mumps

La parotiditis es una enfermedad infecciosa inmunoprevenible  causada por el virus de la parotiditis, miembro del género Rubulavirus, familia Paramyxoviridae, del cual se conocen 12 genotipos confirmados, designados como A-L y otro nuevo genotipo designado como M. Las vacunas anti-parotiditis por lo general,  se fabrican empleando virus vivo atenuado de alguno de estos genotipos y están  disponibles como monovalente (parotiditis) y trivalente (sarampión-rubéola-parotiditis). A pesar de los programas de vacunación implementados por muchos países, se han presentado brotes de parotiditis en forma epidémica en la cual se ha detectado co-circulación de genotipos entre poblaciones vacunadas. Entre las posibles explicaciones  están: el fracaso primario a la vacunación, pérdida de efectividad secundaria e infección por virus heterólogos. Como consecuencia la Organización Mundial de la Salud (OMS) ha recomendado estudios moleculares epidemiológicos, que incluya la genotipificación de cepas circulantes del virus de la parotiditis, como parte del programa de vigilancia. Esto permitirá una mayor información de la distribución de los genotipos en todo el mundo, contribuyendo a la vigilancia de la parotiditis y posiblemente en la reformulación de vacunas más eficaces. Esta revisión muestra la importancia que tiene la caracterización molecular  o genotipificación del virus de la parotiditis, con el propósito de comprender y explicar el comportamiento epidemiológico de esta enfermedad que ha sido ampliamente controlada por la aplicación sistemática de la vacuna a nivel mundial.

Mumps is a vaccine-preventable infectious disease, caused by mumps virus, member of Rubulavirus genus, Paramyxoviridae family, has been classified into 12 confirmed genotypes, designated as A-L and one proposed genotype, M. Usually the anti-mumps vaccines are manufactured using attenuated live virus genotypes and any of these are available as monovalent (mumps) and trivalent (measles-mumps-rubella). Although vaccination programs implemented by many countries, there have been outbreaks of mumps in epidemic form, in which has been detected co-circulation of genotypes among vaccinated populations. Possible explanations are: the primary vaccination failure, loss of high effectiveness and heterologous virus infection. Because of this, the World Health Organization (WHO) has recommended molecular epidemiological studies, including genotyping of circulating strains of mumps virus as part of the monitoring program. This information will allow greater distribution of genotypes worldwide, contributing to monitoring and possibly mumps reformulating more effective vaccines. This review shows the importance of molecular characterization and genotyping of mumps virus, in order to understand and explain the epidemiological behavior of the disease has been largely controlled by the systematic application of the vaccine worldwide.

Structure-function analysis of two variants of mumps virus hemagglutinin-neuraminidase protein

A point mutation from guanine (G) to adenine (A) at nucleotide position 1081 in the hemagglutinin-neuraminidase (HN) gene has been associated with neurovirulence of Urabe AM9 mumps virus vaccine. This mutation corresponds to a glutamic acid (E) to lysine (K) change at position 335 in the HN glycoprotein. We have experimentally demonstrated that two variants of Urabe AM9 strain (HN-A1081 and HN-G1081) differ in neurotropism, sialic acidbinding affinity and neuraminidase activity. In the present study, we performed a structure-function analysis of that amino acid substitution; the structures of HN protein of both Urabe AM9 strain variants were predicted. Based on our analysis, the E/K mutation changes the protein surface properties and to a lesser extent their conformations, which in turn reflects in activity changes. Our modeling results suggest that this E/K interchange does not affect the structure of the sialic acid binding motif; however, the electrostatic surface differs drastically due to an exposed short alpha helix. Consequently, this mutation may affect the accessibility of HN to substrates and membrane receptors of the host cells. Our findings appear to explain the observed differences in neurotropism of these vaccine strains.

Utilização da cultura de células de rim de Hamster para o isolamento do vírus da caxumba (parotidite epidêmica) / Cultures of hamster kidney cells in the isolation of mumps virus

Cultura de células de linhagem contínua de rim de hamster (BHK-21) demonstrou ser um sistema mais sensível e mais rápido para o isolamento do vírus da caxumba, quando comparado com o de ovos embrionados de galinha. Estas células podem ser cultivadas sem dificuldade, em laboratório, ao contrário das células primárias de rim de macaco, igualmente sensíveis mas que são de difícil obtenção. A utilização de células BHK-21 para o isolamento do vírus da caxumba, do material biológico, constitui uma boa alternativa no diagnóstico das infecções causadas por esse vírus (AU).