A Biotechnological Review on Patents Applied to Rubella Diagnosis.

BACKGROUND: Rubella, caused by the Rubella virus (RV), is considered a mild self-limited illness. However, RV has teratogenic potential. Laboratory investigation plays an important role in both diagnosis and surveillance of the disease. The main methods for diagnosing Rubella are serological assays for the detection of specific IgM and molecular assays for detecting viral RNA. However, some laboratories perform IgG avidity testing, virus isolation and analysis of genetic sequence as tools to help Rubella eradication. The importance of the diagnosis of Rubella involves the appropriate treatment of the disease, because the Rubella clinical symptoms may be similar to those of other diseases, and the population monitoring to avoid new emergent cases. This study addresses different methods of diagnosing Rubella and contributes as a source of knowledge to assist health systems in controlling the disease. OBJECTIVE: The main objective of this study was to review the available patents regarding Rubella diagnosis published in intellectual property databases, and provides an overview of the technologies available for the diagnosis of Rubella. METHOD: The search strategy was based on the keywords searched separately or together using a Boolean operator either in the patent title or abstract the time interval was restricted to patents filed or granted from January 2009 until February 2022. The database used was Google Patents. RESULTS: This study analyzed 24 patent documents regarding strategies for the diagnosis of Rubella. Of these, 15 patents disclose strategies for detecting Rubella antibodies, 7 patents the detection of Rubella virus nucleic acid, and 2 patents the production of antibodies applied in Rubella diagnosis. CONCLUSION: Rubella is still a public health problem in some countries, mainly those in development, especially due to congenital Rubella syndrome, which can cause malformation or fetal death. However, its diagnosis is challenging, due to similarity of symptoms with other diseases, and for this reason, laboratory diagnosis is essential. Studies like this encourage researchers and governments to invest in research to continue the development of new products, using different areas of biotechnology, to solve society's problems, especially diseases that have an impact on global health, such as Rubella.

Rubella virus genotype 1G and echovirus 9 as etiologic agents of exanthematous diseases in Brazil: insights from phylogenetic analysis.

The aim of the present study was to identify the rubella virus (RV) and enterovirus (EV) genotypes detected during the Epidemiological Surveillance on Exanthematic Febrile Diseases (VIGIFEX) study and to perform phylogenetic analysis. Ten RV- and four EV-positive oropharyngeal samples isolated from cell culture were subjected to RT-PCR and sequencing. Genotype 1G and echovirus 9 (E-9) was identified in RV- and EV-positive samples, respectively. The RV 1G genotype has been persisting in Brazil since 2000-2001. No evidence of E-9 being involved in exanthematic illness in Brazil has been reported previously. Differential laboratory diagnosis is essential for management of rash and fever disease.

Molecular epidemiology of rubella viruses involved in congenital rubella infections in São Paulo, Brazil, between 1996 and 2009.

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as congenital rubella syndrome (CRS). This retrospective study was conducted between 1996 and 2009 with surveillance specimens collected from patients suspected of congenital rubella infection (CRI) and CRS. The clinical samples (nine amminiotic fluid, eight urine, eight blood, one conception product, and one placenta) were sent for viral isolation and genotyping. Twenty-seven sequences were analysed and four genotypes (1a, 1B, 1G, and 2B) were identified in São Paulo that were involved in congenital infection. To our knowledge, this study is the first report that describes genetic diversity of the circulating rubella strains involved in CRI.

Molecular analysis of rubella virus in travelers suspected of measles infection in São Paulo, Brazil.

OBJECTIVE: To identify measles virus genotypes in three cases of travelers suspected of measles infection. METHODS: Samples (blood and urine) were collected for serology, virus isolation, and genotyping. Sera were analyzed for IgM antibodies against measles virus and rubella virus by enzyme-linked immunosorbent assay (ELISA) (Siemens - Marburg, Germany). Clinical samples (lymphocytes and urine) were inoculated into Statens Serum Institute rabbit corneal epithelial cell line- ATCC CL 60 (SIRC) and Vero Slam cells. RNA was extracted from clinical samples and cell culture was inoculated and processed by polymerase chain reaction (PCR) with oligonucleotides specific for measles virus (MV) and rubella virus (RV). RESULTS: All patients showed IgM negative serology for MV and positive IgM for RV. RV belonging to genotypes 1B, 1C, and 1E were isolated from patients who came from Finland, Peru, and Germany, respectively. Genotype 1B has been found in Europe and on the East Coast of South America; 1C has been found in Peru and the West Coast of South America, and 1E, first identified in 1997, now appears to have worldwide distribution. CONCLUSION: Information about RV and MV genotypes circulating in São Paulo is essential for the control of measles, rubella, and congenital rubella syndrome (CRS) in Brazil.

Molecular analysis of rubella virus in travelers suspected of measles infection in São Paulo, Brazil / Análise molecular do vírus da rubéola em turistas suspeitos de infecção por sarampo em São Paulo, Brasil

OBJECTIVE: To identify measles virus genotypes in three cases of travelers suspected of measles infection. METHODS: Samples (blood and urine) were collected for serology, virus isolation, and genotyping. Sera were analyzed for IgM antibodies against measles virus and rubella virus by enzyme-linked immunosorbent assay (ELISA) (Siemens - Marburg, Germany). Clinical samples (lymphocytes and urine) were inoculated into Statens Serum Institute rabbit corneal epithelial cell line- ATCC CL 60 (SIRC) and Vero Slam cells. RNA was extracted from clinical samples and cell culture was inoculated and processed by polymerase chain reaction (PCR) with oligonucleotides specific for measles virus (MV) and rubella virus (RV). RESULTS: All patients showed IgM negative serology for MV and positive IgM for RV. RV belonging to genotypes 1B, 1C, and 1E were isolated from patients who came from Finland, Peru, and Germany, respectively. Genotype 1B has been found in Europe and on the East Coast of South America; 1C has been found in Peru and the West Coast of South America, and 1E, first identified in 1997, now appears to have worldwide distribution. CONCLUSION: Information about RV and MV genotypes circulating in São Paulo is essential for the control of measles, rubella, and congenital rubella syndrome (CRS) in Brazil.

OBJETIVO: Identificar o genótipo do vírus do sarampo em três viajantes suspeitos de infecção por sarampo. MÉTODOS: Amostras (sangue e urina) foram coletadas para sorologia, isolamento viral e genotipagem. As sorologias para pesquisa de IgM para o vírus do sarampo e da rubéola foi realizada utilizando-se o kit de ELISA (Siemens - Marburg, Alemanha). As amostras clínicas (linfócito e urina) foram inoculadas na SIRC (Statens Serum Institute rabbit corneal epithelial cell line-ATCC CL 60) e nas células Vero Slam. O RNA foi extraído das amostras clínicas e das células inoculadas e processadas por PCR, utilizando oligonucleotideos específicos para sarampo e rubéola. RESULTADOS: Todos os pacientes apresentaram sorologia IgM negativa para sarampo e positivo para rubéola. Os vírus da rubéola isolados dos pacientes que vieram da Finlândia, Peru e Alemanha pertencem aos genótipos 1B, 1C e 1E, respectivamente. O genótipo 1B foi encontrado na Europa e na costa oriental da América do Sul, o genótipo 1C foi encontrado no Peru e na costa oeste da América do Sul e o genótipo 1E, identificado pela primeira vez em 1997, agora aparenta ser um genótipo com distribuição mundial. CONCLUSÃO: O conhecimento dos genótipos de sarampo e rubéola que circulam em São Paulo é essencial para o controle do sarampo, rubéola e síndrome da rubéola congênita.

Epidemiological and molecular characterization of rubella virus isolated in São Paulo, Brazil during 1997-2004.

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 1997-2004 were isolated in cell culture and genotyped. Twenty-eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in São Paulo, Brazil.

Phylogenetic analysis of rubella virus strains during an outbreak in São Paulo, 2007-2008.

Rubella virus (RV) is an important human pathogen that causes rubella, an acute contagious disease. It also causes severe birth defects collectively known as congenital rubella syndrome when infection occurs during the first trimester of pregnancy. Here, we present the phylogenetic analysis of RV that circulated in São Paulo during the 2007-2008 outbreak. Samples collected from patients diagnosed with rubella were isolated in cell culture and sequenced. RV RNA was obtained from samples or RV-infected cell cultures and amplified by reverse transcriptase-polymerase chain reaction. Sequences were assigned to genotypes by phylogenetic analysis using RV reference sequences. Seventeen sequences were analyzed, and three genotypes were identified: 1a, 1G, and 2B. Genotypes 1a and 1G, which were isolated in 2007, were responsible for sporadic rubella cases in São Paulo. Thereafter, in late 2007, the epidemiological conditions changed, resulting in a large RV outbreak with the clear dominance of genotype 2B. The results of this study provide new approaches for monitoring the progress of elimination of rubella from São Paulo, Brazil.

Isolation and genotyping of rubella virus from a case of congenital infection in Brazil.

The incidence of CRS and CRI has decreased markedly worldwide with the implementation of efficient vaccination programs. We report a congenital rubella case with fetal death occurred at 29th week of gestation. RV was confirmed in placenta. The results of phylogenetic analysis showed that the RVs/SaoPaulo01.- BRA/08.CRI belongs to the genotype 2B of RV.

Acute liver failure associated with rubella virus in a child.

Acute liver failure is a syndrome with a wide range of etiologic possibilities in children, but in up to 50% of the cases in the literature no diagnosis is established. This case report adds rubella virus to the list of possible causes of acute liver failure. This association was made by serologic, cell culture, molecular, histopathologic, and immunohistochemical methods.

Phylogenetic analysis of rubella viruses isolated in 2008 outbreak in Argentina.

BACKGROUND: A rubella outbreak was recorded in Buenos Aires during 2008. OBJECTIVES: The objective of this communication is to present the genetic and phylogenetic analyses of wild-type RUBV circulating in Buenos Aires during the 2008 outbreak. STUDY DESIGN: Throat swab samples collected from patients diagnosed with rubella between June 2008 and December 2008 were inoculated in cell culture and 23 isolates were sequenced. RESULTS: Phylogenetic analysis of the WHO-recommended window (nt 8731-9469) of the E1 envelope glycoprotein was performed and all isolates clustered with the 2B genotype. CONCLUSIONS: Genotype 2B seems to be endemically circulating in the Southern cone of Latin America, thus causing recent outbreaks.

Confirmation of rubella within 4 days of rash onset: comparison of rubella virus RNA detection in oral fluid with immunoglobulin M detection in serum or oral fluid.

Rubella virus infection is typically diagnosed by the identification of rubella virus-specific immunoglobulin M (IgM) antibodies in serum, but approximately 50% of serum samples from rubella cases collected on the day of rash onset are negative for rubella virus-specific IgM. The ability to detect IgM in sera and oral fluids was compared with the ability to detect rubella virus RNA in oral fluids by reverse transcription-PCR (RT-PCR) by using paired samples taken within the first 4 days after rash onset from suspected rubella cases during an outbreak in Perú. Sera were tested for IgM by both indirect and capture enzyme immunoassays (EIAs), and oral fluids were tested for IgM by a capture EIA. Tests for IgM in serum were more sensitive for the confirmation of rubella than the test for IgM in oral fluid during the 4 days after rash onset. RT-PCR confirmed more suspected cases than serum IgM tests on days 1 and 2 after rash onset. The methods confirmed approximately the same number of cases on days 3 and 4 after rash onset. However, a few cases were detected by serum IgM tests but not by RT-PCR even on the day of rash onset. Nine RT-PCR-positive oral fluid specimens were shown to contain rubella virus sequences of genotype 1C. In summary, RT-PCR testing of oral fluid confirmed more rubella cases than IgM testing of either serum or oral fluid samples collected in the first 2 days after rash onset; the maximum number of confirmations of rubella cases was obtained by combining RT-PCR and serology testing.

Isolation and genotype analysis of rubella virus from a case of Guillain-Barré syndrome.

Rubella virus (RV) infection has sporadically been linked to Guillain-Barré syndrome (GBS), but the association with RV has been based only on clinical and/or serological backgrounds. In the present case it was possible to isolate RV (genotype 1a) from cerebrospinal fluid and peripheral blood mononuclear cells of an 18-year-old woman diagnosed with GBS after clinical manifestations of rubella. This report contributes to confirm RV as one of the triggering pathogens of this peripheral nervous system disease.

Analysis of gene expression in fetal and adult cells infected with rubella virus.

Congenital infection with rubella virus (RUB) leads to persistent infection and congenital defects and we showed previously that primary human fetal fibroblasts did not undergo apoptosis when infected with RUB, which could promote fetal virus persistence [Adamo, P., Asís, L., Silveyra, P., Cuffini, C., Pedranti, M., Zapata, M., 2004. Rubella virus does not induce apoptosis in primary human embryo fibroblasts cultures: a possible way of viral persistence in congenital infection. Viral Immunol. 17, 87-100]. To extend this observation, gene chip analysis was performed on a line of primary human fetal fibroblasts (10 weeks gestation) and a line of human adult lung fibroblasts (which underwent apoptosis in response to RUB infection) to compare gene expression in infected and uninfected cells. A total of 632 and 516 genes were upregulated or downregulated in the infected fetal and adult cells respectively in comparison to uninfected cells, however only 52 genes were regulated in both cell types. Although the regulated genes were different, across functional gene categories the patterns of gene regulation were similar. In general, regulation of pro- and anti-apoptotic genes following infection appeared to favor apoptosis in the adult cells and lack of apoptosis in the fetal cells, however there was a greater relative expression of anti-apoptotic genes and reduced expression of pro-apoptotic genes in uninfected fetal cells versus uninfected adult cells and thus the lack of apoptosis in fetal cells following RUB infection was also due to the prevailing background of gene expression that is antagonistic to apoptosis. In support of this hypothesis, it was found that of a battery of five chemicals known to induce apoptosis, two induced apoptosis in the adult cells, but not in fetal cells, and two induced apoptosis more rapidly in the adult cells than in fetal cells (the fifth did not induce apoptosis in either). A robust interferon-stimulated gene response was induced following infection of both fetal and adult cells and many of the genes upregulated in both cell types were those involved in establishment of an antiviral state; this is the first demonstration of an interferon response at this early stage of human embryonic development. In both fetal and adult cells, interferon controlled but did not eliminate virus spread and apoptosis was not induced in infected fetal cells in the absence of interferon. In addition to the interferon response, chemokines were induced in both infected fetal and adult cells. Thus, it is possible that fetal damage following congenital RUB infection, which involves cell proliferation and differentiation, could be due to induction of the innate immune response as well as frank virus infection.

Campaña nacional de vacunación para la eliminación de la rubéola y del síndrome de rubéola congénita en Bolivia, 2006 / National vaccination campaign for the elimination of rubella and congenital rubella syndrome in Bolivia, 2006

En mayo 2006, Bolivia llevó a cabo una campaña nacional de vacunación en su población de 15 a 39 años para eliminar la rubéola y el síndrome de rubéola congénita del país. Durante seis semanas el país sumó los esfuerzos de los políticos, aliados nacionales e internacionales, trabajadores de salud y organizaciones sociales para alcanzar la meta. La cobertura nacional validada por una encuesta post campaña fue de 94%, sin diferencias significativasde edad, sexo o en la distribución rural urbana. La encuesta probó también un alto nivel de confianza de la población en los servicios de vacunación y el rol importante del personal de salud para informar a la población en el medio rural. Se demuestra que actividades de vacunación masivas en la población adulta y de ambos sexos pueden ser exitosas con una preparación minuciosa y un plan de comunicación estudiado.

Estudio de un mecanismo de persistencia del virus Rubéola en la infección congénita / Study of a mechanism of persistence of the virus Rubella in the congenital infection

Se investigó el rol de la apoptosis en un posible mecanismo de persistencia del virus Rubéola (Rub) en la infección congénita, utilizando cultivos primarios de fibroblastos fetales humanos (FFH) en activa proliferación. Se demostró que, en contraste con lo que se observa en células Vero, RK13, placenta humana a término, y fibroblastos diploides de adulto humano (Hs888Lu), Rub no induce apoptosis en FFH. Por inmunohistoquímica se detectó la actividad de JUN, gen que promueve la proliferación celular, en FFH pero no en Hs888Lu. El análisis de expresión de genes en FFH y Hs888Lu con microarreglos de ADN mostró que en FFH están sobre-expresados genes que estimulan la supervivencia y proliferación celular, como factores de crecimiento, PI3K, y oncogenes de la familia Ras. Las kinasas p-Akt y p-ERK se detectaron en FFH por western blotting. A continuación se demostró que Rub sí induce apoptosis en FFH, cuando se suprimen las vías PI3K¹Akt y Ras¹Raf¹MEK¹ERK. Significativamente, la infección de las células fetales no prospera en estas condiciones. Así, la persistencia de Rub en la infección congénita puede estar inversamente relacionada con la ocurrencia de apoptosis en las células hospedadoras. También se analizó la expresión de genes en células infectadas por Rub, identificándose más de 1500 genes celulares inducidos o suprimidos por el virus, muchos de ellos relacionados con la respuesta inmune, las vías metabólicas, y las cascadas de transmisión de señales. En FFH, Rub modifica la expresión de diferentes genes del desarrollo (genes Hox), que establecen el patrón corporal y promueven la proliferación y la diferenciación celular durante la morfogénesis. Estos datos representan nuevos puntos de partida para postular hipótesis sobre el modo en que el virus ejerce su efecto teratogénico, y brindan un amplio panorama para estudiar la relación entre Rub y la célula.(AU)

Estudio de un mecanismo de persistencia del virus Rubéola en la infección congénita / Study of a mechanism of persistence of the virus Rubella in the congenital infection

Se investigó el rol de la apoptosis en un posible mecanismo de persistencia del virus Rubéola (Rub) en la infección congénita, utilizando cultivos primarios de fibroblastos fetales humanos (FFH) en activa proliferación. Se demostró que, en contraste con lo que se observa en células Vero, RK13, placenta humana a término, y fibroblastos diploides de adulto humano (Hs888Lu), Rub no induce apoptosis en FFH. Por inmunohistoquímica se detectó la actividad de JUN, gen que promueve la proliferación celular, en FFH pero no en Hs888Lu. El análisis de expresión de genes en FFH y Hs888Lu con microarreglos de ADN mostró que en FFH están sobre-expresados genes que estimulan la supervivencia y proliferación celular, como factores de crecimiento, PI3K, y oncogenes de la familia Ras. Las kinasas p-Akt y p-ERK se detectaron en FFH por western blotting. A continuación se demostró que Rub sí induce apoptosis en FFH, cuando se suprimen las vías PI3K–Akt y Ras–Raf–MEK–ERK. Significativamente, la infección de las células fetales no prospera en estas condiciones. Así, la persistencia de Rub en la infección congénita puede estar inversamente relacionada con la ocurrencia de apoptosis en las células hospedadoras. También se analizó la expresión de genes en células infectadas por Rub, identificándose más de 1500 genes celulares inducidos o suprimidos por el virus, muchos de ellos relacionados con la respuesta inmune, las vías metabólicas, y las cascadas de transmisión de señales. En FFH, Rub modifica la expresión de diferentes genes del desarrollo (genes Hox), que establecen el patrón corporal y promueven la proliferación y la diferenciación celular durante la morfogénesis. Estos datos representan nuevos puntos de partida para postular hipótesis sobre el modo en que el virus ejerce su efecto teratogénico, y brindan un amplio panorama para estudiar la relación entre Rub y la célula.

The genomic analysis of rubella virus detected from outbreak and sporadic cases in Rio de Janeiro state, Brazil.

BACKGROUND: The molecular epidemiology of rubella virus (RV) based on the analysis of the viral E1 gene sequences indicated the existence of two genotypes that differ from each other by 8 to 10% in their nucleotide sequences: genotype I is present in Europe, North America and Asia; and genotype II is present only in Asia. OBJECTIVES: The purpose of the study was to identify the RV genotypes circulating in Brazil. STUDY DESIGN: In this study, we analysed 86 clinical samples collected between 1996 and 1999 during a rubella outbreak and from sporadic cases of rubella in Rio de Janeiro State. For the molecular characterisation of RV strains we have used PCR/nested amplification and direct sequencing of a 513-nucleotide region of the E1 gene. RESULTS: The E1 gene sequences of 14 RVs were obtained and were assigned to two lineages, both within genotype I. The percentage divergence of nucleotide sequence ranged from 3.4 to 5.1% between these two lineages. These results were in agreement with the pattern of variation observed among the sequences obtained from other lineages of RV. CONCLUSIONS: This work demonstrated that two new lineages of RV circulated simultaneously between the years 1996 and 1999 in the state of Rio de Janeiro. These results provided new approaches for monitoring the progress of vaccination efforts in Brazil.