At present, 56 types of human adenovirus (HAdVs) have been identified and found to be associated with a variety of clinical features in the respiratory tract, eye, gastrointestinal tract, and other organs. In additions, HAdVs are able to establish persistent and latent infections in humans. Most of the work which has been carried out recently is related to adenovirus vectors and little has been done in other areas such as the nature and mechanisms of adenovirus persistence and latency in human tissues. Another area needing more investigation is the stability of the adenovirus genome which is useful for the development of adenovirus vectors and vaccines and for better understanding of adenovirus evolution especially with conflicting views about this issue.Recombination between two types of adenovirus can happen when the hexon epitope from one type and the fibre epitope from another type are found (intermediate strains). These recombinants can be detected by the conflicting results for serum neutralization (SN) and haemagglutination inhibition (HI) tests or by sequencing and phylogenetic analysis of the hexon and fibre regions of the adenovirus genome. The first part of this study is related to the stability and evolution of different adenovirus species. A total of 31 clinical isolates from AIDS patients previously typed in the hexon L2 region and the fibre knob region were analysed. These isolates were found to be from species D adenovirus (HAdV-D) and 28 of them had contradictory typing results in these two regions so they are clearly intermediate strains. Two isolates appear to be completely new and one isolate (Aids32) was typed as HAdV-D23 variant in both hexon L2 and fibre knob regions. Sequencing and phylogenetic analysis of the hexon L1, fibre shaft and penton regions of these adenoviruses revealed that no intragene recombination events occurred between the hexon L1 and L2 regions or between the fibre knob and fibre shaft regions. Sequencing and phylogenetic analysis of the penton showed that some of the intermediate strains had sequences from a third type of adenovirus in these regions. The penton analysis showed also that intragene recombination between penton HVR and RGD loop regions was common. New types of adenovirus were detected and sequential infection with different adenovirus variants was observed in some patients which indicates that the genome of HAdV-D from AIDS patients are not stable. Full genome sequencing and analysis was carried out for three isolates, two of them appeared to be new types of HAdV-D and the result of multi-recombination events and the third isolate appeared to be a variant of HAdV-D23.The stability of species B adenovirus (HAdV-B) was also analysed. A total of 96 isolates collected from the Manchester area typed previously by serum neutralization (SN) were analysed in five genome regions. Most of these isolates were HAdV-B3 and HAdV-B7 collected during a 15 months outbreak. The rest of the isolates were HAdV-B types 3 and 7 collected in different years following the outbreak in addition to other adenovirus types isolated from different years. The phylogenetic analysis results of all the isolates in the structural regions: hexon L2, penton and fibre knob were found to be consistent and no mismatches (hexon from one type and fiber from another type) were observed. Most of the isolates in the DNA polymerase and E1A regions had the same clustering patterns as the structural regions. However, one HAdV-B7 and one HAdV-B11 isolate changed their clustering patterns in the DNA polymerase region. In addition, HAdV-B16 isolates changed their clustering patterns in both DNA polymerase and E1A regions. The changes of the clustering patterns of some isolates is more likely related to natural variations rather than recombination which indicate that species B adenovirus genome is stable in general. The last part of this study is investigating adenovirus persistence and latency in human tissues. Tonsils and adenoids (106 right and left tonsils and 10 adenoids) were obtained from 57 patients who underwent routine tonsillectomies and/or adenoidectomies. Eighty four (72.41%) tonsils and adenoids samples were positive for HAdV by real-time PCR. The viral load was not the same in the right and left tonsils in most of the cases and ranged from 280 to more than 2.6 x 106 copies/107 cells. Seventy eight of 84 positive samples could be typed by sequencing of the hexon L1 region. Species C types were detected in 82% of the samples followed by species B (7.7%), HAdV-E4 (7.7%) and HAdV-F41 (2.56%). No DNA methylation was detected in the major late promoter (MLP) and E1A promoter regions of six tonsils and adenoids samples and two clinical isolates.

Acute gastroenteritis (AGE) is a global public health problem causing considerable morbidity and mortality among infants and children, especially in low-income settings. Viruses including group A rotaviruses (RVA), noroviruses (NoV), adenoviruses (AdV), sapoviruses (SaV) and astroviruses (AstV) are widely acknowledged to be the most common cause of AGE in children. The importance of newly recognised viruses such as human bocavirus (HBoV) as an aetiological agent of AGE is becoming increasingly evident. The aim of this study was to investigate the molecular epidemiology of HAdV and HBoV in children aged ≤5 years hospitalised for AGE in South Africa (SA) from April 2009 to April 2015. Clinical and demographic data, along with stool specimens were collected from hospitalised children who presented with AGE. Real-time polymerase chain reaction (PCR) was used to screen for the presence of enteric DNA viruses. Genotyping was achieved by nucleotide sequence analysis or multiplex PCR. Whole genome sequencing was performed on selected strains to characterise their genetic variation and evolution. Between April 2009 and December 2014, the prevalence of HAdV in hospitalised children with AGE in SA was 18.1% (656/3623); 62.3% of the HAdV_positive children were 7–24 months of age. Human AdV was detected year round. Co-infections were found in 76.3% (222/291) cases of the HAdV_positive specimens with full enteric screening and AstV was detected most frequently as a co-infecting pathogen. Prolonged hospital stay was observed in human immunodeficiency virus (HIV)-infected children with HAdV. Human AdV-F was the most common species identified (254/603, 42.1%), with almost equally distribution of -40 and -41. Recombination breakpoints of the five HAdV41 strains varied in the number and location, indicating different evolution origins. Between April 2009 and April 2015, the prevalence of HBoV in hospitalised children with AGE in SA was 5.6% (212/3765); the majority of which were from children ≤2-year of age (92%, 195/212). Viral co-infections were found in 67% (142/212) of HBoV cases, while in fully screened specimens (virus, bacteria and parasites), 83.1% (74/89) had evidence of co-infections. In all co-infections, only HAdV was significantly associated with HBoV (adjusted Odds Ratio (aOR))=1.68; (95% CI 1.10-2.52; p=0.015) in multivariate analysis. Human BoV infections were reported throughout the year. All four HBoV genotypes were detected with HBoV1 being the most prevalent (79.6% (152/191). The variation in total number of specimens screened for HAdV and HBoV is because HAdV screening was done until December 2014; while HBoV screening was done until April 2015. The current study highlights the genetic diversity of HAdV-40 and -41 strains circulating in SA and suggests possible evolution from inter-strain recombination. Furthermore, the present study highlights the wide spectrum of HBoV genotypes in children with AGE in SA. This study presents the most comprehensive recent data on HAdV diversity in SA, and new baseline data on a HBoV-associated gastroenteritis in a country where no previous report is available. / Thesis (PhD (Medical Virology))--University of Pretoria, 2019. / Medical Virology / PhD (Medical Virology) / Unrestricted

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Os adenovÃrus (Ad) sÃo agentes importantes de infecÃÃes respiratÃrias agudas (IRAs) em crianÃas, especialmente entre seis meses e cinco anos de idade. Este foi um estudo retrospectivo que teve como objetivos: estabelecer o perfil epidemiolÃgico e clÃnico das infecÃÃes respiratÃrias agudas causadas por Ad em crianÃas atendidas no Hospital Infantil Albert Sabin, na cidade de Fortaleza, no perÃodo de janeiro de 2001 a dezembro de 2006; observar a freqÃÃncia de detecÃÃo de Ad em casos de IRAs em crianÃas atendidas nesse hospital; descrever as principais caracterÃsticas clÃnicas das infecÃÃes respiratÃrias causadas por Ad; pesquisar a existÃncia de padrÃo de sazonalidade do Ad na cidade de Fortaleza; determinar a taxa de isolamento do Ad em cultura de cÃlulas HEp-2 inoculadas com amostras de secreÃÃes de nasofaringe (SNF) consideradas positivas para Ad por imunofluorescÃncia indireta (IFI); e criar um banco de cepas de Ad para futuros estudos sobre diversidade antigÃnica e genÃmica desses agentes. Foram coletadas nesse perÃodo, amostras de SNF de 3070 crianÃas com atà sete dias de sintomas de IRA. Todas as amostras foram submetidas a IFI e, dessas, 54 se mostraram positivas para Ad. Quarenta e uma dessas amostras positivas foram inoculadas em monocamadas de cÃlulas HEp-2, obtendo-se isolamento viral em 32 delas. Outras 103 amostras negativas por IFI, escolhidas aleatoriamente, foram inoculadas, resultando em trÃs isolamentos adicionais, totalizando 57 casos confirmados de infecÃÃo por Ad. A freqÃÃncia de detecÃÃo de Ad foi de 1,86% em relaÃÃo ao total de casos de IRAs e 6,1% das IRAs virais. NÃo foi observado um padrÃo de sazonalidade nem correlaÃÃo com perÃodo chuvoso ou seco. A maior parte das IRAs por Ad ocorreu em crianÃas na faixa etÃria entre sete e 24 meses com 63,15% dos casos. As IRAs por Ad foram observadas principalmente em crianÃas atendidas em ambulatÃrios (50,88%), e o diagnÃstico predominante foi a infecÃÃo de vias aÃreas superiores (70,18%). Os principais sintomas e sinais apresentados pelos pacientes com IRA por Ad foram febre, tosse, coriza, anorexia, vÃmitos e/ou diarrÃia, obstruÃÃo nasal e dispnÃia. A principal conduta terapÃutica aplicada em casos de IRAs por Ad foi o uso de aerossol / salbutamol (42,11%) / Adenoviruses (Ad) are important etiological agents of acute respiratory infections (ARI) in children, particularly between six months and five years old. This was a retrospective study, whose objectives were: to perform clinical and epidemiologic profile of adenoviral respiratory infections in children attended in Hospital Infantil Albert Sabin, in the city of Fortaleza, from January 2001 to December 2006; to observe the detection frequency of Ad in cases of ARI in children attended in that hospital; to describe the main clinical features of adenoviral respiratory infections; to search for the existence of a seasonal pattern of adenoviral infections in the city of Fortaleza; to determine the isolation rate of Ad in HEp-2 cell lines inoculated with samples of nasopharyngeal secretions (NPS) considered positive to Ad by indirect immunofluorescence (IIF); and to create a collection of Ad strains for future studies on antigenic and genomic diversity of these agents. NPS samples of 3,070 children with ARI up to seven days of the onset of symptoms were collected. IIF was applied to all of the samples, and among them, 54 were positive to Ad. Forty one of those positive samples were inoculated into HEp-2 cells, resulting in 32 viral isolations. Other 103 randomly choosen negative samples were also inoculated, resulting in more three isolations, reaching the number of 57 confirmed cases of Ad infections. The detection frequency of Ad was 1.86% of total number of cases of ARI and 6.1% of cases of viral ARI. It was not observed any seasonal pattern or correlation to rainy or dry season. Most cases of adenoviral ARI occurred in children aged seven to 24 months, representing 63.15% of cases. Ad ARI was observed mainly in children attended in the out-patient facility (50.88%), and the predominant diagnosis was upper respiratory tract infection (70.18%). The main clinical features in Ad patients were fever, cough, rhinorrhea, anorexia, vomiting or diarrhea, nasal obstruction, and dyspnea. The main therapeutic management for Ad patients was use of nebulization (about 42% of patients)

The purpose of this study was to assess the effect of early region 1 functions of Ad5 on the ability of viruses containing a selectable marker in early region 3 (E3) to transform mammalian cells. To do this I have constructed and characterized five recombinant viruses containing the thymidine kinase (tk) gene from Herpes Simplex Virus type 1 (HSV1) inserted in E3. The biochemical transformation assay performed using these recombinant viruses allowed the separation of the selection process (incorporation and expression of tk) from the requirement for expression of E1 functions. The method of isolating the desired recombinant viruses was in vivo recombination following mixed infection of 293 cells. The parental viruses used were: hr 1 which expresses a truncated version of the E1a 243R product lacking amino acids 166 to 2437 pm975 which fails to express the E1a 289R product d1312 from which the majority of the E1a coding region has been deleted: hr6 which fails to express a wild type E1b 496R protein; and wild type Ad 5. Each coinfection was done with d1E1,3tk, a previously constructed recombinant virus (Haj-Ahmad and Graham, 1986). Using the resulting recombinant viruses, semipermissive tk- Rat 2 cells were infected and selected for conversion to the tk+ phenotype, as well as being assayed for viability post infection. Comparisons were made of tk transformation frequencies with and without correction for differential cell viability measured after infection with different viruses. Correction for differential cell viability greatly reduced the differences in transformation frequencies observed directly. However hr1tk remained able to induce tk+ transformation at a significantly greater frequency than hr6tk, pm975tk, or Ad5tk. The mutants d131,2tk and d1E1,3tk gave statistically indistinguishable results corresponding to an intermediate level of transformation, while hr6tk, pm975tk, and Ad5tk were grouped together as being least efficient at transformation. The infected Rat2 cell viability assays provide evidence of a correlation involving expression of the early region la (Ela) 289 residue product, efficient viral DNA replication, and cell death. Recombination frequencies obtained during the isolation of the recombinant viruses varied greatly depending on the combination of infecting parental viruses. The following factors appeared to affect recombination frequency: 1. the input ratio of the coinfecting viruses; 2. interference in the replication of d1E1,3tk relative to the other virus present; and 3. the presence of small numbers of mismatched base pairs (seven) near the left terminus of some of the viruses used in coinfection with d1E1,3tk. / Thesis / Master of Science (MS)

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-29T11:10:52Z No. of bitstreams: 2 Dissertação - Hugo César Pereira Santos - 2015.pdf: 3535564 bytes, checksum: 4fc7d88b1da6008bc42c01f46e4f8d7a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-10-29T11:20:29Z (GMT) No. of bitstreams: 2 Dissertação - Hugo César Pereira Santos - 2015.pdf: 3535564 bytes, checksum: 4fc7d88b1da6008bc42c01f46e4f8d7a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-10-29T11:20:29Z (GMT). No. of bitstreams: 2 Dissertação - Hugo César Pereira Santos - 2015.pdf: 3535564 bytes, checksum: 4fc7d88b1da6008bc42c01f46e4f8d7a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-19 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / The human adenoviruses (HAdV) infect people of all ages worldwide, causing a wide range of clinical syndromes, depending on the viral type. In immunocompromised hosts, as transplanted patients, HAdV infection can result in a bad prognosis. Some aspects of viral pathogenesis, such as the association between viremia and/or viral load with disseminated disease and the optimal moment to start the therapy, are still not well established in adult patients that have undergone allogeneic hematopoietic stem cell transplantation (ASCT). Therefore, the main objective of this study was to monitor ASCT recipients for HAdV occurrence, and also correlate viral positivity, viral load and molecular variant with clinical symptoms and patients’ prognosis. For this, stool and serum from 21 patients were monitored in a 2 years period (from October/2012 to October/2014). Serum and fecal samples were screened by Nested-PCR, using primers targeting a partial region of the hexon gene (143bp). Fecal samples were further screened by a commercial enzyme immunoassay (EIA). In total, 57% of the patients had at least one positive sample (serum or stool) for HAdV. Patients presented high viral load (varying from 7,7x103 to 2x108 copies/mL), with a higher viral load in stool when compared to serum. Positive samples were submitted to genomic sequencing, revealing the occurrence of HAdV from C, D and F species. The main clinical symptom presented by infected patients was diarrhea, and graft-versus-host disease was the main intercurrence; however it was not possible to directly associate viral positivity to cause of death. We hope to contribute for a better understanding of the HAdV infection pattern in patients submitted to ASCT. Our data highlights the importance of the inclusion of HAdV testing in the routine laboratory exams of this group of patients. / Os adenovírus humanos (HAdV) podem infectar pessoas de todas as idades, causando uma ampla gama de quadros clínicos. Em pacientes imunocomprometidos, como os indivíduos que receberam transplante, a infecção por esses vírus pode resultar em pior prognóstico para o paciente. Determinados aspectos da patogenia viral, como a associação entre viremia e/ou carga viral com a doença disseminada, bem como o melhor momento para o início da terapia, não estão ainda bem esclarecidos em pacientes que foram submetidos ao transplante de células progenitoras hematopoiéticas (TACPH), principalmente em indivíduos adultos. Dessa forma, o principal objetivo deste estudo foi realizar o monitoramento da infecção por HAdV e a caracterização molecular das variantes virais em pacientes submetidos ao TACPH, bem como determinar a carga viral e correlacionar a infecção com o quadro clínico e prognóstico dos pacientes. Foram analisadas amostras de soro e fezes de 21 pacientes submetidos ao TACPH no período de dois anos (de outubro/2012 a outubro/2014). As amostras de fezes foram triadas por ensaio imunoenzimático e por Nested-PCR, enquanto as amostras de soro foram triadas somente por Nested-PCR de uma região parcial do gene hexon (produto esperado de 143 pb). Foram detectadas amostras positivas de soro e/ou fezes de 57% dos pacientes. De forma geral, os pacientes do presente estudo apresentaram elevadas cargas virais (variando de 7,7x103 à 2x108 CG/mL), que foram mais elevadas nas fezes. As amostras positivas foram submetidas ao sequenciamento genômico e resultados revelaram a ocorrência de adenovírus das espécies C, D e F. O principal quadro clínico apresentado pelos pacientes foi a diarreia e a principal intercorrência observada, a doença do enxerto contra o hospedeiro (DECH). Dez pacientes foram a óbito durante o período de estudo, entretanto, não foi possível associar a infecção por HAdV diretamente à causa mortis. Esperamos que os dados obtidos possam auxiliar no melhor entendimento do padrão da infecção dos HAdVs em pacientes submetidos ao TACPH, de forma a contribuir para que a pesquisa de adenovírus seja incluída na rotina de exames desses pacientes.

RNA splicing is a process where introns are removed and exons are joined together. Human adenovirus type 2 pre-mRNAs undergoes intensive alternative splicing and produce more than 40 differently spliced mRNAs.  This thesis work is focused on the identification of new splice sites in adenovirus. By virtue of Illumina mRNA sequencing technology we have identified 255 splice sites. Splice site analysis of the introns revealed the presence of three types of splice sites GT-AG (61.2%), GC-AG (25.9%) and AT-AC (12.9%). Among 255 splice sites, 224 were new. Significantly, more than 50% of the new splice sites were located in the major late transcription unit on the positive strand of adenovirus DNA. Three new splice sites; 17452-29489 (GC-AG) located on the negative strand of adenovirus DNA in the E2 region, 9668-20346 (AT-AC) and 9699-30505 (GC-AG) on the positive strand of adenovirus DNA in the major late transcription unit were further confirmed by PCR analysis. / Adenovirus replication and transcriptome

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-30T20:05:29Z No. of bitstreams: 1 thaisaparecidavieirareis.pdf: 1630302 bytes, checksum: d9e1d09066119dbc3c4a339f7ccc6564 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T15:59:16Z (GMT) No. of bitstreams: 1 thaisaparecidavieirareis.pdf: 1630302 bytes, checksum: d9e1d09066119dbc3c4a339f7ccc6564 (MD5) / Made available in DSpace on 2016-07-13T15:59:16Z (GMT). No. of bitstreams: 1 thaisaparecidavieirareis.pdf: 1630302 bytes, checksum: d9e1d09066119dbc3c4a339f7ccc6564 (MD5) Previous issue date: 2012-06-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A doença diarréica aguda (DDA) é, ainda hoje, uma das principais causas de morbidade e mortalidade infantil, nos países em desenvolvimento. Na diarreia aguda não bacteriana, os adenovírus entéricos constituem um dos importantes agentes etiológicos da doença. Os adenovírus humanos (HAdV) pertencem à família Adenoviridae e ao gênero Mastadenovirus, cujos membros estão classificados em 7 espécies (A-G) e 54 sorotipos. Dentre estes, os sorotipos 40 e 41, ambos da espécie F, são os mais comumente associados com a DDA. Considerando-se, então, a importância da DDA em países em desenvolvimento, o grande número de casos que não são esclarecidos e o pouco conhecimento sobre a infecção e a participação dos HAdV na gênese da DDA, em Juiz de Fora, Minas Gerais, foi realizado o presente estudo. Entre janeiro de 2007 e dezembro de 2010, foram analisados 395 espécimes fecais diarréicos, provenientes de indivíduos de várias idades, atendidos em serviços ambulatoriais e hospitalizados. A presença dos HAdV foi detectada pela reação de PCR , utilizando-se os iniciadores específicos e a caracterização molecular das amostras positivas foi feita pelo seqüenciamento e análise filogenética das sequências parciais do gene do Hexon. Para as análises estatísticas, foi utilizado o programa SPSS versão 13.0, tendo se adotado um valor de significância como p<0,05. A prevalência da infecção por HAdV, no período 2007-2010, foi de 10,9% (43/395). Os resultados mostraram que não houve correlação significante entre a procedência da amostra (ambulatorial X hospitalar) e a ocorrência da infecção (p=0,152), o mesmo tendo sido observado em relação ao gênero do indivíduo infectado (p=0,393). Por outro lado, a maioria dos casos positivos foi detectada em crianças de até 24 meses de idade, mostrando uma correlação estatisticamente significante entre a idade dos indivíduos infectados e a ocorrência da infecção (p=0,007). Na maioria dos casos de infecção pelo HAdV (36/43), este foi o único agente viral detectado, no entanto, foram observados casos de coinfecção HAdV/Rotavirus (5/43) e HAdV/Norovirus (2/43). A análise filogenética das sequências parciais do gene do Hexon, de 35 amostras positivas, revelou que todas agruparam com amostras de HAdV da espécie F, sorotipo 41, confirmando assim, a associação de HAdV entéricos, nos casos estudados. Este levantamento epidemiológico revelou a presença e a circulação destes vírus na população de Juiz de Fora, no período avaliado, bem como sua importante participação na gênese da DDA, permitindo assim, esclarecer uma boa parte dos casos da doença, que normalmente ficaria sem definição etiológica. / Acute diarrheal disease (ADD) is still the major cause of child morbidity and mortality in developing countries. Among the non-bacterial diarrhea, enteric adenoviruses are one of the most important etiologic agents of disease. Human adenoviruses (HAdV) belongs to the Adenoviridae family and Mastadenovirus genus. The virus are classified into seven species (A-G) and 54 serotypes. Among them, serotypes 40 and 41, both of the species F, are the most commonly associated with ADD. Taking in consideration the importance of the DDA in developing countries, the large number of cases that don’t have the etiologic agent identified and the lack of knowledge about the participation of HAdV infection and the pathogenesis of ADD, we performed this study in Juiz de Fora, Minas Gerais. Between January of 2007 and December of 2010 395 diarrheal fecal specimens originating from individuals of various ages treated in ambulatory and hospitalized were analyzed. The presence of HAdV was detected by PCR, using specific primers, and molecular characterization of positive samples was performed by sequencing and phylogenetic analysis of partial sequences of the hexon gene. For statistical analyzes, we used SPSS version 13.0, adopting a value of p <0.05 as significant. The prevalence of infection by HAdV between 2007-2010 was 10.9% (43/395). The results showed no significant correlation between the origin of the sample (hospital X ambulatory) and the occurrence of infection (p = 0.152), and the same was observed in relation to gender of the infected person (P=0,393). Moreover, the majority of positive cases was detected in children under 24 months of age, showing a statistically significant correlation between the age of the infected individuals and the occurrence of infection (p=0,007). In most cases of infection HAdV (36/43), this was the only viral agent detected, however, cases of co-infection HAdV / Rotavirus (5/43) and HAdV /Norovirus (2/43) were identified. Phylogenetic analysis of partial sequences of the hexon gene from 35 positive samples revealed that all samples clustered with HAdV species F, serotypes 41, confirming the association of enteric HAdV in the cases of this study. This epidemiological survey revealed the presence and circulation of these viruses in the population of Juiz de Fora in the period studied, as well as its important role in the genesis of the DDA, our data identified a good number of cases of the disease, which normally remains unidentified.