Metagenomic analysis reveals Hepatitis A virus in suspected yellow fever cases in Brazil

In tropical countries,acute febrile illnesses (AFIs) have a broad spectrum of possible aetiologies.In Brazil, Dengue virus (DENV), Chikungunya virus (CHIKV), Zika virus (ZIKV),and Yellow fever virus (YFV) are currently the major arbovirus infections associatedwith AFIs. DENV became endemic following its re-emergence in 1986, and CHIKVand ZIKV were introduced in the region in the last three years (Figueiredo 2015 ⁷ ).More recently, YFV showed an upsurge, representing the most severe YFV epidemicin the country in the last 50 years (Bonaldo et al. 2017 ² ). Given such a complexsituation, establishment of an AFI laboratory-confirmed diagnosis is fundamentalto determine the accurate epidemiological scenario as well as initiate effectiveand timely control measures and treatment. However, misidentifications are quitecommon due to the high cross-reactivity between flaviviruses and co-infectingviruses (Felix et al. 2017 ⁶ ). Moreover, negative cases represent an additionalthreat to public health, since they can be due to unsuspected infectious agentswith the potential to disseminate silently, as was the case in the early stageof the recent ZIKV epidemic in Brazil (Campos et al. 2015 ³ ).

In this study,we applied a metagenomic approach to elucidate the prevalence of AFI cases occurringin Brazil in 2008-2012 that were suspected to be YF given that the patientshad travelled to sylvatic YFV endemic regions. During the 20th century, YFVwas endemic to the northern and central-western Brazilian regions. However,between 2007 and 2009, the YFV circulation area expanded and reached the southeasternand southern regions. Since 2016, YFV has been causing outbreaks that affectnon-human primates and unvaccinated human populations from rural areas of southeasternBrazilian states (Minas Gerais, Espírito Santo, Rio de Janeiro and SãoPaulo) (Mir et al. 2017 ⁸ ).

The cases studiedherein were all deemed to be negative for the major arboviruses (DENV and YFV)occurring in Minas Gerais in 2012 based on serological and specific reversetranscription-polymerase chain reaction (RT-PCR) analyses. To determine thepresence of infectious agents in these cases, we applied metagenomics as reportedpreviously (Conteville et al. 2015 ⁴ ). The DNA and RNA from five serum sampleswere amplified by random RT-PCR, mixed as a pool of samples, and sequenced onan Illumina HiSeq 2500 platform system (Oswaldo Cruz Foundation, high-throughputsequencing platform) using 2 × 100-bp paired-end reads generated withNextera XT libraries. The metagenomic sequences were analysed with referenceto the Kraken (Wood & Salzberg 2014) standard database that includes bacteria,archaea, and viral genomes.

Surprisingly, theanalysis resulted in the identification of only one infectious agent: hepatitisA virus (HAV). The reads assigned as HAV comprised 941 bp of the reference genome(EU526088) encompassing the following partial genomic regions: the 5?untranslated region (UTR), VP3, VP1, 2A, 2C, and 3D. To confirm this result,a specific PCR assay targeting the HAV 5?UTR (Endo et al. 2007 ⁵ ) was performed, and Sanger sequencing of the ampliconsconfirmed the presence of HAV in two of the five samples. Phylogenetic analysisof the HAV 5?UTR sequences (403 bp) further revealed that the HAV isolated from both casesbelong to the sub-genotype 1A (Figure), which is the mostprevalent genotype in Brazil and other South American countries (Fernándezet al. 2012). Both patients were residents of Rio de Janeiro and presented thefollowing symptoms after returning from Caxambu, Minas Gerais: fever, nausea,and abdominal discomfort. One of the patients also presented thrombocytopenia,leucopoenia, and hepatosplenomegaly, while the other patient also presentedretro-orbital pain and myalgia.

HAV infection haslong been endemic in Brazil, and in 2012, when the two cases from the presentstudy occurred, 600 cases were reported in the country (MS 2015 ⁹ ). Viral hepatitismight lead to severe liver failure; however, acute hepatitis A is generallyself-limited, and in complex epidemiological scenarios, with the co-occurrenceof arboviruses, this infection would not be the main suspicion. Therefore, mostHAV infections may be not investigated, leading to underestimation of the prevalenceof infections caused by this virus. In fact, ongoing outbreaks of HAV are currentlyimpacting European countries. Interestingly, these outbreaks are attributedto HAV genotype 1A (Werber et al. 2016), which is the same genotype identifiedin the present Brazilian cases. AFIs can be associated with several types ofinfections, including those caused by viruses, bacteria, and parasites, andeach type of infection requires distinct treatment and control measures. Therefore,this study reinforces the importance of establishing unbiased methods for diagnosisconsidering the challenges of routine clinical practice, particularly when newcomplex epidemiological scenarios occur.

ACKNOWLEDGEMENTS

To the IOC/FIOCRUZhigh-throughput sequencing platform.

AUTHORS' CONTRIBUTION

LCC performed thedata analysis; LCC and ACPV wrote the manuscript; LCC, ACPV and MCLM discussedthe results; AMBF and RMRN supervised the study; MCLM and ACPV conceived anddesigned the study. All authors read and approved the final manuscript.