First discovered in 19751, B19V is a common human pathogen responsible for many diseases ranging from benign childhood condition such as erythema infectiosum, also known as fifth disease2 to fetal death3. B19V is a single-stranded DNA virus which belongs to the Parvoviridae family, genus Erythrovirus and is predominantly transmitted via respiratory secretions such as saliva, sputum, or nasal mucus. The virus is recognized by only one serotype which can be further divided into three genotypes with relevant biological properties. B19V prototype-like isolates are known as genotype 1, LaLi and A6 variants as genotype 2, while V9 and D91.1-like isolates are grouped as genotype 34,5.
At present, B19V genotype 1 is predominantly found worldwide5. On the other hand, genotype 2 is nearly extinct and can only be found in subjects born before 19726. Genotype 3 is prevalent in West Africa, and occasionally detected in France and United States5,7. Furthermore, all the three genotypes have already been identified in Brazil8.
Many viral agents such as rubella virus, measles virus, and arboviruses like dengue usually cause symptoms similar to B19V infection which includes rash and joint pain3,9,10,11. However, it is unknown what percentage of acute febrile illness cases among children from arboviral endemic areas may be due to B19V infection. Accordingly, during the first dengue epidemics in Manaus, Amazonas State of Brazil; IgM antibodies against B19V were detected in the serum of patients with skin rash but negative for dengue12. Thus, laboratorial validation is critical for precise diagnosis of patients suffering from acute febrile illness with rash, particularly when multiple viruses are reported to co-circulate in the same region.
In this study, we investigated B19V infection in children with acute febrile illness that were negative for malaria and dengue, and belonged to a mid-size city, Tefé, in the Amazonas state of Brazil. Furthermore, we performed molecular characterization of the B19V in order to determine the genotypes that are in circulation in Tefé.
The city of Tefé (03° 21′ 14″ S; 64° 42′ 39″ W) is located 523 km from Manaus with approximately 62,000 inhabitants of which about 88% reside in urban areas. Routinely, a considerable number of people migrate from nine other different municipalities to Tefé for work, commerce, and further travel purposes. Tefé is the main fishing port in the Amazonas State region and is an important route in the Solimões river during transport. The busiest airport of this region is also located in this city13 (https://cidades.ibge.gov.br/brasil/am/tefe/panorama).
Sample collection, molecular testing, and sequence analysis
Patients enrolled in this study had been attended at Regional Hospital of Tefé (HRT) between January and July 2013 and were initially evaluated for malaria by blood smear test. All 341 malaria-negative serum samples from patients aging from six months to 83 years old were collected between the first and fourth day after the onset of symptoms. Further, the serum samples were transferred to the reference hospital for infectious diseases Fundação de Medicina Tropical – Dr. Heitor Vieira Dourado (FMT-HVD) situated in Manaus, the capital of the Amazonas State.
At FMT-HVD, RNA was extracted from the samples using QIAamp viral RNA Mini-Kit (Qiagen, Hilden, Germany) followed by semi-nested multiplex PCR protocol for DENV RNA detection14. Furthermore, all 44 samples belonging to children between 1 year and 15 years old that were found to be negative for malaria and dengue were assessed for B19V DNA. Total nucleic-acid was isolated from the samples using DNA PureLink viral RNA/DNA Mini-Kit (Invitrogen, Carlsbad, CA, USA) followed by nested-PCR protocol for the detection of B19V DNA15. Briefly, the first PCR reactions was performed with partial major capsid protein 1 (PVP1) (5′- ACAAGCCTGGGCAAGTTAGC-3′) as forward primer and PVP2 (5′- CTGCACCAGTGCTGGCTTCT-3′) as reverse primer. Further, 10% (by volume) of the first PCR reaction product was used for the nested-PCR reaction with primers, PVP2 and PVP3 (5′- TGGGCCTGGCAATGAGCTAC-3′). All nested-PCR products were electrophoresed on agarose gel with ethidium bromide and visualized under UV light. Amplicons were precipitated with solution containing 20% w/v polyethylene glycol (PEG) 8000 (Promega, WI, USA) and 2.5 M NaCl as described by Lis and Schleif 16. Nucleotide sequencing was performed using the same primers of the nested-PCR reaction and BigDye v3.1 (Applied Biosystems, USA) at the genomics platform of Instituto Leônidas e Maria Deane (ILMD), Fiocruz Amazônia.
All trace files were inspected for quality, trimmed for primer removal, and used for contig assembly using human parvovirus B19 RefSeq Genome (NC_000883.2) as reference. BLAST analysis was performed on the consensus nucleotide sequence obtained from the patient samples of Tefé (Tefé samples)17 using MegaBLAST algorithm against entire non-redundant nucleotide collection (nr/nt) of GenBank + EMBL + DDBJ + PDB + RefSeq sequences with few exclusions.
Further, the final nucleotide sequence of each sample in this study was aligned using MAFFT v7.22218 representing each B19V genotype (genotype 1 or G1, 24 sequences; genotype 2 or G2, 7 sequences; genotype 3 or G3, 15 sequences) and a simian parvovirus sequence (U26342) as negative control. Lastly, this dataset was used for viral genotyping based on phylogenetic analysis using neighbor-joining clustering method and 2,000 bootstrap replicates. All bioinformatic analyses were performed with Geneious software version 9.1.519. The final edition of the phylogenetic tree that was made using FigTree v1.4.3 (http:// bio.ed.ac.uk/software/figtree/) and GIMP v2.8.10 (www.gimp.org).
Among the 44 samples evaluated for B19V, eight (18.2%) samples that were collected between March and July 2013 were found to be positive by nested-PCR. The samples belonged to patients who primarily reported symptoms such as fever, headache, ocular pain, and/or muscle pain; and none of them were diagnosed with rash at the time of sample collection (Table 1). The pediatric patients belong to the age group of 6 to 14 years and all of them residing in Tefé with no travel history within 15 days before the onset of the symptoms.
|Sample ID||Gender||Age (years)||Symptoms Onset||Fever||Headache||Ocular pain||Muscle pain||Vomit|
After performing quality trimming and primer removal, a fragment consisting of 285 nucleotides from each sequence was analyzed. Among the B19V nucleotide sequences of Tefé, seven out of eight were found to be identical. TF195 was an exception which had an adenine instead of cytosine (silent mutation) at position 3,047 when compared to the B19V reference sequence (NC_000883). BLAST analysis returned 22 identical sequences (query cover = 100%, identity = 100%). Among the obtained sequences, 15 sequences corresponded to Brazilian patients with leukemia between 2007 and 201020 while one was from a fatal case of a 12 year-old boy21. One sequence was from a sample collected in USA in 1994 from a dead fetus22. We could not find complete records for the other six sequences.
Further, all Tefé sample sequences were aligned with other 46 B19V sequences available at GenBank representing the three genotypes and one simian parvovirus sequence, strain B20 obtained from Macaca fascicularis, that was used as an outgroup. Phylogenetic analyses showed that all eight sequences from Tefé samples belonged to genotype 1 (Figure 1).
In the present study, we performed nested-PCR assay to detect B19V DNA in serum samples of patients with acute febrile syndrome from Tefé, a mid-sized city located in the countryside of the Amazonas state, Brazil. From the 44 patient samples evaluated, we found eight children in the range of 6 to 14 years to be positive for B19V. Several other studies investigating B19V in the Brazilian population living in other different regions found that a prevailing number of B19V positive cases were associated with the same age group10. Accordingly, we aimed our present study to investigate the samples from this pediatric (6 to 14 years old) group. Furthermore, the first study to assess B19V-specific antibodies in the samples (collected between January 1999 and December 2003) from patients residing in the Amazonas state predominantly included pediatric patients under 15 years23. Consequently, our results suggest that B19V detection should be considered as a differential diagnosis at least in pediatric patients with acute febrile illness.
We detected B19V infections in Tefé between March and July which is also the middle and end phase of monsoon in this region. Although our results were consistent with the previously published data by Figueiredo et al. (2005), we were unable to reveal the seasonal pattern as samples were not collected throughout the year. In regions with temperate climate, B19V infections occur mainly in seasonal periods which includes late winter and early spring5. In Brazil, a study performed in Belém, Pará State between 1988-1989 showed that most cases of B19V infection occur in the dry season24. Different studies conducted in Rio Janeiro9 and Espírito Santo25 have revealed variation in B19V infection with season. However, such observation was not reported by Wermelinger et al. (2002). Thus, further longitudinal studies covering all the regions of the country are required to determine the seasonal pattern of B19V transmission in Brazil. Such a study is highly significant as it will help determine the specific seasons that are at high-risk for parvovirus in each region. Further, this would assist physicians in diagnosing precisely when laboratorial confirmation is unavailable.
Nucleotide sequence analysis of B19V-positive Tefé samples showed eight highly similar sequences, at least in the analyzed target sequence, with only one variation that was observed in sample, TF195. Furthermore, BLAST analysis showed 22 identical sequences that were obtained from patients with varying illness in Brazil and other countries. Phylogenetic analysis of the genome region coding for VP2 and VP3 showed that Tefé sample sequences from Tefé samples were associated with genotype 1, the most common genotype worldwide and among all age groups5. Moreover, the sequences of the present study were found to be closely associated with the subtype 1A. However, the relative small length of the analyzed sequence region was not enough to support the findings.
Initial clinical symptoms caused by B19V and other viruses can be easily misdiagnosed with malaria, dengue, and other arbovirus related diseases that are endemic in the Amazon region. Thus, laboratorial confirmation in such cases is highly significant and much needed26–27. Often, rash is not found in adult patients infected with B19V. However, arthropathy which can be symmetrical and involve multiple joints, is prevalent specifically among infected middle-aged women28. Other clinical conditions such as acute or persistent arthropathy, transient aplastic crisis, hydrops fetalis, and fetal death can occur due to infections during the second trimester of pregnancy3. Thus, differential diagnosis of B19V infections during gestation preferably by molecular methods should be recognized as to evaluate the real burden of this disease especially in cases involving Zika virus endemicity. Moreover, in this study, B19V detection was performed in samples collected during the acute phase of illness and before the emergence of rash. Most common symptoms reported by patients in our study was fever, headache, and ocular globe pain. Thus, without specific laboratory assessments, such cases could be easily misdiagnosed as dengue, Zika, or other type of acute febrile illness.
In summary, our results emphasize the importance of considering B19V in differential diagnosis of cases with acute febrile illness. Moreover, the unique epidemiological setting in the Amazon region with several pathogens that can cause acute febrile illness strengthens the necessity of reliable differential diagnosis to include other less-known viruses endemic in the region such as Mayaro virus and Oropouche virus29. Furthermore, due to difficulty in accessing laboratories in the remotes areas of the Amazon, the development and use of more field-friendly molecular tests like Loop mediated isothermal amplification (LAMP)30 should be encouraged.