Fernando Lucas de Melo

High Rates of Molecular Evolution in Hantaviruses

Hantaviruses are rodent-borne Bunyaviruses that infect the Arvicolinae, Murinae, and Sigmodontinae subfamilies of Muridae. The rate of molecular evolution in the hantaviruses has been previously estimated at approximately 10(-7) nucleotide substitutions per site, per year (substitutions/site/year), based on the assumption of codivergence and hence shared divergence times with their rodent hosts. If substantiated, this would make the hantaviruses among the slowest evolving of all RNA viruses. However, as hantaviruses replicate with an RNA-dependent RNA polymerase, with error rates in the region of one mutation per genome replication, this low rate of nucleotide substitution is anomalous. Here, we use a Bayesian coalescent approach to estimate the rate of nucleotide substitution from serially sampled gene sequence data for hantaviruses known to infect each of the 3 rodent subfamilies: Araraquara virus (Sigmodontinae), Dobrava virus (Murinae), Puumala virus (Arvicolinae), and Tula virus (Arvicolinae). Our results reveal that hantaviruses exhibit short-term substitution rates of 10(-2) to 10(-4) substitutions/site/year and so are within the range exhibited by other RNA viruses. The disparity between this substitution rate and that estimated assuming rodent-hantavirus codivergence suggests that the codivergence hypothesis may need to be reevaluated.

Syphilis at the Crossroad of Phylogenetics and Paleopathology

The origin of syphilis is still controversial. Different research avenues explore its fascinating history. Here we employed a new integrative approach, where paleopathology and molecular analyses are combined. As an exercise to test the validity of this approach we examined different hypotheses on the origin of syphilis and other human diseases caused by treponemes (treponematoses). Initially, we constructed a worldwide map containing all accessible reports on palaeopathological evidences of treponematoses before Columbuss return to Europe. Then, we selected the oldest ones to calibrate the time of the most recent common ancestor of Treponema pallidum subsp. pallidum, T. pallidum subsp. endemicum and T. pallidum subsp. pertenue in phylogenetic analyses with 21 genetic regions of different T. pallidum strains previously reported. Finally, we estimated the treponemes evolutionary rate to test three scenarios: A) if treponematoses accompanied human evolution since Homo erectus; B) if venereal syphilis arose very recently from less virulent strains caught in the New World about 500 years ago, and C) if it emerged in the Americas between 16,500 and 5,000 years ago. Two of the resulting evolutionary rates were unlikely and do not explain the existent osseous evidence. Thus, treponematoses, as we know them today, did not emerge with H. erectus, nor did venereal syphilis appear only five centuries ago. However, considering 16,500 years before present (yBP) as the time of the first colonization of the Americas, and approximately 5,000 yBP as the oldest probable evidence of venereal syphilis in the world, we could not entirely reject hypothesis C. We confirm that syphilis seems to have emerged in this time span, since the resulting evolutionary rate is compatible with those observed in other bacteria. In contrast, if the claims of precolumbian venereal syphilis outside the Americas are taken into account, the place of origin remains unsolved. Finally, the endeavor of joining paleopathology and phylogenetics proved to be a fruitful and promising approach for the study of infectious diseases.

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in São Paulo state, Brazil. We show that different viral genotypes entered São Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in São Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.

Introduction of Dengue Virus 4 (DENV-4) Genotype I into Brazil from Asia?

Dengue fever is the most important viral neglected tropical disease [1], and during 2008 more than 865,000 cases of dengue infection were reported in the American continent. Brazil is experiencing severe yearly outbreaks of dengue virus (DENV) serotypes 1 to 3 [2]. Since its brief incursion into the eastern Brazilian Amazon in 1982 [3], DENV-4 has not been detected or known to have caused outbreaks in Brazil; as a result, its inclusion in routine epidemiological surveillance protocols was deemed unnecessary by Brazilian health authorities. In 2008, however, DENV-4 was reported in three patients without any history of traveling outside Manaus (Brazil) by academic researchers who did not have any DENV-4 strains in their laboratories [4]. The Brazilian health authorities dismissed the work, not only by not being able to reproduce the findings in their own laboratories, but also by arguing that ultimately there is no apparent DENV-4 outbreak in Brazil. n nBut what could the available sequences from two of the patients actually be showing? To their credit, Figueiredo et al. [4] identified the sequences as DENV-4 using a coarse-grained similarity search with BLAST [5]. Nevertheless, sequences sharing the highest bit-scores in BLAST are not necessarily the closest phylogenetic relatives [6]. Therefore, we have undertaken a more detailed phylogenetic analysis of the controversial samples from Manaus. An initial dataset including reference sequences from all four serotypes was used to classify the samples from Manaus (dataset available upon request). The results supported the previous finding that samples AM750 and AM1619 were indeed DENV-4. A second data set was then assembled containing 34 DENV-4 sequences, including all three DENV-4 genotypes (I, II, and III) known to cause outbreaks around the world [7] (GenBank accession numbers {type:entrez-nucleotide,attrs:{text:EU127900,term_id:159170579,term_text:EU127900}}EU127900, {type:entrez-nucleotide,attrs:{text:EU127899,term_id:159170576,term_text:EU127899}}EU127899, {type:entrez-nucleotide,attrs:{text:AY947539,term_id:61652904,term_text:AY947539}}AY947539, {type:entrez-nucleotide,attrs:{text:AY618992,term_id:53653750,term_text:AY618992}}AY618992, {type:entrez-nucleotide,attrs:{text:AY618991,term_id:53653748,term_text:AY618991}}AY618991, {type:entrez-nucleotide,attrs:{text:AY618990,term_id:53653746,term_text:AY618990}}AY618990, {type:entrez-nucleotide,attrs:{text:AY618989,term_id:53653744,term_text:AY618989}}AY618989, {type:entrez-nucleotide,attrs:{text:AY618988,term_id:53653742,term_text:AY618988}}AY618988, {type:entrez-nucleotide,attrs:{text:AY152360,term_id:28171535,term_text:AY152360}}AY152360, {type:entrez-nucleotide,attrs:{text:AY152316,term_id:28171436,term_text:AY152316}}AY152316, {type:entrez-nucleotide,attrs:{text:AY152312,term_id:28171427,term_text:AY152312}}AY152312, {type:entrez-nucleotide,attrs:{text:AY152304,term_id:28171409,term_text:AY152304}}AY152304, {type:entrez-nucleotide,attrs:{text:AY152300,term_id:28171400,term_text:AY152300}}AY152300, {type:entrez-nucleotide,attrs:{text:AY152292,term_id:28171382,term_text:AY152292}}AY152292, {type:entrez-nucleotide,attrs:{text:AY152260,term_id:28171310,term_text:AY152260}}AY152260, {type:entrez-nucleotide,attrs:{text:AY152252,term_id:28171292,term_text:AY152252}}AY152252, {type:entrez-nucleotide,attrs:{text:AY152212,term_id:28171202,term_text:AY152212}}AY152212, {type:entrez-nucleotide,attrs:{text:AY152148,term_id:28171058,term_text:AY152148}}AY152148, {type:entrez-nucleotide,attrs:{text:AY152144,term_id:28171049,term_text:AY152144}}AY152144, {type:entrez-nucleotide,attrs:{text:AY152132,term_id:28171022,term_text:AY152132}}AY152132, {type:entrez-nucleotide,attrs:{text:AY152112,term_id:28170977,term_text:AY152112}}AY152112, {type:entrez-nucleotide,attrs:{text:AY152108,term_id:28170968,term_text:AY152108}}AY152108, {type:entrez-nucleotide,attrs:{text:AY152104,term_id:28170959,term_text:AY152104}}AY152104, {type:entrez-nucleotide,attrs:{text:AY152100,term_id:28170950,term_text:AY152100}}AY152100, {type:entrez-nucleotide,attrs:{text:AY152096,term_id:28170941,term_text:AY152096}}AY152096, {type:entrez-nucleotide,attrs:{text:AY152092,term_id:28170932,term_text:AY152092}}AY152092, {type:entrez-nucleotide,attrs:{text:AY152088,term_id:28170923,term_text:AY152088}}AY152088, {type:entrez-nucleotide,attrs:{text:AY152084,term_id:28170914,term_text:AY152084}}AY152084, {type:entrez-nucleotide,attrs:{text:AY152076,term_id:28170896,term_text:AY152076}}AY152076, {type:entrez-nucleotide,attrs:{text:AY152064,term_id:28170869,term_text:AY152064}}AY152064, {type:entrez-nucleotide,attrs:{text:AY152060,term_id:28170860,term_text:AY152060}}AY152060, {type:entrez-nucleotide,attrs:{text:AY152052,term_id:28170842,term_text:AY152052}}AY152052, {type:entrez-nucleotide,attrs:{text:AF326573,term_id:12659201,term_text:AF326573}}AF326573, and {type:entrez-nucleotide,attrs:{text:AF289029,term_id:11096032,term_text:AF289029}}AF289029). Crucially, both samples from Manaus were sister taxa and nested within Asian genotype I (Figure 1, see legend for methods used) using a genetic algorithm–based method [8]. This result was also obtained more than 91% of the time during maximum likelihood non-parametric bootstrap with the PhyML program [9] (Figure 1). Moreover, it also had a high Bayesian posterior probability (0.99) while using a Markov chain Monte Carlo inference-based method in the MrBayes program [10] (Figure 1). Remarkably, the samples isolated in Manaus in 2007 were distinct, which would be expected from viruses isolated from different individuals in a web of transmission. We further substantiated our findings by comparing the likelihood of the observed tree (lnLu200a=u200a−1588.53) to those estimated for alternative groupings into genotype II (lnLu200a=u200a−1705.85) and III (lnLu200a=u200a−1702.88). We argue that the likelihood differences in combination with high non-parametric bootstrap values and high posterior probabilities (Figure 1) constitute considerable evidence that DENV-4 genotype I strains may be in fact circulating in Brazil. Nevertheless, even with the high levels of support obtained during our analysis, we would argue that additional sequence information is necessary to further describe these strains. n n n nFigure 1 n nPhylogenetic Tree for DENV-4 Samples from Manaus, Brazil. n n n nWhile genotype II has been present in the Americas for over two decades, the presence of DENV-4 genotype I in Brazil was a rather unexpected result, since genotype I is not known to be circulating in the West [3],[11],[12]. Moreover, we would expect its emergence in Central America and the Caribbean first, following the pattern of introduction of many other DENVs [13], but the intensification of economic activities between Brazil and other Asian countries (especially China) could explain a direct introduction to Brazil that bypasses the Caribbean. These are preliminary, albeit potentially troublesome, results, and it begs checking if these strains entered Brazil directly or if DENV-4 genotype I is already experiencing cryptic circulation elsewhere in the Americas. Although DENV-4 is less prevalent (2%), it has been shown to be involved in 10% of dengue hemorrhagic fever cases following secondary infections in children in Thailand [14]. In sum, the presence of DENV-4 in the Americas should be taken with great caution and warrant an intensification of the ongoing surveillance activities in Brazil in particular, where routine serology and nucleic acid detection methodology that includes DENV-4 should be considered.

Molecular characterization of human erythrovirus B19 strains obtained from patients with several clinical presentations in the Amazon region of Brazil

BACKGROUNDnHuman erythrovirus B19, endemic in the Amazon region since 1990, is associated with a wide spectrum of clinical presentations.nnnOBJECTIVESnTo assess the prevalence of erythrovirus B19 infection and the relative frequency of erythrovirus B19 genotypes in patients in the Amazon region with various clinical presentations.nnnSTUDY DESIGNnA total of 487 clinical samples obtained from patients with symptoms suggestive of erythrovirus infection were tested using specific IgM and IgG antibody assays (ELISA) and PCR for viral DNA detection. Partial VP1 and VP2 regions were sequenced and genotyped by phylogenetic reconstruction.nnnRESULTSnB19 DNA was detected in 117 (24%) of 487 samples. Of these, 106 (91%) isolates were genotype 1 and 11 (9%) were genotype 3. No genotype 2 was found. Genotype 1 had three clusters (A1, A2 and B) and all genotype 3 sequences were subtype 3b. All patients with hematological disorders within cluster B of genotype 1 were infected by the same B19 lineage, suggesting that this lineage of B19 may have been transmitted via transfusion of blood products.nnnCONCLUSIONnWe reported two genotypes, 1 and 3b, with three genotype 1 clusters co-circulating in the Amazon region during the past 10 years.

Phylogenetic analysis of a near-full-length sequence of an erythrovirus genotype 3 strain isolated in Brazil

Human parvovirus B19 is the only member of the genus Erythrovirus that causes human disease. Recent findings of several strains with considerable sequence divergence from B19 have suggested a new classification for parvovirus genotypes as 1 (B19), 2 (A-6 and LaLi) and 3 (V9). In their overall DNA sequence, the three genotypes differ by ~10%. Here, we report the isolation of a genotype-3-related strain named BR543 during a prospective study conducted in Sao Paulo, Brazil. Analysis of the nearly full-length genome sequence of BR543 indicates that this B19 variant sequence clusters with Gh2768, a strain from Ghana belonging to subtype 3b, and showed mostly synonymous substitutions.

One-step protocol for amplification of near full-length cDNA of the rabies virus genome

Full-length genome sequencing of the rabies virus is not a routine laboratory procedure. To understand fully the epidemiology, genetic variation and evolution of the rabies virus, full-length viral genomes need to be obtained. For rabies virus studies, cDNA synthesis is usually performed using nonspecific oligonucleotides followed by cloning. When specific primers are used, the cDNA obtained is only partial and is limited to the coding regions. Therefore, the development of methods for synthesizing long cDNA using rabies virus-specific primers is of fundamental importance. A new protocol for the synthesis of long cDNA and the development of 19 new primers are described in this study. This procedure allowed the efficient amplification of the full-length genome of the rabies virus variant maintained by hematophagous bat (Desmodus rotundus) populations following the synthesis of a complete long cDNA. Partial sequencing of the rabies virus genome was performed to confirm rabies-specific PCR amplification. Because degenerate primers were employed, this technique can be adapted easily to other variants. Importantly, this new method is faster and less expensive than cloning methods.

Demographic Histories of ERV-K in Humans, Chimpanzees and Rhesus Monkeys

We detected 19 complete endogenous retroviruses of the K family in the genome of rhesus monkey (Macaca mulatta; RhERV-K) and 12 full length elements in the genome of the common chimpanzee (Pan troglodytes; CERV-K). These sequences were compared with 55 human HERV-K and 20 CERV-K reported previously, producing a total data set of 106 full-length ERV-K genomes. Overall, 61% of the human elements compared to 21% of the chimpanzee and 47% of rhesus elements had estimated integration times less than 4.5 million years before present (MYBP), with an average integration times of 7.8 MYBP, 13.4 MYBP and 10.3 MYBP for HERV-K, CERV-K and RhERV-K, respectively. By excluding those ERV-K sequences generated by chromosomal duplication, we used 63 of the 106 elements to compare the population dynamics of ERV-K among species. This analysis indicated that both HERV-K and RhERV-K had similar demographic histories, including markedly smaller effective population sizes, compared to CERV-K. We propose that these differing ERV-K dynamics reflect underlying differences in the evolutionary ecology of the host species, such that host ecology and demography represent important determinants of ERV-K dynamics.

BF Integrase Genes of HIV-1 Circulating in São Paulo, Brazil, with a Recurrent Recombination Region

Although some studies have shown diversity in HIV integrase (IN) genes, none has focused particularly on the gene evolving in epidemics in the context of recombination. The IN gene in 157 HIV-1 integrase inhibitor-naïve patients from the São Paulo State, Brazil, were sequenced tallying 128 of subtype B (23 of which were found in non-B genomes), 17 of subtype F (8 of which were found in recombinant genomes), 11 integrases were BF recombinants, and 1 from subtype C. Crucially, we found that 4 BF recombinant viruses shared a recurrent recombination breakpoint region between positions 4900 and 4924 (relative to the HXB2) that includes 2 gRNA loops, where the RT may stutter. Since these recombinants had independent phylogenetic origin, we argue that these results suggest a possible recombination hotspot not observed so far in BF CRF in particular, or in any other HIV-1 CRF in general. Additionally, 40% of the drug-naïve and 45% of the drug-treated patients had at least 1 raltegravir (RAL) or elvitegravir (EVG) resistance-associated amino acid change, but no major resistance mutations were found, in line with other studies. Importantly, V151I was the most common minor resistance mutation among B, F, and BF IN genes. Most codon sites of the IN genes had higher rates of synonymous substitutions (dS) indicative of a strong negative selection. Nevertheless, several codon sites mainly in the subtype B were found under positive selection. Consequently, we observed a higher genetic diversity in the B portions of the mosaics, possibly due to the more recent introduction of subtype F on top of an ongoing subtype B epidemics and a fast spread of subtype F alleles among the B population.

Structural and phylogenetic relationship of ORF 31 from the Anticarsia gemmatalis MNPV to poly (ADP-ribose) polymerases (PARP)

ORF 31 is a unique baculovirus gene in the genome of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D). It encodes a putative polypeptide of 369 aa homologous to poly (ADP-ribose) polymerase (PARP) found in the genomes of several organisms. Moreover, we found a phylogenetic association with Group I PARP proteins and a 3D homology model of its conserved PARP C-terminal catalytic domain indicating that had almost an exact spatial superimposition of <1xa0Å with other PARP available structures. The 5′ end of ORF 31 mRNA was located at the first nucleotide of a CATT motif at position −27. Using real-time PCR we detected transcripts at 3xa0h post-infection (p.i.) increasing until 24xa0h p.i., which coincides with the onset of DNA replication, suggestive of a possible role in DNA metabolism.