Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil.

Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.

Bifidobacteria define gut microbiome profiles of golden lion tamarin (Leontopithecus rosalia) and marmoset (Callithrix sp.) metagenomic shotgun pools.

Gut microbiome disruptions may lead to adverse effects on wildlife fitness and viability, thus maintaining host microbiota biodiversity needs to become an integral part of wildlife conservation. The highly-endangered callitrichid golden lion tamarin (GLT-Leontopithecus rosalia) is a rare conservation success, but allochthonous callitrichid marmosets (Callithrix) serve as principle ecological GLT threats. However, incorporation of microbiome approaches to GLT conservation is impeded by limited gut microbiome studies of Brazilian primates. Here, we carried out analysis of gut metagenomic pools from 114 individuals of wild and captive GLTs and marmosets. More specifically, we analyzed the bacterial component of ultra filtered samples originally collected as part of a virome profiling study. The major findings of this study are consistent with previous studies in showing that Bifidobacterium, a bacterial species important for the metabolism of tree gums consumed by callitrichids, is an important component of the callitrichid gut microbiome - although GTLs and marmosets were enriched for different species of Bifidobacterium. Additionally, the composition of GLT and marmoset gut microbiota is sensitive to host environmental factors. Overall, our data expand baseline gut microbiome data for callitrichids to allow for the development of new tools to improve their management and conservation.

The characterization of two novel neotropical primate papillomaviruses supports the ancient within-species diversity model.

Papillomaviruses (PVs) are non-enveloped icosahedral viruses with a circular double-stranded DNA genome of ∼8,000 base pairs (bp). More than 200 different PV types have been identified to date in humans, which are distributed in five genera, with several strains associated with cancer development. Although widely distributed in vertebrates, Neotropical Primates (NP) PV infection was described for the first time only in 2016. Currently, four complete genomes of NP PVs have been characterized, three from Saimiri sciureus (SscPV1 to SscPV3) and one from Alouatta guariba (AgPV1). In this work, we describe two novel PV strains infecting Callithrix penicillata (provisionally named CpenPV1 and CpenPV2), using anal swab samples from animals residing at the Brasilia Primatology Center and next generation sequencing. The genomes of CpenPV1 (7,288 bp; 41.5% guanine-cytosine content - GC) and CpenPV2 (7,250 bp; 40.7% GC) contain the characteristic open reading frames (ORFs) for the early (E6, E7, E1, E2, and E4) and late (L2 and L1) PV genes. The L1 ORFs, commonly used for phylogenetic identification, share 76 per cent similarity with each other and differ 32 per cent from any other known PV, indicating that these new strains meet the criteria for defining novel species. PV genes phylogenetic variance was analyzed and different degrees of saturation revealed similar levels of topological heterogeneity, ruling out saturation as primary etiological factor for this phenomenon. Interestingly, the two CpenPV strains form a monophyletic clade within the Gammapapillomavirus genus (provisionally named gammapapillomavirus 32). Unlike for other NP PV strains, which grouped into a new sister genus of Alphapapillomavirus, this is the first report of NP PV strains grouping into a genus previously considered to exclusively comprise Old World Primates (OWP) PVs, including human PVs. These findings confirm the existence of a common ancestor for Gammapapillomavirus already infecting primates before the split of OWP and NP at ∼40 million years ago. Finally, our findings are consistent with an ancient within-species diversity model and emphasize the importance of increasing sampling to help understanding the PV-primate codivergence dynamics and pathogenic potential.

Effect of natural polymorphisms in the HIV-1 CRF02_AG protease on protease inhibitor hypersusceptibility.

Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02_AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02_AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02_AG. In direct head-to-head virus competitions, CRF02_AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02_AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.

The impact of the nelfinavir resistance-conferring mutation D30N on the susceptibility of HIV-1 subtype B to other protease inhibitors.

The human immunodeficiency virus type 1 (HIV-1) protease mutation D30N is exclusively selected by the protease inhibitor (PI) nelfinavir and confers resistance to this drug. We demonstrate that D30N increases the susceptibility to saquinavir (SQV) and amprenavir in HIV-1 subtype B isolates and that the N88D mutation in a D30N background neutralizes this effect. D30N also suppresses indinavir (IDV) resistance caused by the M46I mutation. Interestingly, in patients with viruses originally containing the D30N mutation who were treated with IDV or SQV, the virus either reversed this mutation or acquired N88D, suggesting an antagonistic effect of D30N upon exposure to these PIs. These findings can improve direct salvage drug treatment in resource limited countries where subtype B is epidemiologically important and extend the value of first and second line PIs in these populations.

Conservation patterns of HIV-1 RT connection and RNase H domains: identification of new mutations in NRTI-treated patients.

BACKGROUND: Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. METHODS AND FINDINGS: We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naïve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher's exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. CONCLUSIONS: This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.

Differential drug resistance acquisition in HIV-1 of subtypes B and C.

BACKGROUND: Subtype C is the most prevalent HIV-1 subtype in the world, mainly in countries with the highest HIV prevalence. However, few studies have evaluated the impact of antiretroviral therapy on this subtype. In southern Brazil, the first developing country to offer free and universal treatment, subtypes B and C co-circulate with equal prevalence, allowing for an extensive evaluation of this issue. METHODS AND FINDINGS: Viral RNA of 160 HIV-1+ patients was extracted, and the protease and reverse transcriptase genes were sequenced, subtyped and analyzed for ARV mutations. Sequences were grouped by subtype, and matched to type (PI, NRTI and NNRTI) and time of ARV exposure. Statistical analyses were performed to compare differences in the frequency of ARV-associated mutations. There were no significant differences in time of treatment between subtypes B and C groups, although they showed distinct proportions of resistant strains at different intervals for two of three ARV classes. For PI, 26% of subtype B strains were resistant, compared to only 8% in subtype C (p = 0.0288, Fisher's exact test). For NRTI, 54% of subtype B strains were resistant versus 23% of subtype C (p = 0.0012). Differences were significant from 4 years of exposure, and remained so until the last time point analyzed. The differences observed between both subtypes were independent of time under rebound viremia in cases of virologic failure and of the number of HAART regimens used by treated patients. CONCLUSIONS: Our results pointed out to a lower rate of accumulation of mutations conferring resistance to ARV in subtype C than in subtype B. These findings are of crucial importance for current initiatives of ARV therapy roll-out in developing countries, where subtype is C prevalent.

HIV-1 subtype C dissemination in southern Brazil.

OBJECTIVES: To describe the molecular and epidemiological profile of HIV-1 in patients followed at the University Hospital of Rio Grande, Brazil. DESIGN AND METHODS: A cross-sectional study was conducted from September to December 2002. Plasma viral RNA of 85 patients was extracted and protease and reverse transcriptase genes were polymerase chain reaction-amplified and sequenced. Sequences were subtyped and examined to antiretroviral resistance mutations. Laboratory data and past history of antiretroviral treatment were also collected. RESULTS: Most viruses were either subtype B (42%) or subtype C (45%). No risk behaviour, sexual orientation or laboratory parameter was associated with any specific subtype, but subtype C tended to be more frequently found in women (P = 0.06). The prevalence of subtype C has increased over the HIV/AIDS epidemic, accounting for almost 60% of cases diagnosed in 2002. Intra-subtype genetic distances were smaller in subtype C than in subtype B, suggesting a more recent introduction of the former in the epidemic. Of patients under treatment, 60% had at least one antiretroviral drug resistance mutation, but no mutation was specifically associated with any HIV-1 subtype. Only one resistance mutation each was found in drug-naive patients with subtypes B and C. CONCLUSION: Despite the fact that subtype C appeared in southern Brazil more recently than subtype B, it is now the predominant strain in Rio Grande. The epidemic spread of subtype C could be taking place in Brazil, and possibly in south America, a phenomenon similar to that seen in other countries where this subtype is now totally dominant.