The Relationship between HERV, Interleukin, and Transcription Factor Expression in ZIKV Infected versus Uninfected Trophoblastic Cells.

Zika virus (ZIKV) is an arbovirus with maternal, sexual, and TORCH-related transmission capabilities. After 2015, Brazil had the highest number of ZIVK-infected pregnant women who lost their babies or delivered them with Congenital ZIKV Syndrome (CZS). ZIKV triggers an immune defense in the placenta. This immune response counts with the participation of interleukins and transcription factors. Additionally, it has the potential involvement of human endogenous retroviruses (HERVS). Interleukins are immune response regulators that aid immune tolerance and support syncytial structure development in the placenta, where syncytin receptors facilitate vital cell-to-cell fusion events. HERVs are remnants of ancient viral infections that integrate into the genome and produce syncytin proteins crucial for placental development. Since ZIKV can infect trophoblast cells, we analyzed the relationship between ZIKV infection, HERV, interleukin, and transcription factor modulations in the placenta. To investigate the impact of ZIKV on trophoblast cells, we examined two cell types (BeWo and HTR8) infected with ZIKV-MR766 (African) and ZIKV-IEC-Paraíba (Asian-Brazilian) using Taqman and RT2 Profiler PCR Array assays. Our results indicate that early ZIKV infection (24-72 h) does not induce differential interleukins, transcription factors, and HERV expression. However, we show that the expression of a few of these host defense genes appears to be linked independently of ZIKV infection. Future studies involving additional trophoblastic cell lineages and extended infection timelines will illuminate the dynamic interplay between ZIKV, HERVs, interleukins, and transcription factors in the placenta.

Proposal of Model for Evaluation of Viral Kinetics of African/Asian/Brazilian-Zika virus Strains (Step Growth Curve) in Trophoblastic Cell Lines.

The Zika virus (ZIKV) epidemic brought new discoveries regarding arboviruses, especially flaviviruses, as ZIKV was described as sexually and vertically transmitted. The latter shows severe consequences for the embryo/fetus, such as congenital microcephaly and deficiency of the neural system, currently known as Congenital ZIKV Syndrome (CZS). To better understand ZIKV dynamics in trophoblastic cells present in the first trimester of pregnancy (BeWo, HTR-8, and control cell HuH-7), an experiment of viral kinetics was performed for African MR766 low passage and Asian-Brazilian IEC ZIKV lineages. The results were described independently and demonstrated that the three placental cells lines are permissive and susceptible to ZIKV. We noticed cytopathic effects that are typical in in vitro viral infection in BeWo and HTR-8. Regarding kinetics, MR766lp showed peaks of viral loads in 24 and 48 hpi for all cell types tested, as well as marked cells death after peak production. On the other hand, the HTR-8 lineage inoculated with ZIKV-IEC exhibited increased viral production in 144 hpi, with a peak between 24 and 96 hpi. Furthermore, IEC had peak variations of viral production for BeWo in 144 hpi. Considering such in vitro results, the hypothesis that maternal fetal transmission is probably a way of virus transmission between the mother and the embryo/fetus is maintained.

Rabies virus diversification in aerial and terrestrial mammals.

Rabies is a fatal zoonotic infection of the central nervous system of mammals and has been known to humans for millennia. The etiological agent, is a neurotropic RNA virus in the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus. There are currently accepted to be two cycles for rabies transmission: the urban cycle and the sylvatic cycle. The fact that both cycles originated from a common RABV or lyssavirus ancestor and the adaptive divergence that occurred since then as this ancestor virus adapted to a wide range of fitness landscapes represented by reservoir species in the orders Carnivora and Chiroptera led to the emergence of the diverse RABV lineages currently found in the sylvatic and urban cycles. Here we study full genome phylogenies and the time to the most recent common ancestor (TMRCA) of the RABVs in the sylvatic and urban cycles. Results show that there were differences between the nucleotide substitution rates per site per year for the same RABV genes maintained independently in the urban and sylvatic cycles. The results identify the most suitable gene for phylogenetic analysis, heterotachy among RABV genes and the TMRCA for the two cycles.

Granzyme a in Chikungunya and other arboviral infections

Granzyme A (GzmA) is secreted by cytotoxic lymphocytes and has traditionally been viewed as a mediator of cell death. However, a growing body of data suggests the physiological role of GzmA is promotion of inflammation. Here, we show that GzmA is significantly elevated in the sera of chikungunya virus (CHIKV) patients and that GzmA levels correlated with viral loads and disease scores in these patients. Serum GzmA levels were also elevated in CHIKV mouse models, with NK cells the likely source. Infection of mice deficient in type I interferon responses with CHIKV, Zika virus, or dengue virus resulted in high levels of circulating GzmA. We also show that subcutaneous injection of enzymically active recombinant mouse GzmA was able to mediate inflammation, both locally at the injection site as well as at a distant site. Protease activated receptors (PARs) may represent targets for GzmA, and we show that treatment with PAR antagonist ameliorated GzmA- and CHIKV-mediated inflammation.

Granzyme a in Chikungunya and other arboviral infections

Granzyme A (GzmA) is secreted by cytotoxic lymphocytes and has traditionally been viewed as a mediator of cell death. However, a growing body of data suggests the physiological role of GzmA is promotion of inflammation. Here, we show that GzmA is significantly elevated in the sera of chikungunya virus (CHIKV) patients and that GzmA levels correlated with viral loads and disease scores in these patients. Serum GzmA levels were also elevated in CHIKV mouse models, with NK cells the likely source. Infection of mice deficient in type I interferon responses with CHIKV, Zika virus, or dengue virus resulted in high levels of circulating GzmA. We also show that subcutaneous injection of enzymically active recombinant mouse GzmA was able to mediate inflammation, both locally at the injection site as well as at a distant site. Protease activated receptors (PARs) may represent targets for GzmA, and we show that treatment with PAR antagonist ameliorated GzmA- and CHIKV-mediated inflammation.

Yellow fever and orthotopic liver transplantation: new insights from the autopsy room for an old but re-emerging disease.

AIMS: The clinical spectrum of yellow fever (YF) ranges from asymptomatic to fulminant hepatitis. During the sylvatic YF epidemic in the metropolitan area of São Paulo, Brazil in 2018, seven orthotopic liver transplantations (OLTs) were performed in our institution to treat fulminant YF hepatitis. Three patients recovered, while four patients died following OLT. The autopsy findings of all these cases are presented herein as the first description of YF in transplanted patients. METHODS AND RESULTS: All patients were men, aged 16-40 years, without vaccination to YF virus (YFV). All organs were examined, with tissue sampling for histopathological analysis. Detection of YF virus antigens (YFV Ag) was performed with two primary antibodies (mouse polyclonal anti-YFV antibody directed to wild strain and a goat anti-YF virus antibody), and RT-PCR assays were utilised to detect YFV-RNA. All the cases depicted typical findings of YF hepatitis in the engrafted liver. The main extrahepatic findings were cerebral oedema, pulmonary haemorrhage, pneumonia, acute tubular necrosis and ischaemic/reperfusion pancreatitis. Of the four cases, the YVF Ag was detected in the heart in one case, liver and testis in three cases, and the kidney and spleen in all four cases. All four cases had YF virus RNA detected by RT-PCR in the liver and in other organs. CONCLUSIONS: Infection of the engrafted liver and other organs by YFV, possibly combined with major ischaemic systemic lesions, may have led to the death of four of the seven patients undergoing OLT.

Zika Virus in Peridomestic Neotropical Primates, Northeast Brazil.

Zika virus (ZIKV) is a mosquito-borne viral disease associated with fetal microcephaly and other central nervous system (CNS) symptomatology. It was first identified in a Rhesus macaque in Uganda in 1947 and later in humans (Zika fever). In 2015, ZIKV was notified in Northeast Brazil where it was associated with CNS alterations and with rapid epidemic spread. Considering that ZIKV infects Old World monkeys, the aim of this study was to follow its potential in neotropical primates. Here, we show the detection of ZIKV in marmosets and capuchin monkeys captured in Ceara state, Northeast Brazil. Nine (9/132) samples were positive by quantitative RT-PCR assay. Neutralizing antibodies in primates for ZIKV were also detected by PRNT. The ZIKV-positive samples were obtained from peridomestic animals captured in proximity to humans in areas with reports of ZIKV-associated microcephaly cases during the epidemic period. These results reiterate the molecular evidence of ZIKV infection in neotropical primates, and the temporal detection suggests that detection in primates occurred during the epidemic period in humans. However, a continuous surveillance is necessary to exclude the possibility of virus circulation and transmission in wild environments.

Granzyme A in Chikungunya and Other Arboviral Infections.

Granzyme A (GzmA) is secreted by cytotoxic lymphocytes and has traditionally been viewed as a mediator of cell death. However, a growing body of data suggests the physiological role of GzmA is promotion of inflammation. Here, we show that GzmA is significantly elevated in the sera of chikungunya virus (CHIKV) patients and that GzmA levels correlated with viral loads and disease scores in these patients. Serum GzmA levels were also elevated in CHIKV mouse models, with NK cells the likely source. Infection of mice deficient in type I interferon responses with CHIKV, Zika virus, or dengue virus resulted in high levels of circulating GzmA. We also show that subcutaneous injection of enzymically active recombinant mouse GzmA was able to mediate inflammation, both locally at the injection site as well as at a distant site. Protease activated receptors (PARs) may represent targets for GzmA, and we show that treatment with PAR antagonist ameliorated GzmA- and CHIKV-mediated inflammation.

Persistence and Intra-Host Genetic Evolution of Zika Virus Infection in Symptomatic Adults: A Special View in the Male Reproductive System.

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Persistence and intra-host genetic evolution of zika virus infection in symptomatic adults: A special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus

Characterisation of Zika virus infection in primary human astrocytes.

BACKGROUND: The recent Zika virus (ZIKV) outbreak has linked ZIKV with microcephaly and other central nervous system pathologies in humans. Astrocytes are among the first cells to respond to ZIKV infection in the brain and are also targets for virus infection. In this study, we investigated the interaction between ZIKV and primary human brain cortical astrocytes (HBCA). RESULTS: HBCAs were highly sensitive to representatives of both Asian and African ZIKV lineages and produced high viral yields. The infection was associated with limited immune cytokine/chemokine response activation; the highest increase of expression, following infection, was seen in CXCL-10 (IP-10), interleukin-6, 8, 12, and CCL5 (RANTES). Ultrastructural changes in the ZIKV-infected HBCA were characterized by electron tomography (ET). ET reconstructions elucidated high-resolution 3D images of the proliferating and extensively rearranged endoplasmic reticulum (ER) containing viral particles and virus-induced vesicles, tightly juxtaposed to collapsed ER cisternae. CONCLUSIONS: The results confirm that human astrocytes are sensitive to ZIKV infection and could be a source of proinflammatory cytokines in the ZIKV-infected brain tissue.

Yellow fever virus RNA in urine and semen of convalescent patient, Brazil

Yellow fever virus RNA is usually detected in blood of infected humans. We detected virus RNA in urine and semen samples from a convalescent patient. A complete virus genome was sequenced for an isolate from a urine sample. This virus had a South American I genotype and unique synapomorphic changes

Yellow Fever Virus RNA in Urine and Semen of Convalescent Patient, Brazil.

Yellow fever virus RNA is usually detected in blood of infected humans. We detected virus RNA in urine and semen samples from a convalescent patient. A complete virus genome was sequenced for an isolate from a urine sample. This virus had a South American I genotype and unique synapomorphic changes.

Modeling neuro-immune interactions during Zika virus infection.

Although Zika virus (ZIKV) infection is often asymptomatic, in some cases, it can lead to birth defects in newborns or serious neurologic complications in adults. However, little is known about the interplay between immune and neural cells that could contribute to the ZIKV pathology. To understand the mechanisms at play during infection and the antiviral immune response, we focused on neural precursor cells (NPCs)-microglia interactions. Our data indicate that human microglia infected with the current circulating Brazilian ZIKV induces a similar pro-inflammatory response found in ZIKV-infected human tissues. Importantly, using our model, we show that microglia interact with ZIKV-infected NPCs and further spread the virus. Finally, we show that Sofosbuvir, an FDA-approved drug for Hepatitis C, blocked viral infection in NPCs and therefore the transmission of the virus from microglia to NPCs. Thus, our model provides a new tool for studying neuro-immune interactions and a platform to test new therapeutic drugs.

Persistence and intra-host genetic evolution of Zika virus infection in symptomatic adults: a special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Yellow fever virus RNA in urine and semen of convalescent patient, Brazil

Yellow fever virus RNA is usually detected in blood of infected humans. We detected virus RNA in urine and semen samples from a convalescent patient. A complete virus genome was sequenced for an isolate from a urine sample. This virus had a South American I genotype and unique synapomorphic changes.

Persistence and intra-host genetic evolution of Zika virus infection in symptomatic adults: a special view in the male reproductive system

We followed the presence of Zika virus (ZIKV) in four healthy adults (two men and two women), for periods ranging from 78 to 298 days post symptom onset. The patients were evaluated regarding the presence of the virus in different body fluids (blood, saliva, urine and semen), development of immune responses (including antibodies, cytokines and chemokines), and virus genetic variation within samples collected from semen and urine during the infection course. The analysis was focused primarily on the two male patients who shed the virus for up to 158 days after the initial symptoms. ZIKV particles were detected in the spermatozoa cytoplasm and flagella, in immature sperm cells and could also be isolated from semen in cell culture, confirming that the virus is able to preserve integrity and infectivity during replication in the male reproductive system (MRS). Despite the damage caused by ZIKV infection within the MRS, our data showed that ZIKV infection did not result in infertility at least in one of the male patients. This patient was able to conceive a child after the infection. We also detected alterations in the male genital cytokine milieu, which could play an important role in the replication and transmission of the virus which could considerably increase the risk of ZIKV sexual spread. In addition, full genome ZIKV sequences were obtained from several samples (mainly semen), which allowed us to monitor the evolution of the virus within a patient during the infection course. We observed genetic changes over time in consensus sequences and lower frequency intra-host single nucleotide variants (iSNV), that suggested independent compartmentalization of ZIKV populations in the reproductive and urinary systems. Altogether, the present observations confirm the risks associated with the long-term replication and shedding of ZIKV in the MRS and help to elucidate patterns of intra-host genetic evolution during long term replication of the virus.

Yellow fever virus RNA in urine and semen of convalescent patient, Brazil

Yellow fever virus RNA is usually detected in blood of infected humans. We detected virus RNA in urine and semen samples from a convalescent patient. A complete virus genome was sequenced for an isolate from a urine sample. This virus had a South American I genotype and unique synapomorphic changes.

First Genome Sequences of Dengue Virus (DENV) Strains Isolated during the First DENV-4 Outbreak in São Paulo, Brazil.

Dengue virus (DENV) is an arbovirus belonging to the genus Flavivirus, family Flaviviridae In Brazil, the reemergence and spread of DENV type 4 (DENV-4) across the country were responsible for a significant outbreak in Guarujá, São Paulo, Brazil. Here, we report the first genomic sequences of DENV strains circulating in Guarujá during the 2013 outbreak.