Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 Replication by Hypertonic Saline Solution in Lung and Kidney Epithelial Cells.

An unprecedented global health crisis has been caused by a new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We performed experiments to test if a hypertonic saline solution was capable of inhibiting virus replication. Our data show that 1.2% NaCl inhibited virus replication by 90%, achieving 100% of inhibition at 1.5% in the nonhuman primate kidney cell line Vero, and 1.1% of NaCl was sufficient to inhibit the virus replication by 88% in human epithelial lung cell line Calu-3. Furthermore, our results indicate that the inhibition is due to an intracellular mechanism and not to the dissociation of the spike SARS-CoV-2 protein and its human receptor. NaCl depolarizes the plasma membrane causing a low energy state (high ADP/ATP concentration ratio) without impairing mitochondrial function, supposedly associated with the inhibition of the SARS-CoV-2 life cycle. Membrane depolarization and intracellular energy deprivation are possible mechanisms by which the hypertonic saline solution efficiently prevents virus replication in vitro assays.

Inhibition of severe acute respiratory syndrome coronavirus 2 replication by hypertonic saline solution in lung and kidney epithelial cells

An unprecedented global health crisis has been caused by a new virus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We performed experiments to test if a hypertonic saline solution was capable of inhibiting virus replication. Our data show that 1.2% NaCl inhibited virus replication by 90%, achieving 100% of inhibition at 1.5% in the nonhuman primate kidney cell line Vero, and 1.1% of NaCl was sufficient to inhibit the virus replication by 88% in human epithelial lung cell line Calu-3. Furthermore, our results indicate that the inhibition is due to an intracellular mechanism and not to the dissociation of the spike SARS-CoV-2 protein and its human receptor. NaCl depolarizes the plasma membrane causing a low energy state (high ADP/ATP concentration ratio) without impairing mitochondrial function, supposedly associated with the inhibition of the SARS-CoV-2 life cycle. Membrane depolarization and intracellular energy deprivation are possible mechanisms by which the hypertonic saline solution efficiently prevents virus replication in vitro assays.

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.

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

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.

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.

Rabies virus related to vampire bats (Desmodus rotundus) isolated from a crab-eating fox (Cerdocyon thous) in Southeast Brazil

In this study, rabies virus was isolated from a naturally infected crab-eating fox [Cerdocyon thous] in a rural area of São Paulo State, Brazil. The sample was positive for the standard techniques used for rabies diagnosis. The isolate was antigenically characterized using anti-nucleoprotein monoclonal antibodies. Studies on molecular epidemiology by patterns of nucleotide substitution in the nucleoprotein gene showed that the isolate was genetically homologous to the rabies variant maintained and transmitted by the common vampire bat Desmodus rotundus. This is the first report of detection of this variant from a crab-eating fox in Brazil and represents evidence of cross-species transmission. This was the unique report where this variant was isolated from a crab eating fox in Brazil.

Antibodies to rabies virus in terrestrial wild mammals in native rainforest on the north coast of São Paulo State, Brazil.

Rabies causes thousands of human and animal deaths worldwide each year. The emergent importance of rabies in wild animals demonstrates the necessity of epidemiologic studies of infection in these species toward the development of better strategies for prevention and control of rabies. We analyzed the circulation of rabies virus among wildlife species from a native rainforest in São Paulo State, Brazil. We used the rapid fluorescent focus inhibition test (RFFIT) to test for rabies virus-neutralizing antibodies in 139 captured terrestrial mammals and the fluorescent antibody test (FAT), mouse inoculation test (MIT), and reverse-transcriptase (RT)-PCR to test for virus in samples from the central nervous system of 53 animals found dead. The percentage of samples positive by RFFIT was 10.8%. All samples tested by FAT, MIT, and RT-PCR were negative. Research should be continued to obtain a better understanding of the role of wildlife in the circulation and transmission of rabies virus.

Heminested reverse-transcriptase polymerase chain reaction (hnRT-PCR) as a tool for rabies virus detection in stored and decomposed samples.

BACKGROUND: The use of methods, both sensitive and specific, for rabies diagnosis are important tools for the control and prophylaxis of the disease. Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) has been used in rabies diagnosis with good results, even in decomposed materials. Additionally, molecular techniques have been used for epidemiological studies and to gain a better knowledge of viral epidemiology. FINDINGS: The aim of this work was to evaluate the RT-PCR and hnRT-PCR for rabies virus detection in original tissues stored at -20 degrees C for different periods considering their use for rabies virus detection in stored and decomposed samples. RT-PCR and hnRT-PCR were evaluated in 151 brain samples from different animal species, thawed and left at room temperature for 72 hours for decomposition.The RT-PCR and hnRT-PCR results were compared with previous results from Direct Fluorescent Antibody Test and Mouse Inoculation Test. From the 50 positive fresh samples, 26 (52%) were positive for RT-PCR and 45 (90%) for hnRT-PCR. From the 48 positive decomposed samples, 17 (34, 3%) were positive for RT-PCR and 36 (75%) for hnRT-PCR. No false-positives results were found in the negatives samples evaluated to the molecular techniques. CONCLUSION: These results show that the hnRT-PCR was more sensitive than RT-PCR, and both techniques presented lower sensibility in decomposed samples. The hnRT-PCR demonstrated efficacy in rabies virus detection in stored and decomposed materials suggesting it's application for rabies virus retrospective epidemiological studies.