Antimicrobial Activity of Some Medicinal Herbs to the Treatment of Cutaneous and Mucocutaneous Infections: Preliminary Research.

(1) Background: Superficial, including cutaneous and mucocutaneous infections are a current public health problem with universal distribution. One of the main concerns, in the present/future, is fungal/bacterial infections by resistant microorganisms. This study aimed to verify if decoctions of coptidis (Coptis chinensis, Ranunculaceae family), neem (Azadirachta indica, Meliaceae family), and their essential oils (EOs), as well as the EO of manuka (Leptospermum scoparium, Myrtaceae family) have antimicrobial activity against prevalent species of microorganisms responsible for superficial infections. (2) Methods: The antimicrobial activity was determined by the minimum inhibitory concentration (MIC), using broth microdilution method, and minimum lethal concentration (MLC) was determined from subculture of MIC plates. (3) Results: C. chinensis EO and decoction demonstrated some antifungal action against the yeasts and dermatophytes tested. Greatest bactericidal effect against Propionibacterium acnes and some action against Staphylococcus aureus was observed. For A. indica only EO proved activity against dermatophytes and P. acnes. L. scoparium EO showed the broadest antimicrobial spectrum with activity against bacteria, yeasts, and dermatophytes showing greater activity against P. acnes and S. aureus. (4) Conclusions: C. chinensis (EO/decoction), EOs of L. scoparium and A. indica proved in vitro efficacy against fungal, bacterial, or mixed agents of superficial infections, either by sensitive or resistant strains.

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern.

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/µL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Educational Approach to Prevent the Burden of Vaccinia Virus Infections in a Bovine Vaccinia Endemic Area in Brazil.

Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonotic disease characterized by exanthematous lesions on the teats of dairy cows and the hands of milkers, and is an important public health issue in Brazil and South America. BV also results in economic losses to the dairy industry, being a burden to the regions involved in milk production. In the past 20 years, much effort has been made to increase the knowledge regarding BV epidemiology, etiologic agents, and interactions with the hosts and the environment. In the present study, we evaluated milking practices that could be associated with VACV infections in an endemic area in Brazil and proposed an educational tool to help prevent VACV infections. In our survey, 124 individuals (51.7%) from a total of 240 had previously heard of BV, 94 of which knew about it through BV outbreaks. Although most individuals involved in dairy activities (n = 85/91) reported having good hygiene practices, only 29.7% used adequate disinfecting products to clean their hands and 39.5% disinfected cows' teats before and after milking. Furthermore, 46.7% of individuals reported having contact with other farm and domestic animals besides dairy cattle. We also evaluated the presence of IgG and IgM antibodies in the surveyed population. Overall, 6.1% of likely unvaccinated individuals were positive for anti-Orthopoxvirus IgG antibodies, and 1.7% of all individuals were positive for IgM antibodies. Based on our findings, we proposed educational materials which target individuals with permanent residence in rural areas (mainly farmers and milkers), providing an overview and basic information about preventive measures against VACV infections that could enhance BV control and prevention efforts, especially for vulnerable populations located in endemic areas.

Exposure of free-ranging capybaras (Hydrochoerus hydrochaeris) to the vaccinia virus.

The aim of this study was to evaluate the possibility of free-ranging animals/hunting dogs as sources of infection in the vaccinia virus (VACV) transmission chain. Serological, cell culture and molecular assays were conducted in 56 free-ranging animals and 22 hunting dogs. ELISA/neutralizing assays showed that two (2.5%) capybaras (Hydrochoerus hydrochaeris) had anti-OPV positive antibodies, while all samples tested negative through PCR/cell culture. After being hit by cars on roads, capybaras that exhibited neither clinical signs nor any association with bovine outbreaks had neutralizing antibodies against the Orthopoxvirus, as detected through plaque-reduction neutralizing tests and ELISA. Evidence exists regarding peridomestic capybaras acting as a source of the virus and serving as a link between wild and urban environments, thus contributing to viral maintenance.

Silent Circulation of the Saint Louis Encephalitis Virus among Humans and Equids, Southeast Brazil.

Saint Louis encephalitis virus (SLEV) is a mosquito-borne flavivirus that occurs throughout the Americas, and is considered a public health threat. In Brazil, SLEV has been detected from human cases associated with dengue-like disease, but no neurological symptoms were reported. Furthermore, the epidemiology of SLEV in human populations is still poorly explored in the country. We reported serological and molecular detection of SLEV in a healthy population of equids and humans from rural areas in Southeast Brazil. A plaque reduction neutralization test was applied, and neutralizing antibodies were detected in 11 individuals (4.6%) and 60 horses (21.5%). A qPCR targeting the 5'UTR region and reverse transcription-PCR (RT-PCR) targeting the non-structural protein (NS5) gene were performed and three individuals tested positive in both assays. Subsequent phylogenetic analysis confirmed SLEV circulation and its findings suggest the occurrence of an asymptomatic or subclinical presence in human and animal cases, correlating with the risks for outbreaks and consequently burden of SLEV infections to public health. Preventive strategies should include improved surveillance in regions with a high probability of SLEV occurrence, improvement in diagnostic methods, and evaluation of exposure/risk factors that can favor SLEV emergence.

Trapping the Enemy: Vermamoeba vermiformis Circumvents Faustovirus Mariensis Dissemination by Enclosing Viral Progeny inside Cysts.

Viruses depend on cells to replicate and can cause considerable damage to their hosts. However, hosts have developed a plethora of antiviral mechanisms to counterattack or prevent viral replication and to maintain homeostasis. Advantageous features are constantly being selected, affecting host-virus interactions and constituting a harsh race for supremacy in nature. Here, we describe a new antiviral mechanism unveiled by the interaction between a giant virus and its amoebal host. Faustovirus mariensis infects Vermamoeba vermiformis, a free-living amoeba, and induces cell lysis to disseminate into the environment. Once infected, the cells release a soluble factor that triggers the encystment of neighbor cells, preventing their infection. Remarkably, infected cells stimulated by the factor encyst and trap the viruses and viral factories inside cyst walls, which are no longer viable and cannot excyst. This unprecedented mechanism illustrates that a plethora of antiviral strategies remains to be discovered in nature.IMPORTANCE Understanding how viruses of microbes interact with its hosts is not only important from a basic scientific point of view but also for a better comprehension of the evolution of life. Studies involving large and giant viruses have revealed original and outstanding mechanisms concerning virus-host relationships. Here, we report a mechanism developed by Vermamoeba vermiformis, a free-living amoeba, to reduce Faustovirus mariensis dissemination. Once infected, V. vermiformis cells release a factor that induces the encystment of neighbor cells, preventing infection of further cells and/or trapping the viruses and viral factories inside the cyst walls. This phenomenon reinforces the need for more studies regarding large/giant viruses and their hosts.

Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles.

Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.

Silent Orthohantavirus Circulation Among Humans and Small Mammals from Central Minas Gerais, Brazil.

New World orthohantaviruses are emerging RNA viruses that cause hantavirus cardiopulmonary syndrome (HCPS). These viruses are a burden to public health around the world with a lethality rate of around 60%. In South America, rodents of Sigmodontinae subfamily are the main reservoirs of orthohantaviruses. We described a serosurvey for orthohantaviruses circulation in an apparently healthy human population and small mammals from rural areas in Central Minas Gerais State, Brazil. A total of 240 individuals and 50 small mammals (26 rodents belonging to 10 different species and 24 marsupials from 4 different species) were sampled during 2012-2013. The seroprevalence rates of IgG/IgM antibodies in humans were 7.1 and 1.6%, respectively. Only one rodent, an Oligoryzomys nigripes captured in peridomestic area, tested positive for IgG antibodies and viral RNA. Our findings suggest a silent circulation of orthohantaviruses in a region of intensive agriculture production. The detection of seropositive humans in an area with a lack of previous HCPS reports highlights potential oligosymptomatic cases and the need for surveillance strategies that could reduce the risk of future outbreaks.

Serological Evidence of Orthopoxvirus Circulation Among Equids, Southeast Brazil.

Since 1999 Vaccinia virus (VACV) outbreaks involving bovines and humans have been reported in Brazil; this zoonosis is known as Bovine Vaccinia (BV) and is mainly an occupational disease of milkers. It was only in 2008 (and then again in 2011 and 2014) however, that VACV was found causing natural infections in Brazilian equids. These reports involved only equids, no infected humans or bovines were identified, and the sources of infections remain unknown up to date. The peculiarities of Equine Vaccinia outbreaks (e.g., absence of human infection), the frequently shared environments, and fomites by equids and bovines in Brazilian farms and the remaining gaps in BV epidemiology incited a question over OPV serological status of equids in Brazil. For this report, sera from 621 equids - representing different species, ages, sexes and locations of origin within Minas Gerais State, southeast Brazil - were examined for the presence of anti-Orthopoxvirus (OPV) antibodies. Only 74 of these were sampled during an Equine Vaccinia outbreak, meaning some of these specific animals presented typical lesions of OPV infections. The majority of sera, however, were sampled from animals without typical signs of OPV infection and during the absence of reported Bovine or Equine Vaccinia outbreaks. Results suggest the circulation of VACV among equids of southeast Brazil even prior to the time of the first VACV outbreak in 2008. There is a correlation of OPVs outbreaks among bovines and equids although many gaps remain to our understanding of its nature. The data obtained may even be carefully associated to recent discussion over OPVs history. Moreover, data is available to improve the knowledge and instigate new researches regarding OPVs circulation in Brazil and worldwide.

Ubiquitous giants: a plethora of giant viruses found in Brazil and Antarctica.

BACKGROUND: Since the discovery of giant viruses infecting amoebae in 2003, many dogmas of virology have been revised and the search for these viruses has been intensified. Over the last few years, several new groups of these viruses have been discovered in various types of samples and environments.In this work, we describe the isolation of 68 giant viruses of amoeba obtained from environmental samples from Brazil and Antarctica. METHODS: Isolated viruses were identified by hemacolor staining, PCR assays and electron microscopy (scanning and/or transmission). RESULTS: A total of 64 viruses belonging to the Mimiviridae family were isolated (26 from lineage A, 13 from lineage B, 2 from lineage C and 23 from unidentified lineages) from different types of samples, including marine water from Antarctica, thus being the first mimiviruses isolated in this extreme environment to date. Furthermore, a marseillevirus was isolated from sewage samples along with two pandoraviruses and a cedratvirus (the third to be isolated in the world so far). CONCLUSIONS: Considering the different type of samples, we found a higher number of viral groups in sewage samples. Our results reinforce the importance of prospective studies in different environmental samples, therefore improving our comprehension about the circulation anddiversity of these viruses in nature.

Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles

ABSTRACT Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.

Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles

ABSTRACT Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.

Serologic and Molecular Evidence of Vaccinia Virus Circulation among Small Mammals from Different Biomes, Brazil.

Vaccinia virus (VACV) is a zoonotic agent that causes a disease called bovine vaccinia, which is detected mainly in milking cattle and humans in close contact with these animals. Even though many aspects of VACV infection have been described, much is still unknown about its circulation in the environment and its natural hosts/reservoirs. To investigate the presence of Orthopoxvirus antibodies or VACV DNA, we captured small rodents and marsupials in 3 areas of Minas Gerais state, Brazil, and tested their samples in a laboratory. A total of 336 animals were tested; positivity ranged from 18.1% to 25.5% in the 3 studied regions located in different biomes, including the Atlantic Forest and the Cerrado. Analysis of nucleotide sequences indicated co-circulation of VACV groups I and II. Our findings reinforce the possible role played by rodents and marsupials in VACV maintenance and its transmission chain.

Evaluating anti-Orthopoxvirus antibodies in individuals from Brazilian rural areas prior to the bovine vaccinia era.

Vaccinia virus naturally circulates in Brazil and is the causative agent of a zoonotic disease known as bovine vaccinia (BV). We retrospectively evaluated two populations from the Amazon and Southeast Regions. BV outbreaks had not been reported in these regions before sample collection. Neutralising antibodies were found in 13 individuals (n = 132) with titres ranging from 100 ≥ 6,400 neutralising units/mL. Univariate analysis identified age and vaccination as statistically significant risk factors in individuals from the Southeast Region. The absence of detectable antibodies in vaccinated individuals raises questions about the protection of smallpox vaccine years after vaccination and reinforces the need for surveillance of Orthopoxvirus in Brazilian populations without evidence of previous outbreaks.

Group 2 vaccinia virus, Brazil.

In 2011, vaccinia virus caused an outbreak of bovine vaccinia, affecting dairy cattle and dairy workers in Brazil. Genetic and phenotypic analyses identified this isolate as distinct from others recently identified, thereby reinforcing the hypothesis that different vaccinia virus strains co-circulate in Brazil.

Perfil sorológico, viremia e eliminação do circovírus suíno tipo 2 em animais naturalmente infectados pertencentes a granjas com ou sem a síndrome da refugagem multissistêmica / Serological profile, viremia and PCV2 shedding in naturally infected pigs of herds with or without postweaning multisystemic wasting syndrome

Um estudo virológico e sorológico seccional (E1) e outro longitudinal (E2) foram realizados em granjas com (G2 e G3) e sem (G1) a síndrome de refugagem multissitêmica (SRM) no Brasil. Foram coletadas amostras de sangue, soro, swabs nasal e retal de animais de cada categoria do ciclo produtivo: porcas, leitões maternidade, creche, recria e terminação. Em E1, nas granjas G1a e G2, foram amostrados 40 animais de cada categoria. Em E2, nas granjas G1b e G3, 35 leitões na maternidade foram identificados e amostrados ao longo do ciclo produtivo. O soro foi avaliado para presença de anticorpos contra circovírus suíno tipo 2 (CVS2) e sangue e swabs para presença do ácido nucléico viral. Em E1, a categoria porcas possuía altas taxas de animais virêmicos e soropositivos, com porcentagem de porcas com títulos altos superior a G2. Em G1a a queda de imunidade passiva ocorreu entre o final da fase de creche e início da recria com aumento da eliminação viral em swabs e subsequente soroconversão. Em G2 a queda ocorreu entre a fase final da maternidade e início da creche, com diminuição da eliminação viral. Em E2, a queda da imunidade materna ocorreu entre a 1ª e 2ª coleta em G1b; e em G3, entre a 2ª e 3ª coleta. Em ambas as granjas, a queda de imunidade passiva coincidiu com o aumento da viremia e eliminação viral e a soroconversão ocorreu entre a 3ª e 4a coleta em ambas as granjas com aumento da média de título de anticorpos e declínio da viremia. Viremia e eliminação viral foram detectadas em todas as coletas realizadas; 42% dos animais amostrados em E2 foram virêmicos em todas as coletas e todas as amostras de tecido coletadas no abate foram positivas para o CVS2. Este estudo confirma a persistência da viremia mesmo em presença de altos títulos de anticorpos e que o perfil sorológico em um rebanho com e sem a presença da síndrome pode ser diferente, principalmente em relação à duração da imunidade passiva.

A virological and serological cross-sectional study (E1) and a longitudinal study (E2) were performed on herds with (G2 and G3) and without (G1) post weaning multisystemic syndrome (PMWS) in Brazil. Blood, serum, nasal and rectal swabs samples were collected of sows, farrowing piglets, nursery, growing and finishing pigs. In E1, were sampled 40 animals in each category (G1a and G2). In E2, (G1b and G3), 35 farrowing piglets were identified and sampled along the production cycle. Porcine circovirus type 2 (PCV2) antibodies were assayed. A PCR was used to detected PCV2 genome in blood and swabs. In E1, sows had high rates of viremic and seropositives animals, with percentage of sows with high antibodies titers greater than G2. Passive antibodies decline occurred between nursery and growing area with increased viral shedding in swabs and subsequent seroconversion in G1. In G2, the passive antibodies decay occurred in nursery, with a reduction in viral shedding. In E2, the decline of maternal immunity occurred between the 1st and 2nd collection in G1b, and between 2nd and 3rd collections in G3. In both herds, the decay of passive immunity coincided with increased viremia and viral shedding; and seroconversion occurred between the 3rd and 4th collection in both herds with decline of viremia. Viremia and viral shedding was detected in all samples days, 42% of animals sampled in E2 were viremic and all tissue samples collected at slaughterwere positive for PCV2. This study confirms the persistence of viremia even in the presence of high titers of antibodies and the serological profile in a herd with or without PMWS may be different, especially with regard to the passive immunity duration.

Vaccinia virus infection in monkeys, Brazilian Amazon.

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil.