Retrospective Investigation in Horses with Encephalitis Reveals Unnoticed Circulation of West Nile Virus in Brazil.

During these past years, several studies have provided serological evidence regarding the circulation of West Nile virus (WNV) in Brazil. Despite some reports, much is still unknown regarding the genomic diversity and transmission dynamics of this virus in the country. Recently, genomic monitoring activities in horses revealed the circulation of WNV in several Brazilian regions. These findings on the paucity of genomic data reinforce the need for prompt investigation of WNV infection in horses, which may precede human cases of encephalitis in Brazil. Thus, in this study, we retrospectively screened 54 suspicious WNV samples collected between 2017 and 2020 from the spinal cord and brain of horses with encephalitis and generated three new WNV genomes from the Ceará and Bahia states, located in the northeastern region of Brazil. The Bayesian reconstruction revealed that at least two independent introduction events occurred in Brazil. The first introduction event appears to be likely related to the North American outbreak, and was estimated to have occurred in March 2013.The second introduction event appears to have occurred in September 2017 and appears to be likely related to the South American outbreak. Together, our results reinforce the importance of increasing the priority of WNV genomic monitoring in equines with encephalitis in order to track the dispersion of this emerging pathogen through the country.

West Nile Virus in Brazil.

Background: West Nile virus (WNV) was first sequenced in Brazil in 2019, when it was isolated from a horse in the Espírito Santo state. Despite multiple studies reporting serological evidence suggestive of past circulation since 2004, WNV remains a low priority for surveillance and public health, such that much is still unknown about its genomic diversity, evolution, and transmission in the country. Methods: A combination of diagnostic assays, nanopore sequencing, phylogenetic inference, and epidemiological modeling are here used to provide a holistic overview of what is known about WNV in Brazil. Results: We report new genetic evidence of WNV circulation in southern (Minas Gerais, São Paulo) and northeastern (Piauí) states isolated from equine red blood cells. A novel, climate-informed theoretical perspective of the potential transmission of WNV across the country highlights the state of Piauí as particularly relevant for WNV epidemiology in Brazil, although it does not reject possible circulation in other states. Conclusion: Our output demonstrates the scarceness of existing data, and that although there is sufficient evidence for the circulation and persistence of the virus, much is still unknown on its local evolution, epidemiology, and activity. We advocate for a shift to active surveillance, to ensure adequate preparedness for future epidemics with spill-over potential to humans.

Swine influenza A virus subtypes circulating in Brazilian commercial pig herds from 2012 to 2019.

The swine influenza A virus (SIAV) subtypes/lineages H1N1pdm09, H3N2, H1N2, and H1N1 of seasonal human origin are widespread in Brazilian swine herds. A monovalent inactivated H1N1pdm09 vaccine was licensed in Brazil in 2014. However, there are concerns about its efficacy due to the limited vaccine cross-protection against heterologous viruses and the potential for exacerbated reactions against vaccine strains. Thus, monitoring SIAVs subtypes/lineages that are circulating in the Brazilian swine population is important, by applying a fast and efficient diagnostic test in herd field samples. A RT-PCR assay was developed, using primers specific for HA subtyping of Brazilian SIAV, and was used to evaluate the occurrence of subtypes from samples collected between 2012 and 2019. From 167 field samples positive for influenza A, 117 were subtyped by nested RT-PCR assay. A higher occurrence of H1N1pdm was observed from 2012 to 2015, H3N2 in 2017, and H1hu in 2017 to 2019. A hemagglutination inhibition test was performed in serum samples received from 2017 to 2019, confirming these data. The molecular data highlights the importance of H1hu and H3N2 detection since there are no vaccines available for the subtypes/lineages and raises an alert of H1hu for its potential to infect humans. Serological data suggest a cyclical profile of occurrence between the H3N2 and H1N1pdm over time. Monitoring SIAVs circulating in Brazilian swine herds is necessary, which provides the relevant information for field veterinarians to apply effective control measures on the properties.

Enteric Organisms Detected in Feces of Dogs With Bloody Diarrhea: 45 Cases.

Bloody diarrhea is a common condition in dogs, but studies evaluating the enteropathogens involved specifically in adult dogs are scarce. In the present study, stool samples from 45 adult dogs with bloody diarrhea were evaluated for the four enteric organisms mainly reported in these cases: canine parvovirus type 2 (CPV-2), Clostridioides difficile, Clostridium perfringens, and Salmonella spp. In addition, the samples were also tested for coronavirus, rotavirus, Giardia spp., and Escherichia coli pathotypes to provide a better understanding of possible co-occurrence. Vaccination status, diet, and clinical outcome were also obtained when available. CPV-2b was identified in 17 dogs (37.8%), being the most frequent cause of bloody diarrhea, including completely vaccinated adult dogs. Toxigenic C. difficile and C. perfringens netF+ were detected in 6 (13.3%) and 5 (11.1%) dogs, in some cases in a co-occurrence with other enteric organisms. Three fatal cases of salmonellosis were identified in dogs fed a raw meat-based diet, raising the risks associated with this increasing practice.

Viability of SARS-CoV-2 in river water and wastewater at different temperatures and solids content.

COVID-19 patients can excrete viable SARS-CoV-2 virus via urine and faeces, which has raised concerns over the possibility of COVID-19 transmission via aerosolized contaminated water or via the faecal-oral route. These concerns are especially exacerbated in many low- and middle-income countries, where untreated sewage is frequently discharged to surface waters. SARS-CoV-2 RNA has been detected in river water (RW) and raw wastewater (WW) samples. However, little is known about SARS-CoV-2 viability in these environmental matrices. Determining the persistence of SARS-CoV-2 in water under different environmental conditions is of great importance for basic assumptions in quantitative microbial risk assessment (QMRA). In this study, the persistence of SARS-CoV-2 was assessed using plaque assays following spiking of RW and WW samples with infectious SARS-CoV-2 that was previously isolated from a COVID-19 patient. These assays were carried out on autoclaved RW and WW samples, filtered (0.22 µm) and unfiltered, at 4 °C and 24 °C. Linear and nonlinear regression models were adjusted to the data. The Weibull regression model achieved the lowest root mean square error (RMSE) and was hence chosen to estimate T90 and T99 (time required for 1 log and 2 log reductions, respectively). SARS-CoV-2 remained viable longer in filtered compared with unfiltered samples. RW and WW showed T90 values of 1.9 and 1.2 day and T99 values of 6.4 and 4.0 days, respectively. When samples were filtered through 0.22 µm pore size membranes, T90 values increased to 3.3 and 1.5 days, and T99 increased to 8.5 and 4.5 days, for RW and WW samples, respectively. Remarkable increases in SARS-CoV-2 persistence were observed in assays at 4 °C, which showed T90 values of 7.7 and 5.5 days, and T99 values of 18.7 and 17.5 days for RW and WW, respectively. These results highlight the variability of SARS-CoV-2 persistence in water and wastewater matrices and can be highly relevant to efforts aimed at quantifying water-related risks, which could be valuable for understanding and controlling the pandemic.

High positivity values for bovine leukemia virus in human breast cancer cases from Minas Gerais, Brazil.

Bovine leukemia virus (BLV) is a retrovirus that causes lymphoma in cattle worldwide and has also been associated with breast cancer in humans. The mechanism of BLV infection in humans and its implication as a primary cause of cancer in women are not known yet. BLV infection in humans may be caused by the consumption of milk and milk-products or meat from infected animals. Breast cancer incidence rates in Brazil are high, corresponding to 29.5% a year of cancer cases among women. In 2020, an estimated 66,280 new cases of breast cancer are expected, whereas in 2018 breast cancer has led to 17,572 deaths, the highest incidence and lethality among cancers in women in this country that year. BLV infection occurrence ranges from 60 to 95% in dairy herds. In addition, there are some regions, such as the Minas Gerais State, southeastern Brazil, where the population traditionally consume unpasteurized dairy products. Taken together, this study aimed to verify if there is a higher association between breast cancer and the presence of BLV genome in breast tissue samples within this population that consumes raw milk from animals with high rates of BLV infection. A molecular study of two BLV genes was carried out in 88 breast parenchyma samples, between tumors and controls. The amplified fragment was subjected to BLV proviral sequencing and its identity was confirmed using GenBank. BLV proviral genes were amplified from tumor breast parenchyma samples and healthy tissue control samples from women, revealing a 95.9% (47/49) and 59% (23/39) positivity, respectively. Our results show the highest correlation of BLV and human breast cancer found in the world to date within the population of Minas Gerais, Brazil.

Parrot bornavirus in naturally infected Brazilian captive parrots: Challenges in viral spread control.

Psittaciform orthobornaviruses are currently considered to be a major threat to the psittacine bird population worldwide. Parrot bornavirus (PaBV) was identified recently in Brazil and, since then, few studies have been conducted to understand the epidemiology of PaBV in captive psittacine birds. In the present study, natural infections by PaBV in South American parrots were investigated in two breeding facilities: commercial (A) and conservationist (B). Thirty-eight psittacine of 21 different species were presented for postmortem examination. Tissue samples were collected and investigated for the presence of PaBV-RNA using RT-PCR. In addition, clinical information about these birds was used when available. PaBV infection was detected in 73.7% of all birds investigated, indicating a wide dissemination of this virus in both facilities. From birds investigated in aviary A, 66.7% showed clinical signs, 100% had typical lesions of proventricular dilatation disease (PDD), 100% had mild to severe proventricular dilatation and 88.9% were PaBV-positive. In birds from aviary B, 27.6% showed clinical signs, 65.5% had typical lesions of PDD, 62% had mild to severe proventricular dilatation and 69% were PaBV-positive. Neurological disease was observed more frequently than gastrointestinal disease. Sequencing analysis of the matrix gene fragment revealed the occurrence of genotype 4 (PaBV-4) in both places. About 15.8% of birds in this study are threatened species. We discussed the difficulties and challenges for controlling viral spread in these aviaries and implications for South American psittacine conservation. These results emphasize the urgent need to develop a national regulatory and health standard for breeding psittacine birds in the country.

West Nile virus associated with equid encephalitis in Brazil, 2018.

Mosquito-borne arboviruses are a major public health concern worldwide and are responsible for emerging and re-emerging diseases. Taken together, the arboviruses have a strong impact on public health and are the most common causes of equine encephalitis. In-depth diagnostic investigation of equine viral encephalitis is of utmost importance for the epidemiological surveillance and control of this disease. Regarding neurological disorders in equids, in April-May 2018, at least 12 cases of equid mortality with acute neurological signs were reported in six farms from Espirito Santo state, Brazil. To investigate the aetiological agent of this neurological disease outbreak, central nervous system (CNS) fragments from two horses and two donkeys were submitted for virologic diagnosis. Rabies, equine herpesvirus-1, and arbovirus-associated encephalomyelitis were investigated using differential diagnosis techniques. West Nile virus (WNV) was detected by nested RT-PCR in CNS fragments from each of the four animals in the study and confirmed by nucleotide sequencing. This is the first case of neurological disease in equids confirmed to be associated with WNV infection in Brazil. This finding unveils a new and urgent field of research and the need to understand the epidemiological and clinical characteristics of the disease and the risk to public health.

Clostridium perfringens and C. difficile in parvovirus-positive dogs.

The aim of this study was to investigate Clostridium difficile and Clostridium perfringens in 82 diarrheic dogs positive for canine parvovirus type 2 (CPV). Enterotoxigenic C. perfringens type A was isolated from three (3.6%) dogs. One (1.2%) strain was also positive for NetE- and NetF-encoding genes, which are commonly associated with diarrhea in dogs. Toxigenic C. difficile was isolated from one animal (1.2%), which was also positive for A/B toxins. The present study identified C. difficile and C. perfringens infection in CPV-positive dogs. Further studies are necessary to clarify if clostridial infections may predispose or potentiate CPV-infection in dogs or vice versa.

Bovine vaccinia: Inactivated Vaccinia virus vaccine induces protection in murine model.

Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions on the teats of dairy cows and the milkers' hands. Since 1999, due to the occurrence of many BV outbreaks in dairy farms across all Brazilian regions, there is a need to improve the control and prevention measures of the disease. Vaccination is one of the major tools to prevent viral diseases, and it could be an alternative for BV prevention. The main objective of this study was the development of vaccine formulations against BV using the inactivated VACV strain GP2 as antigen combined with different adjuvants. Potency tests were performed in mice, which were vaccinated with two doses at a 21-day interval, and then challenged with the vaccine homologous virus. VACV strain GP2 inactivated by beta-propiolactone (BPL) in association with adjuvants was effective in inducing a humoral immune response against VACV, as measured by neutralizing antibody (NA) titers, and was variable depending on the adjuvant used in each vaccine formulation. The vaccine formulation containing aluminum hydroxide (AH) associated with saponin as adjuvant induced the production of high NA titers in all vaccinated mice, giving 100% protection in Balb/c murine model after challenge with homologous virus.

Epidemiology of a Bluetongue outbreak in a sheep flock in Brazil.

In January 2013, an outbreak of Bluetongue (BT) a ecting a Lacaune sheep ock occurred in Vassouras, Rio de Janeiro state, Brazil. From March to August 2013, blood samples collection and clinical examination were performed monthly, in order to monitor the epidemiological pro le of Bluetongue virus (BTV) circulation and clinical disease in the ock. Agar gel immunodi usion (AGID) and reverse transcription polymerase chain reaction (RT-PCR) targeting BTV segment 10 were used as diagnostic assays. Additionally, insect trapping was conducted in the farm from May to July 2013. The ock serological prevalence to BTV was 80% since the rst month of monitoring, with a variation in the serological rate depending on the sheep age categories. The number of susceptible lambs increased with time, probably due to the decrease of passive immunity. Viral RNA was detected in blood samples, demonstrating viral circulation, prolonged viraemia, and potential source for virus transmission in the region, even in a dry and cool season. The presence of Culicoides pusillus and Culicoides insignis was con rmed in the farm. The emergence of this outbreak in a Brazilian endemic area for BTV emphasises an urgent need of animal surveillance and BTV epidemiological studies.

Genome Sequence of Bluetongue virus Serotype 17 Isolated in Brazil in 2014.

The complete genome sequence of Bluetongue virus (BTV) serotype 17 strain 17/BRA/2014/73, isolated from a sheep in Brazil in 2014, is reported here. All segments clustered with western topotype strains and indicated reassortment events with other BTV from the Americas. The strain 17/BRA/2014/73 represents a novel reference strain for BTV-17 from South America.

Protective immunity and safety of a genetically modified influenza virus vaccine.

Recombinant influenza viruses are promising viral platforms to be used as antigen delivery vectors. To this aim, one of the most promising approaches consists of generating recombinant viruses harboring partially truncated neuraminidase (NA) segments. To date, all studies have pointed to safety and usefulness of this viral platform. However, some aspects of the inflammatory and immune responses triggered by those recombinant viruses and their safety to immunocompromised hosts remained to be elucidated. In the present study, we generated a recombinant influenza virus harboring a truncated NA segment (vNA-Δ) and evaluated the innate and inflammatory responses and the safety of this recombinant virus in wild type or knock-out (KO) mice with impaired innate (Myd88 -/-) or acquired (RAG -/-) immune responses. Infection using truncated neuraminidase influenza virus was harmless regarding lung and systemic inflammatory response in wild type mice and was highly attenuated in KO mice. We also demonstrated that vNA-Δ infection does not induce unbalanced cytokine production that strongly contributes to lung damage in infected mice. In addition, the recombinant influenza virus was able to trigger both local and systemic virus-specific humoral and CD8+ T cellular immune responses which protected immunized mice against the challenge with a lethal dose of homologous A/PR8/34 influenza virus. Taken together, our findings suggest and reinforce the safety of using NA deleted influenza viruses as antigen delivery vectors against human or veterinary pathogens.

Isolation of saint louis encephalitis virus from a horse with neurological disease in Brazil.

St. Louis encephalitis virus (SLEV) is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil.

Transcription of pattern recognition receptors and abortive agents induced chemokines in the bovine pregnant uterus.

Pattern recognition receptors (PRRs) are important components of the innate immune system whose ligands are specific pathogen associated molecular patterns (PAMPs). Considering the scarcity of studies on transcription of PRRs in the pregnant uterus of cows, and its response to PAMPs and microorganisms that cause abortion in cattle, this study aimed to characterize the transcription of TLR1-10, NOD1, NOD2 and MD2 in bovine uterus throughout gestation and to investigate the sensitivity of different uterine tissues at third trimester of pregnancy to purified TLR ligands or heat-killed Brucella abortus, Salmonella enterica serotype Dublin (S. Dublin), Listeria monocytogenes, and Aspergillus fumigatus, by assessing chemokine transcription. RNA extracted from endometrium, placentome and intercotiledonary region of cows at the first (n=6), second (n=6), and third (n=6) trimesters of pregnancy were subjected to real time RT-PCR. After stimulation of endometrium and intercotiledonary regions with purified TLR ligands or heat-killed microorganisms, gene transcription was assessed by real time RT-PCR. In the placentome, there was no significant variation in TLRs transcription throughout the three trimesters of pregnancy. In the endometrium, there was significant variation in TLR4 and TLR5 transcription during the three stages of gestation; i.e. TLR4 transcription was higher during the third trimester, whereas TLR5 transcription was higher during the last two trimesters. In the intercotiledonary region, there was significant variation in transcription of TLR1/6, TLR7, and TLR8, which were more strongly expressed during the first trimester of pregnancy. At the third trimester of gestation, significant transcription of CXCL6 and CXCL8 was detected mostly in endometrial tissues in response to purified TLR4 and TLR2 ligands. Transcription of these chemokines was induced in the endometrium and intercotiledonary region at the third trimester of pregnancy when stimulated with heat-killed B. abortus or S. Dublin. Therefore, this study demonstrates that some PRRs are expressed in the uterus during pregnancy, which coincides with its ability to respond to stimulation with TLRs ligands as well as heat-killed organisms known to cause abortion in cattle.

Sorologia, reação em cadeia da polimerase e histopatologia para leptospirose em amostras coletadas de vacas leiteiras, em matadouro da região de Parnaíba, Piauí / Serology, polymerase chain reaction and histopathology for leptospirosis in samples collected at slaughter from dairy cows of Parnaiba region, state of Piauí, Brazil

The presence of anti leptospiral agglutinins (microscopic agglutination test - MAT) and DNA of leptospires was investigated in the kidney and urine (Polymerase Chain Reaction - PCR) in samples collected at the time of slaughter of cattle originating from the dairy basin of Parnaíba, Piauí, Brazil, as also the lesions in kidney, lung, liver, uterus, ovary and placenta (histopathology and immunohistochemistry). In the MAT, Hardjo was the predominant serovar with the highest number of reagent animals for the strain Hardjobovis/Sponselee. Anti-leptospiral antigens were scored in epithelial cells, interstitial vascular endothelium, endothelium of glomerular capillaries and Bowman's capsule of 20 positive animals. Inflammatory cells were more common in the kidney. PCR was positive in urine and kidney tissue.

Foi investigada a presença de aglutininas anti leptospiras (reação de soroaglutinação microscópica - SAM), de DNA de leptospiras no rim e na urina (reação de cadeia pela polimerase - PCR), bem como de lesões no rim, pulmão, fígado, útero, ovário e placenta (histopatologia e imunohistoquímica), em materiais colhidos, por ocasião do abate, de bovinos originários da bacia leiteira da região de Parnaíba, Piauí, Brasil. Na SAM o sorovar predominante foi o Hardjo com maior número de animais reagentes para a estirpe Hardjobovis/Sponselee. Antígenos anti leptospira foram marcados em 20 animais positivos nas células epiteliais e do endotélio vascular, endotélio dos capilares glomerulares e na cápsula de Bowman, somente nos animais infectados. O infiltrado inflamatório foi maior no rim do que nos demais órgãos. A PCR foi positiva em amostras de urina e tecido renal.

Outbreak of human herpesvirus type 1 infection in nonhuman primates (Callithrix penincillata).

Human herpesvirus type 1 (HHV-1) is widely dispersed among the human population. Although infection is often asymptomatic in humans, nonhuman primates develop a severe and often fatal infection. In August 2006, 13 black-tufted marmosets (Callithrix penincillata) from a group of 14 presented with clinical apathy, anorexia, and ataxia. Physical examination revealed conjunctivitis, erosive or ulcerative lesions on the skin, and swollen lymph nodes. Of the 14 animals captured, 10 died. Grossly, ulcers and erosions were observed on the skin of face, nasal planum, lips, and oral mucosa. Histologically, superficial vesicular and erosive stomatitis with associated basophilic intranuclear inclusion bodies in the squamous epithelium were observed. Swabs from oral lesions and tissue samples from necropsied animals were positive for HHV-1 by nested polymerase chain reaction for eight animals.

Postpartum toll-like receptors and ß-defensin 5 mRNA levels in the endometrium of Holstein cows.

Toll-like receptors (TLRs) and ß-defensins are important components of the innate immune system. This study aimed to evaluate endometrial mRNA levels of TLRs (1/6, 2, 4, and 5) and ß-defensin 5 in Holstein cows by quantitative real time RT-PCR. Uterine biopsies were performed from 6 to 12 h after parturition, and cows were divided into two groups: (i) cows with placental retention and clinical signs of uterine infection until 45 days postpartum (n=10) or (ii) cows with normal puerperium (n=10). All cows had detectable levels of TLRs and ß-defensin 5 mRNAs, but these levels did not differ between groups (P>0.05). Levels of TLR4 mRNA had a positive and significant correlation with the time required for uterine involution in both groups.

Naturally acquired visceral leishmaniasis in non-human primates in Brazil.

Visceral leishmaniasis (VL) is a chronic and often fatal protozoal disease that is endemic in Belo Horizonte (State of Minas Gerais, Brazil). Leishmania sp. is an intracellular obligatory parasite of macrophages that can naturally infect several mammalian species. Non-human primates (NHP) have been used as experimental models for infection with Leishmania of the donovani complex. The present report describes a case of visceral leishmaniasis in a black-fronted titi. Among 41 primates kept in captivity in a zoo in Belo Horizonte (State of Minas Gerais, Brazil), one animal, a black-fronted titi (Callicebus nigrifrons), was positive for Leishmania chagasi infection by PCR and immunohistochemistry, and developed a fatal disease with clinical signs and lesions compatible with VL. Other 17 NHP, including six black-fronted titis (C. nigrifrons), one howler monkey (Alouatta guariba), three golden-bellied capuchins (Cebus xanthosternos), one golden-headed lion tamarin (Leontopithecus crysomelas), one black-headed owl monkey (Aotus nigriceps), two Rio Tapajós sakis (Pithecia irrorata) and three emperor tamarins (Saguinus imperator) had blood samples that tested positive for amplification of Leishmania kDNA by PCR, although these NPH had no clinical signs of the disease.