Detection of Vaccinia Virus in Milk: Evidence of a Systemic and Persistent Infection in Experimentally Infected Cows.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects lactating cows and milkers. VACV DNA and infectious particles have been detected in milk of naturally infected cows. However, the period and pattern of VACV shedding in milk is unknown, as is whether the presence of VACV in milk is due to a localized or a systemic infection. To address those questions, eight lactating cows were inoculated with VACV in previously scarified teats. The experiment was divided in two phases. In Phase 1, milk samples were collected daily for 33 days, and in Phase 2, four animals from the first phase were immunosuppressed. In both phases, milk was collected with a sterile catheter on even days and by hand milking on odd days. All animals showed typical BV lesions in the inoculated teats. All milk samples were subjected to nested polymerase chain reaction (PCR) and real-time quantitative PCR to detect VACV DNA. PCR-positive samples were subjected to virus isolation. VACV DNA was intermittently detected in milk in both phases and infectious viral particles could be detected only in phase 2, on the 69th, 73rd, 74th, 77th, 79th, and 81st days postinfection. Despite the possibility of propagation of VACV through milk, it is known that milk continues to be drawn and marketed normally during outbreaks of the disease. The detection of both VACV DNA and infectious particles in milk samples draws attention to the potential public health risk associated with the consumption of milk from BV outbreaks. Detection of VACV in the milk from noninfected teats demonstrated that VACV shedding in milk might be related to a systemic infection. Moreover, it was shown that VACV DNA and viral infectious particles could be detected in milk even after healing of the lesions, demonstrating that VACV may cause a persistent infection in cattle.

33rd Brazilian Society for Virology (SBV) 2022 Annual Meeting.

Each year, the Brazilian Society for Virology promotes a national meeting during the second semester of the year. In October 2022, the 33rd meeting took place at Arraial da Ajuda, Porto Seguro, Bahia, in-person:.this was the first in-person meeting since 2019, as the 2020 and 2021 events occurred online due to the issues imposed by COVID-19. It was a great pleasure for the whole audience to return to an in-person event, which certainly improved the interactions between the attendees in all ways. As usual, the meeting involved massive participation of undergraduate, graduate, and postdoc students, and several noteworthy international researchers were present. During five afternoons and evenings, attendees could discuss and learn about the most recent data presented by distinguished scientists from Brazil and other countries. In addition, young virology researchers from all levels could present their latest results as oral presentations and posters. The meeting covered all virology areas, with conferences and roundtables about human, veterinary, fundamental, environmental, invertebrate, and plant virology. The costs associated with attending the in-person event caused a slight reduction in the number of attendees compared to the two online events. However, even with this issue, the attendance was impressive. The meeting successfully achieved its most important goals: inspiring young and senior scientists and discussing high-quality, up-to-date virology research.

The 32nd Brazilian Society of Virology (SBV) 2021 Annual Meeting.

The Brazilian Society of Virology has been organizing annual meetings for 32 years now. The 32nd annual meeting, which occurred in 2021, was once again an online meeting in consequence of the issues imposed by COVID-19, even with the vaccination advances. As in the 2020 meeting, the number of attendees was high, with considerable participation by undergraduate, graduate, and postdoc students. Distinguished scientists from different countries offered high-quality conferences, and oral presentation sessions were presented by young scientists showing their newest research results. For almost five hours a day during five days, attendees discussed high-quality science related to all areas of virology. Even with the difficulties imposed by another pandemic year, the 32nd SBV annual meeting achieved its most important goal-to inspire young scientists and discuss high-quality virology research.

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.

A-type inclusion bodies: a factor influencing cowpox virus lesion pathogenesis.

The family Poxviridae comprises the most complex animal DNA viruses. During some poxvirus infections, A-type inclusion bodies (ATIs), codified by the ati gene, are produced. Although some studies have compared poxviruses that encode these inclusion bodies with those that do not, the biological function of ATIs is poorly understood. A recombinant ati-deleted cowpox virus was constructed and compared with the wild-type virus in in vitro experiments including electron microscopy and plaque and viral growth assays. No significant differences were observed in vitro. This reinforces the conclusion that the inclusion body is not essential for in vitro viral replication and morphogenesis. Additionally, different lesion progressions in vivo were observed by macroscopic and histological analysis, suggesting that the presence or absence of ATIs could result in different healing dynamics. This is the first time that the role of ATIs during viral replication has been studied based solely on one variable, the presence or absence of ATIs.

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.

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.

Molecular detection and phylogeny of bovine viral diarrhea virus 1 among cattle herds from Northeast, Southeast, and Midwest regions, Brazil.

We examined the circulating BVDV species and genotypes among cattle herds from Northeast, Southeast, and Midwest regions in Brazil. A total of 77 animals tested positive through standard PCR. Phylogenetic analyses revealed the presence of BVDV-1a, highlighting the need for better surveillance strategies to prevent BVDV spread 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.

Bovine Vaccinia: Insights into the Disease in Cattle.

Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions in the teats of dairy cows and the hands of milkers and is an important public health issue. Severe VACV-induced lesions in the teats and udder of cows and buffaloes could lead to mastitis and other secondary infections, thereby reducing productivity and resulting in economic losses to the dairy industry. In Brazil, BV re-emerged in the late 1990s and is now endemic in most of the Brazilian territory. In the last 15 years, much effort has been made to know more about this disease and its epidemiology, etiologic agents, and interactions with the host and the environment. In this review, we describe the known dynamics of VACV infection in cattle and the viral shedding routes, as well as the relevance of BV for animal and public health.

Multiple bluetongue virus serotypes causing death in Brazilian dwarf brocket deer (Mazama nana) in Brazil, 2015-2016.

Bela Vista Biological Sanctuary (RBV) is a protected area of Itaipu Binacional, a hydroelectric power company located on the border of Brazil and Paraguay. A captive population of Brazilian dwarf brocket deer (Mazama nana, Cervidae, Artiodactyla) is maintained for conservation purposes. Despite the reproductive success of the animals, outbreaks of a fatal hemorrhagic disease have been registered over the years, compromising conservation efforts. In order to identify the etiological agents of these hemorrhagic diseases, 32 captive Brazilian dwarf brockets were sampled to investigate bluetongue virus (BTV), epizootic hemorrhagic disease (EHD), and adenovirus hemorrhagic disease (AHD), in 2015. Only one deer (1/32; 3.12%) was seropositive for BTV. After this survey, five animals died in the early autumn of 2015 and 2016, again presenting clinical signs of hemorrhagic disease. Using RT-qPCR, RT-PCR and DNA sequencing, five BTV serotypes (3, 14, 18, 19, and 22) were identified in blood and tissues collected during necropsies. These BTV serotypes had not been previously described or isolated in Brazil, either in wild or domestic ruminants. Additionally, differential diagnosis was performed for EHD and AHD, but all samples were negative for both diseases. The multiple distinct BTV serotypes identified in these outbreaks resulted in a high lethality (100%) of Brazilian dwarf brockets and indicated that various BTV serotypes are circulating in the area.

Subclinical bovine vaccinia: An important risk factor in the epidemiology of this zoonosis in cattle.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that mainly affects lactating cows and dairy farm milkers. The epidemiological role(s) of other cattle categories such as dry cows, bulls, and heifers in BV remains unclear. This study was performed to investigate VACV in affected dairy cattle herds and perifocal farms during an outbreak in Brazil. Crusts from lesions of cows' teats were collected from all farms with BV outbreaks. Milk, feces, blood, and serum were collected from symptomatic and asymptomatic lactating cows. Blood and serum were also sampled from other cattle categories (calves, heifers, dry cows, and bulls). The samples were tested for VACV by PCR, and to confirm VACV viability, VACV-positive samples were inoculated in BSC-40 cells and stained using immunoperoxidase. Neutralizing antibodies were investigated using plaque reduction neutralization test. Viral DNA was detected in milk, blood, and feces samples of symptomatic and asymptomatic dairy cows and in blood samples from other cattle categories on farms with and without confirmed BV outbreak. In affected farms, viable virus was identified in feces and milk samples from lactating cows and in blood samples from asymptomatic dry cows. Viable VACV was also identified in feces from lactating cows and one bull's blood sample from perifocal farms. Neutralizing antibodies were detected in 81.6% of the herds affected by BV and in 53.8% of the herds on perifocal farms. The presented data indicate a potential source of viral dissemination, which contributes to the persistence and spread of VACV in the environment.

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.

Identification of bluetongue virus serotypes 1, 4, and 17 co-infections in sheep flocks during outbreaks in Brazil.

Bluetongue (BT) is a vector-borne viral disease caused by the Bluetongue virus (BTV), an Orbivirus from the Reoviridae family, affecting domestic and wild ruminants. BTV circulation in Brazil was first reported in 1978, and several serological surveys indicate that the virus is widespread, although with varied prevalence. In 2014, BT outbreaks affected sheep flocks in Rio Grande do Sul state, causing significant mortality (18.4%; 91/495) in BTV-infected sheep. In total, seven farms were monitored, and one or two sheep from each farm that died due to clinical signs of BT were necropsied. Apathy, pyrexia, anorexia, tachycardia, respiratory, and digestive disorders were noted. Additionally, an abortion was recorded in one of the monitored farms. The main gross lesions observed were pulmonary edema, anterior-ventral pulmonary consolidation, muscular necrosis in the esophagus and in the ventral serratus muscle, and hemorrhagic lesions in the heart. The blood and tissue samples were tested for BTV RNA detection by RT-qPCR targeting the segment 10. Positive samples were used for viral isolation. The isolated BTVs were typed by conventional RT-PCR targeting the segment 2 of the 26 BTV serotypes, followed by sequencing analysis. BTV-1, BTV-4 and BTV-17 were identified in the analyzed samples. Double or triple BTV co-infections with these serotypes were detected. We report the occurrence of BT outbreaks related to BTV-1, BTV-4 and BTV-17 infections and co-infections causing clinical signs in sheep flocks in Southern Brazil, with significant mortality and lethality rates.

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.

Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model.

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit without exhibiting any clinical signs.

Antimicrobial resistance in bacteria isolated from pigs with respiratory clinical signs in Brazil / Resistência a antimicrobianos em bactérias isoladas de suínos com sinais clínicos respiratórios no Brasil

Antimicrobial resistance is a current and important issue to public health, and it is usually associated with the indiscriminate use of antimicrobials in animal production. This study aimed to evaluate the antimicrobial susceptibility profile in bacterial isolates from pigs with clinical respiratory signs in Brazil. One hundred sixty bacterial strains isolated from pigs from 51 pig farms in Brazil were studied. In vitro disk-diffusion method was employed using 14 antimicrobial agents: amoxicillin, penicillin, ceftiofur, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline, oxytetracycline, tetracycline, erythromycin, tilmicosin, florfenicol, lincomycin, and sulfadiazine/trimethoprim. The majority of isolates were resistant to at least one antimicrobial agent (98.75%; 158/160), while 31.25% (50/160) of the strains were multidrug resistant. Streptococcus suis and Bordetella bronchiseptica were the pathogens that showed higher resistance levels. Haemophilus parasuis showed high resistance levels to sulfadiazine/trimethoprim (9/18=50%). We observed that isolates from the midwestern and southern regions exhibited four times greater chance of being multidrug resistant than the isolates from the southeastern region studied. Overall, the results of the present study showed a great level of resistance to lincomycin, erythromycin, sulfadiazine/trimethoprim, and tetracycline among bacterial respiratory pathogens isolated from pigs in Brazil. The high levels of antimicrobial resistance in swine respiratory bacterial pathogens highlight the need for the proper use of antimicrobials in Brazilian pig farms.(AU)

A resistência antimicrobiana é uma questão atual e muito importante para a saúde pública, geralmente associada ao uso indiscriminado de antimicrobianos na produção animal. Diante disso, foi investigado o perfil de sensibilidade-antimicrobiana em isolados bacterianos de suínos com sinais clínicos respiratórios no Brasil. Foram estudadas 96 isolados provenientes de 51 granjas de suínos do Brasil. O método de disco-difusão foi empregado usando 14 antimicrobianos: amoxicilina, penicilina, ceftiofur, ciprofloxacina, enrofloxacina, clortetraciclina, doxiciclina, oxitetraciclina, tetraciclina, eritromicina, tilmicosina, florfenicol, lincomicina e sulfadiazina/trimetoprim. Streptococcus suis e Bordetella bronchiseptica foram os patógenos que apresentaram maiores níveis de resistência. Haemophilus parasuis apresentou altos níveis de resistência à sulfadiazina/trimetoprim (9/18=50%). Observou-se que isolados das regiões Centro-Oeste e Sul apresentaram quatro vezes mais chance de serem multirresistentes do que os isolados da região Sudeste. A maioria foi resistente a pelo menos um agente antimicrobiano (98,75%; 158/160) e 31,25% (50/160) das estirpes isoladas eram multirresistentes. No geral, os resultados do presente estudo mostraram grande nível de resistência à lincomicina, eritromicina, sulfadiazina/trimetoprim e tetraciclina entre patógenos respiratórios bacterianos isolados de suínos no Brasil. Os altos níveis de resistência antimicrobiana em patógenos bacterianos respiratórios em suínos reforçam a necessidade do uso criterioso de antimicrobianos na suinocultura brasileira.(AU)

Virulence in murine model shows the existence of two distinct populations of Brazilian Vaccinia virus strains.

Brazilian Vaccinia virus had been isolated from sentinel mice, rodents and recently from humans, cows and calves during outbreaks on dairy farms in several rural areas in Brazil, leading to high economic and social impact. Some phylogenetic studies have demonstrated the existence of two different populations of Brazilian Vaccinia virus strains circulating in nature, but little is known about their biological characteristics. Therefore, our goal was to study the virulence pattern of seven Brazilian Vaccinia virus strains. Infected BALB/c mice were monitored for morbidity, mortality and viral replication in organs as trachea, lungs, heart, kidneys, liver, brain and spleen. Based on the virulence potential, the Brazilian Vaccinia virus strains were grouped into two groups. One group contained GP1V, VBH, SAV and BAV which caused disease and death in infected mice and the second one included ARAV, GP2V and PSTV which did not cause any clinical signals or death in infected BALB/c mice. The subdivision of Brazilian Vaccinia virus strains into two groups is in agreement with previous genetic studies. Those data reinforce the existence of different populations circulating in Brazil regarding the genetic and virulence characteristics.

Zoonotic vaccinia virus infection in Brazil: clinical description and implications for health professionals.

Bovine vaccinia virus outbreaks have been occurring in different regions of Brazil. We report here the time course of natural human infection by vaccinia virus and describe important clinical and epidemiological aspects of this zoonotic infection. The diagnosis of vaccinia virus infection was based on clinical, serological, and molecular procedures.