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BACKGROUND Severe acute respiratory syndrome coronavirus (SARS-CoV-2) omicron variant was first detected in South Africa in November 2021. Since then, the number of cases due to this variant increases enormously every day in different parts of the world. Mutations within omicron genome may impair the molecular detection resulting in false negative results during Coronavirus disease 19 (COVID-19) diagnosis. OBJECTIVES To verify if colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) targeting N and E genes would work efficiently to detect omicron SARS-CoV-2 variant and its sub-lineages. METHODS SARS-CoV-2 reverse transcription quantitative polymerase chain reaction (RT-qPCR) positive samples were sequenced by next generation DNA sequencing. The consensus sequences generated were submitted to Pangolin tool for SARS-CoV-2 lineage identification. RT-LAMP reactions were performed at 65ºC/30 min targeting N and E. FINDINGS SARS-CoV-2 omicron can be detected by RT-LAMP targeting N and E genes despite the genomic mutation of this more transmissible lineage. Omicron SARS-CoV-2 sub-lineages were tested and efficiently detected by RT-LAMP. We demonstrated that this test is very sensitive in detecting omicron variant, with LoD as low as 0.4 copies/µL. MAIN CONCLUSIONS Molecular detection of omicron SARS-CoV-2 variant and its sub-lineages can be achieved by RT-LAMP despite the genomic mutations as a very sensitive surveillance tool for COVID-19 molecular diagnosis.

Equine infectious anemia (EIA) is listed by the World Organization for Animal Health (OIE) as one of the equine diseases that must be notified. No effective treatment or vaccine is available. EIA control is based on segregation and euthanasia of positive equids. The disease is caused by the equine infectious anemia virus (EIAV), a member of the genus Lentivirus of the Retroviridae family. Despite the importance of this disease in equids, EIA has been poorly studied in donkeys (Equus asinus). We evaluate the sanitary conditions related to EIAV in donkeys from a shelter of abandoned animals captured on the roads of the Ceara. A total of 124 donkeys were randomly selected, and three horses lived at the same shelter. The animals were clinically evaluated, and a group of the 20 animals was submitted to hematological tests. Three diagnostic tests for EIA were used, agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) using EIAV recombinant protein gp90 (rgp90) and recombinant protein p26 (rp26) ELISA, and polymerase chain reaction (PCR) for detection of the EIAV tat-gag gene. From the donkeys, only 1 animal was positive using AGID 0.81% (1/124), compared to 21.8% (27/124) in the rgp90 and 10.5% (13/124) in the rp26 ELISA. Proviral DNA was detected by PCR tat-gag in 8.8% (11/124), and phylogenetic analysis confirms that the EIAV sequences of donkeys from the Brazilian Northeast grouped with Pantanal Brazilian sequences. Thus, in light of the results, we conclude that donkeys are carriers of EIAV and could be sources of infection.

De maneira geral, o estudo de bioquímica é complexo e abstrato, em que a quantidade de informações e a apresentação das vias metabólicas geralmente assustam o aluno, resultando muitas vezes na perda de interesse e inadequada compreensão do conteúdo. Assim, o aprendizado da bioquímica necessita de uma boa compreensão, raciocínio e abstração, fatores que dificultam ainda mais o sucesso no ensino desta disciplina. Com intuito de facilitar esta tarefa, diferentes abordagens têm sido desenvolvidas com o propósito de estimular e despertar o interesse na aprendizagem. Aqui, apresentamos os Jogos Metabólicos, estratégia lúdica para auxiliar o aprendizado do metabolismo biossintético-bioenergético. Foi observado, ao longo dos vinte anos em que essa estratégia foi utilizada, um aumento no interesse e no desempenho, resultando em uma melhor compreensão da bioquímica metabólica. Além disso, decorrente da contribuição dos estudantes, a estratégia vem sendo inovada a cada edição.

Meningitis is a disease with a global distribution that constitutes a worldwide burden, with viruses as the primary etiologic agents. The range of viral meningitis severity depends mainly on age, immune status and etiological agent. The aim of this work was to investigate the suspected cases of viral meningitis using molecular techniques to confirm the viral infection. The diagnosed virus was correlated with clinical findings and cytochemical parameters in cerebrospinal liquid (CSF) of patients. CSF of 70 children with the presumptive diagnosis of viral meningitis was analyzed by real time PCR (qPCR). Viruses were identified by qPCR in 44 CSF samples (62.9%). Among them, 31 were identified as Enterovirus (ENTV) (70.4%), six as Human herpes virus 3 (HHV-3) (13.6%), five as Dengue virus (DENV) (11.7%), one as Human herpes virus 1-2 (2.3%) and one as Human herpes virus 5 (2.3%). Patients in the HHV-positive groups had increased percentage of polymorphonuclear neutrophils (PMN) (mean of 81%) while the groups of patients with DENV and ENTV had a mean of 30.9%. This study contributes to the knowledge of the epidemiological distribution of viral agents in CNS infections in children. In addition, it raises the relevance of DENV as an agent of CNS infection, and reinforces the importance for molecular in the cases of CNV infection.

Made available in DSpace on 2021-06-25T10:18:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 The aetiological agent of equine infectious anaemia (EIA) is the retrovirus equine infectious anemia virus (EIAV) that infects all members of the Equidae family. The EIA is widely disseminated in the Brazilian territory with a high seroprevalence in the Brazilian Pantanal and is mainly diagnosed using agar gel immunodiffusion (AGID). There are few complete EIAV genome sequences available in GenBank, which had an impact on molecular detection studies. In this study, we conducted molecular detection and sequencing of EIAV proviral DNA from Brazilian horses. We analysed the genomic region from exon 1 of tat to gag (tat-gag). Comparative serological tests, comprising AGID and two enzyme-linked immunosorbent assays (ELISAs), were also conducted. Of the 133 samples, 58 were positive in the tat-gag PCR, and 49 nucleotide sequences of 272 bp were obtained. Using this developed tat-gag PCR EIAV proviral DNA was detected in 7% of the AGID-negative samples and 26% of the AGID-negative samples were positive in at least one of the ELISA tests used. Using phylogenetic analysis, the Brazilian Pantanal EIAV sequences grouped in a different clade of EIAV sequences from other countries. Thus, the EIAV sequences can contribute to the knowledge of the tat-gag genomic region in the circulating viruses in the Brazilian Pantanal, in addition to providing new information about the genetic diversity. In addition, the serological results demonstrate the greater sensitivity of the ELISAs used in this study compared to AGID for EIA diagnosis. Laboratório de Vírus Departamento de Microbiologia Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Embrapa Pantanal Laboratório de Virologia e Rickettsioses Faculdade de Medicina Veterinária Universidade Federal de Mato Grosso Viriontech do Brasil Indústria de Insumos e Serviços em Biotecnologia Instituto René Rachou Fiocruz-Minas Instituto de Biotecnologia Universidade Estadual Paulista (Unesp) Instituto de Biotecnologia Universidade Estadual Paulista (Unesp)

Putative promoter motifs have been described in viruses belonging to the nucleocytoplasmic large DNA viruses (NCLDVs) group; however, few studies have been conducted to search for promoter sequences in newly discovered amoebal giant viruses. Faustovirus and kaumoebavirus are two Asfarviridae-related giant viruses belonging to the NCLDVs group. The phylogenetic relationships among these viruses led us to investigate if the promoter regions previously identified in the asfarvirus genome could be shared by its amoebal virus relatives. Previous studies demonstrated the role of A/T-rich motifs as promoters of asfarvirus. In this study, we reinforce the importance of A/T rich motifs in asfarvirus and show that the TATTT and TATATA motifs are also shared in abundance by faustovirus and kaumoebavirus. Here, we demonstrate that TATTT and TATATA are mostly present in faustovirus and kaumoebavirus genomic intergenic regions (IRs) and that they are widely distributed at 0 to -100 bp upstream to the start codons. We observed that putative promoter motifs are present as one to dozens of repetitions in IRs of faustovirus, kaumoebavirus, and asfarvirus, which is similar to that described previously for marseilleviruses. Furthermore, the motifs were found in most of the upstream regions of the core genes of faustovirus, kaumoebavirus, and asfarvirus, which suggests that the motifs could already be present in the ancestor of these viruses before the irradiation of this group. Our work provides an in-depth analysis of the putative promoter motifs present in asfarvirus, kaumoebavirus, and faustovirus, which reinforces the relationship among these viruses.

Vaccinia virus (VACV), the etiological agent of bovine vaccinia (BV), is widespread in Brazil and present in most of the milk-producing regions. We conducted a horizontal study of BV in Bahia, a state of Brazil in which the production of milk is increasing. During 2011, human and bovine clinical samples were collected during outbreaks for BV diagnosis, virus isolation and molecular analysis. We collected data for epidemiological inferences. Vaccinia virus was detected in 87.7 % of the analyzed outbreaks, highlighting the effective circulation of VACV in Bahia. The molecular data showed the spreading of group 1 Brazilian VACV to Bahia. We observed a seasonal profile of BV, with its peak in the drier and cooler season. Manual milking was observed in 96 % of the visited properties, showing its importance to viral spread in herds. Under-notification of BV, ineffective animal trade surveillance, and bad milking practices have contributed to the spread of VACV in Brazil.

Made available in DSpace on 2018-12-11T17:20:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-09-01 Empresa Brasileira de Pesquisa Agropecuária Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de Mato Grosso Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Equine infectious anemia (EIA) has a worldwide distribution, and is widespread in Brazil. The Brazilian Pantanal presents with high prevalence comprising equine performance and indirectly the livestock industry, since the horses are used for cattle management. Although EIA is routinely diagnosed by the agar gel immunodiffusion test (AGID), this serological assay has some limitations, so PCR-based detection methods have the potential to overcome these limitations and act as complementary tests to those currently used. Considering the limited number of equine infectious anemia virus (EIAV) sequences which are available in public databases and the great genome variability, studies of EIAV detection and characterization molecular remain important. In this study we detected EIAV proviral DNA from 23 peripheral blood mononuclear cell (PBMCs) samples of naturally infected horses from Brazilian Pantanal using a semi-nested-PCR (sn-PCR). The serological profile of the animals was also evaluated by AGID and ELISA for gp90 and p26. Furthermore, the EIAV PCR amplified DNA was sequenced and phylogenetically analyzed. Here we describe the first EIAV sequences of the 5’ LTR of the tat gene in naturally infected horses from Brazil, which presented with 91% similarity to EIAV reference sequences. The Brazilian EIAV sequences also presented variable nucleotide similarities among themselves, ranging from 93,5% to 100%. Phylogenetic analysis showed that Brazilian EIAV sequences grouped in a separate clade relative to other reference sequences. Thus this molecular detection and characterization may provide information about EIAV circulation in Brazilian territories and improve phylogenetic inferences. Laboratório de Vírus Departamento de Microbiologia Instituto de Ciências Biológicas Universidade Federal de Minas Gerais Laboratório de Biologia Molecular Microbiologia e Sorologia Hospital Universitário Universidade Federal de Santa Catarina Viriontech do Brasil Indústria de Insumos e Serviços em Biotecnologia Instituto Rene Rachou Fiocruz-Minas Embrapa Pantanal Departamento de Microbiologia e Imunologia Instituto de Biociências e Instituto de Biotecnologia Universidade Estadual Paulista UNESP Laboratório de Virologia e Rickettsioses Faculdade de Medicina Veterinária Universidade Federal de Mato Grosso Departamento de Microbiologia e Imunologia Instituto de Biociências e Instituto de Biotecnologia Universidade Estadual Paulista UNESP Empresa Brasileira de Pesquisa Agropecuária: 02.12.01.030.00.00 CNPq: 307423/2015-8 Fundação de Amparo à Pesquisa do Estado de Mato Grosso: 330980/2012

International audience; The family Mimiviridae, comprised by giant DNA viruses, has been increasingly studied since the isolation of the Acanthamoeba polyphaga mimivirus (APMV), in 2003. In this work, we describe the genome analysis of two new mimiviruses, each isolated from a distinct Brazilian environment. Furthermore, for the first time, we are reporting the genomic characterization of mimiviruses of group C in Brazil (Br-mimiC), where a predominance of mimiviruses from group A has been previously reported. The genomes of the Br-mimiC isolates Mimivirus gilmour (MVGM) and Mimivirus golden (MVGD) are composed of double-stranded DNA molecules of similar to 1.2 Mb, each encoding more than 1,100 open reading frames. Genome functional annotations highlighted the presence of mimivirus group C hallmark genes, such as the set of seven aminoacyl-tRNA synthetases. However, the set of tRNA encoded by the Br-mimiC was distinct from those of other group C mimiviruses. Differences could also be observed in a genome synteny analysis, which demonstrated the presence of inversions and loci translocations at both extremities of Br-mimiC genomes. Both phylogenetic and phyletic analyses corroborate previous results, undoubtedly grouping the new Brazilian isolates into mimivirus group C. Finally, an updated pan-genome analysis of genus Mimivirus was performed including all new genomes available until the present moment. This last analysis showed a slight increase in the number of clusters of orthologous groups of proteins among mimiviruses of group A, with a larger increase after addition of sequences from mimiviruses of groups B and C, as well as a plateau tendency after the inclusion of the last four mimiviruses of group C, including the Br-mimiC isolates. Future prospective studies will help us to understand the genetic diversity among mimiviruses.

To the Editor: Vaccinia virus (VACV) causes exanthematous disease (bovine vaccinia) in Brazil. Outbreaks of this disease in humans have been reported since the late 1990s and have spread throughout Brazil (1). Natural human infections with VACV occur by close contact with infected cattle during milking. Lesions can spread to secondary body sites (forearms, arms, and face). Thus, person-to-person transmission occurs (1). Moreover, virus can persist in household environments, remain infectious, and be transmitted by fomites (2). Although raw milk and cheese are potential sources of infection, no clinical cases have been associated with this transmission route (3,4). Data for person-to-person transmission in Brazil are scarce, but person-to-person transmission was recently reported (5). We report a possible case of person-to-person transmission of VACV. This study was approved by the Research Ethics Committee of Universidade Federal de Minas Gerais (registration protocol FR-413704). In September 2012, during a serologic survey in a rural area of Serro City (18°36′17″S, 43°22′46″W), Minas Gerais, Brazil (Technical Appendix Figure, panel A), blood samples were obtained from a family of 5 persons (father, mother, and 3 daughters). The father and mother were 48 and 53 years of age, respectively, and had been vaccinated against smallpox. They reported contact with cows and horses (Technical Appendix Table 1). Only the father had milked cows. The 3 daughters (13, 13, and 14 years of age) did not engage in any exposure activity. However, all family members had consumed raw milk and cheese. Bovine vaccinia lesions were observed on the hand of the father (Technical Appendix Figure, panel B). In 2011, he had vesicular disease (no laboratory diagnosis) with clinical and epidemiologic features (lesions) suggestive of bovine vaccinia on his hands and forearms and systemic symptoms (fever, headache, malaise, myalgia, lymphadenopathy, and abdominal pain). His symptoms were mild and without any systemic clinical features. Two lesions developed on his hands and dried swab samples were collected from both lesions. Swab samples were processed as described (2) and used for virus isolation and molecular diagnosis. On the basis of previous studies that detected viral DNA in clinical samples from persons with bovine vaccinia (1), we used a quantitative PCR to amplify the vgf and ha genes of VACV (3–5), a standard PCR to detect the ha gene (3–5), and a seminested PCR to detect the ati gene (F.L. Assis, unpub. data). Serum samples were used for detection of virus-neutralizing antibodies (orthopoxvirus 50% plaque-reduction neutralization test) and molecular diagnostic studies (1). Virus isolation was attempted in Vero cells and chorioallantoic membrane. All results were negative. The 50% plaque-reduction neutralization test showed that the father, mother, and 14-year-old daughter had neutralizing antibodies against orthopoxvirus (titers 800, 3,200, and 800 neutralizing units/mL, respectively). All family members had positive results by molecular diagnostic test for >1 virus gene (Technical Appendix Table 1). To rule out infection with parapopoxvirus, a complementary PCR (6) was also performed, and all family members had negative results. Quantitative PCR products for the ha gene from 3 virus-positive samples were sequenced in both directions in triplicate (Mega BACE Sequencer; GE Healthcare, Little Chalfont, UK). Sequences were aligned by using ClustalW (http://www.genome.jp/tools/clustalw/) and MEGA4.1 (http://www.megasoftware.net/) and showed 100% identity with each other (Figure). A phylogenetic tree was constructed by using the neighbor-joining method and 1,000-bootstrap replicates in the Tamura-3 parameter model (MEGA4.1). Sequences were grouped with VACV group 2 isolates. Sequences obtained were deposited in GenBank under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"KP889223","term_id":"896688431","term_text":"KP889223"}}KP889223–5). Figure A) Nucleotide sequence of vaccinia virus (VACV) hemagglutinin gene and homologous sequences of several orthopoxviruses, Brazil. Dots indicate sequence identity; dashes indicate deletions. VARV, variola virus; MPXV, monkeypox virus; CPXV, cowpox virus. ... In Brazil, outbreaks of bovine vaccinia are associated with rural environments. However, some clinical and epidemiologic aspects remain unclear. The infection of the father was associated with direct contact with cattle. Immunity conferred by smallpox vaccination did not prevent infection; this lack of immune response has been demonstrated in other studies in Brazil (7). Long-term protection might require multiple virus exposures, and severity of poxvirus infections might be influenced by the immunologic state of the host and virulence of virus strains (1,8,9). The mother and 2 daughters with virus DNA in blood samples and the 14-year-old daughter with high titers of virus-neutralizing antibodies suggest that alternative routes (other than milking) for VACV infection of humans should be considered. These alternative routes can include person-to-person or environmental transmission because the 2 daughters did not report any exposure activities related to milking or contact with cattle (Technical Appendix Tables 1, 2). Persistence of VACV in household environments has been reported (2,10). The family also consumed raw milk and cheese, a common practice in the region. Therefore, infection with VACV through raw milk and cheese consumption should also be considered. The patients did not report oral lesions or a history of skin/mucosal lesions. In conclusion, person-to-person transmission of VACV in these cases might have been caused by direct contact between the father and family members, contact with virus in the home, or consumption of unpasteurized milk and cheese. Additional studies are necessary to elucidate the role of these transmission pathways in spread of VACV in Brazil. Technical Appendix. Additional information on alternative routes of zoonotic vaccinia virus transmission, Brazil. Click here to view.(226K, pdf)

Orthopoxviruses (OPV) are emerging viruses with great importance in human and veterinary medicine, such as Vaccinia virus (VACV), which causes outbreaks of bovine vaccinia (BV) in South America. The clinical aspects of BV are similar to other vesicular infections, complicating the clinical diagnosis. This cross-sectional study evaluated the knowledge of Healthcare Professionals about BV and revealed their unpreparedness about BV in a VACV hyper-endemic area in Brazil, highlighting the public health issues associated with VACV infections. This study presents an opportunity to discuss the importance of vaccination for healthcare professionals who work in areas of VACV circulation and brings an educational measure on VACV infections for health professionals around the world.

Natural infections of Vaccinia virus (VACV)-the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae-cause an occupational emergent zoonotic disease that is primarily associated with the handling of infected dairy cattle. In humans, VACV infection is characterized by skin lesions, primarily on the hands, and accompanied by systemic symptoms such as fever, myalgia, headache, and lymphadenopathy. The diagnosis of VACV is usually performed according to the methods described for other orthopoxviruses. This unit describes the methods utilized to obtain clinical samples, the serological and molecular techniques used for diagnosis, and the isolation methods and techniques used for molecular and biological characterization of the viruses. © 2016 by John Wiley & Sons, Inc.

Psittacid herpesvirus (PsHV) was isolated from 41 birds kept in captivity in Belo Horizonte, Minas Gerais/Brazil using chicken embryo fibroblasts (CEF) cell cultures. For this study, leukocytes or cloacal swabs of live birds were used. Also, portions of liver, spleen or kidney from birds collected at necropsy were utilized for these tests. PCR tests confirmed the presence of PsHV in 100% of samples. Thirty-three of the PCR products were sequenced and the results disclosed a 99% and 100% identity when compared with other sequences PsHV-1 (AY372243.1 and AF261756.1), previously deposited in GenBank. In addition, histopathology was performed and 19 of the 29 birds contained random multifocal lymphoplasmacytic hepatitis with necrotic foci, suggestive of viral infection. Three samples were examined by electron microscopy to visualize the viral particles obtained from cell culture. The viral structures measured 269 nm in average, had envelopes with an icosahedral capsid and tegument, consistent with herpesvirus. Thus, a total of 41 isolates were obtained from PsHV cell cultivation in CEF, confirming the circulation of the virus between parrots kept in captivity in Belo Horizonte, and affirming the importance of further studies in this area.

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.

Bovine vaccinia (BV) is an emerging zoonosis caused by the Vaccinia virus (VACV), genus Orthopoxvirus (OPV), Poxviridae family. In general, human cases are related to direct contact with sick cattle but there is a lack of information about human-to-human transmission of VACV during BV outbreaks. In this study, we epidemiologically and molecularly show a case of VACV transmission between humans in São Francisco de Itabapoana County, Rio de Janeiro state. Our group collected samples from the patients, a 49-year-old patient and his son. Our results showed that patients had developed anti-OPV IgG or IgM antibodies and presented neutralizing antibodies against OPV. The VACV isolates displayed high identity (99.9%) and were grouped in the same phylogenetic tree branch. Our data indicate that human-to-human VACV transmission occurred during a BV outbreak, raising new questions about the risk factors of the VACV transmission chain.

Orthobunyaviruses are arboviruses in which at least 30 members are human pathogens. The members of group C orthobunyaviruses were first isolated in the Brazilian Amazon in 1950, since that time little information is accumulated about ecology and the medical impact of these virus groups in Brazil. Herein, we describe the evidence of Apeu virus (APEUV; an Orthobunyavirus member) infection in wild monkeys from the Brazilian Amazon forest. APEUV was detected by using a neutralizing antibody in serum and its RNA, suggesting past and acute infection of Amazonian monkeys by this virus. These results altogether represent an important contribution of orthobunyavirus ecology in the Amazon and an update about recent circulation and risk for humans with expansion of the cities to Amazon forest.

Thirty-six isolates of psittacid herpesvirus (PsHV), obtained from 12 different species of psittacids in Brazil, were genotypically characterized by restriction fragment length polymorphism (RFLP) analysis and PCR amplification. RFLP analysis with the PstI enzyme revealed four distinct restriction patterns (A1, X, W and Y), of which only A1 (corresponding to PsHV-1) had previously been described. To study PCR amplification patterns, six pairs of primers were used. Using this method, six variants were identified, of which, variants 10, 8, and 9 (in this order) were most prevalent, followed by variants 1, 4, and 5. It was not possible to correlate the PCR and RFLP patterns. Twenty-nine of the 36 isolates were shown to contain a 419 bp fragment of the UL16 gene, displaying high similarity to the PsHV-1 sequences available in GenBank. Comparison of the results with the literature data suggests that the 36 Brazilian isolates from this study belong to genotype 1 and serotype 1.

In Brazil, serologic evidence of Orthopoxvirus (OPV) circulation showed positivity around 20% in cattle, humans, monkeys and rodents. Although OPV seropositivity has been described in buffalo herds in southeastern Brazil, no Vaccinia virus (VACV) (member of genus OPV) outbreaks in buffalo herds have been described in this country. This study aimed to investigate the detection of anti-OPV antibodies and to study the OPV genome in Brazilian buffalo herds. Our results demonstrated a high OPV seropositivity in buffalo herds on Marajo Island and molecular data confirmed the circulation of VACV. The geographical isolation conditionmight be a sine qua non condition to explain our results.

To the Editor: Since 1999, several zoonotic outbreaks of vaccinia virus (VACV) infection have been reported in cattle and humans in rural areas of Brazil. The infections have caused exanthematous lesions on cows and persons who milk them, and thus are detrimental to the milk industry and public health services (1,2). In Brazil during the last decade, VACV outbreaks have been detected from the north to the extreme south of the country (1–4). Because Brazil shares extensive boundaries with other South American countries, humans and cattle on dairy and beef-producing farms in those countries may be at risk of exposure to VACV. To determine if VACV has spread from Brazil to Argentina, we investigated the presence of VACV in serum samples from cattle in Argentina. During 2011, we obtained serum samples from 100 animals (50 dairy and 50 beef cattle) on farms in Cordoba, Corrientes, Entre Rios, and Santa Fe Provinces in Argentina (Technical Appendix, panel A). No VACV cases had been reported in humans or cattle in these provinces. However, Corrientes Province borders the Brazilian state of Rio Grande do Sul, where VACVs (Pelotas 1 and Pelotas 2 viruses) were isolated during an outbreak affecting horses in 2008 (2). To determine the presence of neutralizing antibodies in the serum samples, we used an orthopoxvirus 70% plaque-reduction neutralization test as described (4). On the basis of previous studies that detected viral DNA in serum samples (4–6), we used real-time PCR to amplify the highly conserved orthopoxvirus vaccinia growth factor (vgf) gene DNA (P.A. Alves, unpub. methods). To amplify the hemagglutinin (HA) gene DNA from the serum samples, we used real-time PCR with primers as described by de Souza Trindade et al. 2008 (7). The HA PCR products were directly sequenced in both orientations by using specific primers and capillary electrophoresis (Genetic Analyzer 3130; Applied Biosystems, Grand Island, NY, USA). We used ClustalW (http://www.clustal.org) and MEGA4 software (http://megasoftware.net/) to align nucleotide sequences and construct a phylogenetic tree (neighbor-joining method, 1,000 bootstraps) from the obtained HA fragment. Of the 50 dairy cattle samples, 4 (8.0%) had neutralizing antibodies against orthopoxvirus; of these, 3 (75.0%) had titers of 100 neutralizing units (NU)/mL, and 1 (25.0%) had a titer of 400 NU/mL. Of the 50 beef cattle, 8 (16.0%) had antibodies to orthopoxvirus, 1 (12.5%) of which had a titer of 800 NU/mL. Most of the positive samples were from cattle in Corrientes and Entre Rios Provinces (Table). Table Diagnosis of Orthopoxvirus infection in beef and dairy cattle during a study of the spread of vaccinia virus to cattle herds, Argentina, 2011* Of the 100 serum samples, 5 (3 from beef and 2 from dairy cattle) were positive for vgf by real-time PCR. HA DNA was amplified from 2 of the 3 vgf PCR–positive beef cattle samples; plaque-reduction neutralization test results were also positive for the 2 samples (Table). Alignment of the HA fragment nucleotide sequence of the isolates from Argentina showed that the sequence was highly similar to that of the homologous gene of VACV isolates from Brazil. Furthermore, the sequences showed a signature deletion that is also present in the sequences of VACV isolates from Brazil. Compared with sequences for other VACV isolates, those from Argentina had 2 polymorphisms (Technical Appendix, panel C). The HA sequences from the isolates from Argentina demonstrated 100% identity among themselves and exhibited higher identity with group 1 (98.2% identity) versus group 2 (93.6% identity) isolates from Brazil (Technical Appendix, panel D). In the phylogenetic tree based on the HA nucleotide sequences (Technical Appendix, panel B), the VACVs from Argentina clustered with several group 1 VACVs detected during outbreaks in Brazil. Although no outbreaks of exanthematous VACV infection have been described in cattle or humans in Argentina, we detected neutralizing antibodies against orthopoxvirus and detected VACV DNA in serum samples from cattle in the country. Most of the seropositive samples were from cattle in Entre Rios Province, which shares a border with Uruguay, and Corrientes Province, which shares a border with Rio Grande do Sul State in Brazil, where Pelotas VACVs have been isolated (2). We believe that the seropositive cattle in this study may have been exposed to VACV, the only orthopoxvirus known to be circulating in South America (1–4,8–10). Despite veterinary surveillance efforts of border control organizations, VACV control may be hampered by the circulation of infected rural workers and the misdiagnosis of VACV infection; misdiagnoses occur because VACV lesions resemble those of other exanthematous diseases. Moreover, peridomestic rodents have been hypothesized to act as VACV hosts, and could facilitate the spread of VACV in border areas (10). In addition, we could not rule out the circulation of autochthonous VACV in Argentina, but this is a less likely explanation. Our findings suggest that cattle herds in areas of Argentina near the border with Brazil may be exposed to VACV from Brazil and, thus, may be at risk for VACV infection. Further research is needed to determine the risk factors for VACV infection and to assess the circulation of VACV in South America Technical Appendix: Map of Argentina, indicating locations where blood samples were collected from cattle and results of phylogenetic analysis of vaccine virus isolated from the samples. Click here to view.(611K, pdf)

Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

Blood samples from 1,072 domestic cats of nine administrative regions of Belo Horizonte, MG, were collected and tested using PCR nested for the occurrence of feline leukemia virus (FeLV). Overall occurrence was 47.5% (507/1072) being North (68.1%) and East (54.4%) the most prevalent areas. Epidemiological data showed that FeLV infection was very common among examined cats and breed neither gender nor were predisposing factors for FeLV. The results suggest that the agglomeration of a large number of cats in the same environment can be an important factor for the increase in the rate of transmission of this retrovirus among domestic cats in the studied city.

Since 1999, several Vaccinia virus (VACV) isolates, the etiological agents of bovine vaccinia (BV), have been frequently isolated and characterized with various biological and molecular methods. The results from these approaches have grouped these VACV isolates into two different clusters. This dichotomy has elicited debates surrounding the origin of the Brazilian VACV and its epidemiological significance. To ascertain vital information to settle these debates, we and other research groups have made efforts to identify molecular markers to discriminate VACV from other viruses of the genus Orthopoxvirus (OPV) and other VACV-BR groups. In this way, some genes have been identified as useful markers to discriminate between the VACV-BR groups. However, new markers are needed to infer ancestry and to correlate each sample or group with its unique epidemiological and biological features. The aims of this work were to characterize a new VACV isolate (VACV DMTV-2005) molecularly and biologically using conserved and non-conserved gene analyses for phylogenetic inference and to search for new genes that would elucidate the VACV-BR dichotomy. The VACV DMTV-2005 isolate reported in this study is biologically and phylogenetically clustered with other strains of Group 1 VACV-BR, the most prevalent VACV group that was isolated during the bovine vaccinia outbreaks in Brazil. Sequence analysis of C23L, the gene that encodes for the CC-chemokine-binding protein, revealed a ten-nucleotide deletion, which is a new Group 1 Brazilian VACV genetic marker. This deletion in the C23L open reading frame produces a premature stop-codon that is shared by all Group 1 VACV-BR strains and may also reflect the VACV-BR dichotomy; the deletion can also be considered to be a putative genetic marker for non-virulent Brazilian VACV isolates and may be used for the detection and molecular characterization of new isolates.