Genetic characterization of a putative new type of bovine papillomavirus in the Xipapillomavirus 1 species in a Brazilian dairy herd.

Currently, bovine papillomavirus types are divided into five genera, namely, Deltapapillomavirus, Epsilonpapillomavirus, Xipapillomavirus, Dyoxipapillomavirus, and Dyokappapapillomavirus. In the recent decades, the characterization of numerous putative and novel bovine papillomavirus types from cattle in several geographic regions, has revealed the occurrence of a high viral diversity. In this study, we describe the identification and characterization of a putative new bovine papillomavirus type within species Xipapillomavirus 1 of Xipapillomavirus genus. The detection of the viral types identified in the skin warts was obtained by polymerase chain reaction assays targeting the L1 gene, followed by direct sequencing of the generated amplicons. The partial L1 sequences revealed that bovine papillomavirus types 6, 10, and 11, the putative new bovine papillomavirus type designated BPV/CHI-SW2, and an unreported putative new bovine papillomavirus type (named BPV/BR-UEL08) were associated with cutaneous papillomatosis in the cows from the dairy herd investigated. Phylogenetic reconstruction based on the L1 gene revealed that the BPV/BR-UEL08 isolate clustered with other bovine papillomaviruses classified in the Xipapillomavirus genus, being closely related to representatives of the species Xipapillomavirus 1. Investigations focusing on the molecular epidemiology of bovine papillomaviruses related to clinical outcomes in cattle are of fundamental importance to determine the actual genetic diversity and prevalent viral types to be included in vaccines for cattle.

A TaqMan-based qRT-PCR assay for Senecavirus A detection in tissue samples of neonatal piglets.

This study describes a sensitive (1.3 × 101 genomic copies/µL) and specific TaqMan-based qRT-PCR assay able to detect and quantify SVA RNA in porcine biological samples. The technique represents an efficient tool for the virus diagnosis and assessment of SVA load in tissues of infected animals and for epidemiological studies.

Genetic diversity of bovine papillomavirus types, including two putative new types, in teat warts from dairy cattle herds.

Teat papillomatosis affects dairy cows worldwide. Milking can become difficult due to teat warts, and maintaining affected cows in the herds may diminish economic profit in the dairy industry. Currently, 13 bovine papillomavirus (BPV) types have been fully characterized, and numerous putative BPV types have been identified through partial L1 gene PCR. In order to identify the viral types present in warts on the udders of dairy cows, 40 teat lesions from 24 cows from 13 cattle farms in three States of Brazil were evaluated by PV L1 gene PCR. The warts that were evaluated contained sequences from BPVs 6-10, the putative BPV types BAPV9 and BAPV4, and two unreported putative papillomavirus (PV) types, named BPV/BR-UEL6 and BPV/BR-UEL7. In addition, mixed infections and coinfections were identified, since more than one lesion was observed on the udders of 13 cows. Phylogenetic analysis showed that BPV/BR-UEL6 is closely related to BPVs belonging to the genus Xipapillomavirus, while BPV/BR-UEL7 clustered with the previously reported strains Cervus timorensis and Pudu puda PVs, which represent a putative new PV type, and it was only distantly related to xi-, epsilon-, delta- and dyoxi-PVs. These results provide information that will assist in the understanding of the association of BPVs 6, 7, 8, 9, and 10, as well as putative BPV types BAPV4 and BAPV9, with mammary papillomatosis. This is the first characterization of putative novel PV types BPV/BR-UEL6 and BPV/BR-UEL7 in teat warts of dairy cows, highlighting the high genetic diversity of BPVs associated with teat papillomatosis.

Bovine papillomavirus type 13 DNA in equine sarcoids.

Equine sarcoids are locally aggressive fibroblastic neoplasms considered to be the most common skin tumors of horses worldwide. Bovine papillomavirus types 1 and 2 have typically been associated with sarcoids in equids. Investigations aiming to identify papillomavirus strains, aside from bovine papillomaviruses 1 and 2, which might be associated with sarcoid lesions, have been lacking. The aim of this article is to report the identification of a third bovine papillomavirus type, bovine papillomavirus 13, associated with equine sarcoids. Six sarcoid lesions were collected from diverse anatomical sites on two horses from southern Brazil. To detect a broad spectrum of papillomavirus strains, eight degenerate primer pairs designed to detect conserved regions on the L1 and E1 genes were tested on the DNA samples. Direct sequencing was then performed on the obtained amplicons, and sequence identities were compared with sequences from all bovine papillomavirus types. The FAP59/FAP64, MY09/MY11, and AR-E1F2/AR-E1R4 sequences generated from the sarcoids were shown to present 99 to 100% identity with bovine papillomavirus 13, a new bovine papillomavirus type previously described in cattle. The results from this study suggest that there is a need to identify bovine papillomavirus type 13 and other papillomavirus strains that might be associated with sarcoids in diverse geographical areas; such investigations might establish the frequency of occurrence of this viral type in these common tumors of equids.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil.

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

Cynomolgus monkeys are successfully and persistently infected with hepatitis E virus genotype 3 (HEV-3) after long-term immunosuppressive therapy.

Epidemiological studies found that hepatitis E virus genotype 3 (HEV-3) infection was associated with chronic hepatitis and cirrhosis in immunocompromised patients. Our study aimed to investigate the relationship between the host immunosuppressive status and the occurrence of HEV-related chronic hepatitis. Here we describe a successful experimental study, using cynomolgus monkeys previously treated with tacrolimus, a potent calcineurin inhibitor immunosuppressant, and infected with a Brazilian HEV-3 strain isolated from naturally infected pigs. HEV infected monkeys were followed up during 160 days post infection (dpi) by clinical signs; virological, biochemical and haematological parameters; and liver histopathology. The tacrolimus blood levels were monitored throughout the experiment. Immunosuppression was confirmed by clinical and laboratorial findings, such as: moderate weight loss, alopecia, and herpes virus opportunistic infection. In this study, chronic HEV infection was characterized by the mild increase of liver enzymes serum levels; persistent RNA viremia and viral faecal shedding; and liver histopathology. Three out of four immunosuppressed monkeys showed recurrent HEV RNA detection in liver samples, evident hepatocellular ballooning degeneration, mild to severe macro and microvesicular steatosis (zone 1), scattered hepatocellular apoptosis, and lobular focal inflammation. At 69 dpi, liver biopsies of all infected monkeys revealed evident ballooning degeneration (zone 3), discrete hepatocellular apoptosis, and at most mild portal and intra-acinar focal inflammation. At 160 dpi, the three chronically HEV infected monkeys showed microscopic features (piecemeal necrosis) corresponding to chronic hepatitis in absence of fibrosis and cirrhosis in liver parenchyma. Within 4-months follow up, the tacrolimus-immunosuppressed cynomolgus monkeys infected with a Brazilian swine HEV-3 strain exhibited more severe hepatic lesions progressing to chronic hepatitis without liver fibrosis, similarly as shown in tacrolimus-immunosuppressed solid organ transplant (SOT) recipients. The cause-effect relationship between HEV infection and tacrolimus treatment was confirmed in this experiment.

Electrophoretic RNA genomic profiles of Brazilian Picobirnavirus (PBV) strains and molecular characterization of a PBV isolated from diarrheic calf.

Picobirnavirus (PBV) belongs to the family Picobirnaviridae. PBV are a group of emerging non-enveloped viruses, with a bisegmented double-stranded RNA genome that can infect a wide range of hosts. This study reports the occurrence of PBV in fecal samples from five Brazilian dairy cattle herds. From the 289 stool samples of individual calves analyzed by silver-stained polyacrylamide gel electrophoresis (ss-PAGE) the PBV was detected in 8.3 % (24/289), of which 10.2% (18/176) had diarrheic consistency. Of the 24 positive samples in ss-PAGE, 5 (20.8%) of them showed a small electrophoretic profile and 19 (79.2%) samples had large profile. From the 24 positives samples by ss-PAGE, 15 (62.5%) were successfully amplified (201 bp) using GI specific primers targeting the RdRp gene of PBV. The analysis of nucleotide identity matrix revealed that the bovine PBV strain identified in this study, showed the highest nucleotide identity (81%) with PBV strain detected in turkey (MD-2010/HM803965). This is the first nucleotide sequence of a bovine PBV strain in the American continent and the first detection of small genome profile of PBV-like strains in bovine hosts.

Genetic characterization of a novel bovine papillomavirus member of the Deltapapillomavirus genus.

This report describes the complete genomic sequence and taxonomic position of BPV type 13. The BPV13 genome was amplified using the multiply primed rolling-circle amplification technique and long-template PCR employing two specific primers. The two long PCR fragments obtained were cloned and sequenced via primer walking. The complete genomic sequence of the BPV13 contains 7961 bp encoding eight proteins, E1, E2, E4, E5, E6, E7, L1, and L2. Similarly to the E5 gene in BPVs 1 and 2, the putative BPV13 E5 ORF encodes a small transforming protein that contains a hydrophobic transmembrane domain. Meanwhile, the retinoblastoma tumor suppressor-binding domain is absent in the putative BPV13 E7 protein. The presence of these two specific molecular features has been recognized as a distinct marker for the development of fibropapilloma in artiodactyl PV-induced lesions. The phylogenetic analysis demonstrated that BPV13 is a new member of the Deltapapillomavirus genus, to be classified as the third representative of the Delta 4 species. The characterization of the genomic sequence of this novel PV will aid in the interpretation of the pathologies described to be related to this virus and provide support for the development of diagnostic tools for epidemiological surveillance of BPV13 in its potential natural hosts.

Hepatitis E virus in liver and bile samples from slaughtered pigs of Brazil

The objective of this study was to detect and identify hepatitis E virus (HEV) strains in liver and bile samples from slaughtered pigs in the state of Paraná, Brazil. Liver and bile samples were collected from 118 asymptomatic adult pigs at a slaughterhouse in a major Brazilian pork production area. The samples were assayed using a nested reverse transcription-polymerase chain reaction protocol with primer sets targeting open reading frames (ORF)1 and 2 of the HEV genome. HEV RNA was detected in two (1.7%) liver samples and one (0.84%) bile sample using both primers sets. The HEV strains were classified as genotype 3b on the basis of their nucleotide sequences. These data suggest that healthy pigs may be a source of HEV infection for consumers of pig liver and slaughterhouse workers in Brazil.

A bovine teat papilloma specimen harboring Deltapapillomavirus (BPV-1) and Xipapillomavirus (BPV-6) representatives

The common occurrence of multiple papillomavirus infections has been shown in several studies involving the human host. However, investigations with the aim of identifying mixed papillomavirus infections in cattle have been conducted only recently. In the current work we describe a co-infection with two different bovine papillomavirus (BPV) types that was identified in a bovine teat papilloma. The skin wart was obtained from a cow belonging to a Brazilian beef herd. A PCR assay was carried out with the FAP primer pair, which amplifies a partial segment of the L1 gene (approximately 478 bp), and the amplicon was submitted to direct sequencing. Because nucleotide sequences with satisfactory quality scores were not obtained, the amplicon was cloned and further sequencing, involving ten selected clones, was performed. The sequence analysis of the cloned inserts revealed the presence of two different BPV types. BPV-1 (Deltapapillomavirus genus) was detected in six clones, while BPV-6 (Xipapillomavirus genus) was detected in four clones. This finding confirms the presence of BPV co-infection associated with cutaneous papillomatosis in cattle.

Em seres humanos, as infecções múltiplas pelo papilomavírus têm sido demonstradas em vários estudos. Em bovinos, somente recentemente foram conduzidas investigações com o objetivo de avaliar infecções mistas pelo papilomavírus. O presente trabalho teve como objetivo descrever a co-infecção por dois tipos de papilomavírus bovino (BPV) em um papiloma de teto. A amostra clínica foi obtida de uma vaca pertencente a um rebanho de corte localizado na região norte do estado do Paraná, Brasil. Inicialmente, a técnica de PCR foi realizada com o par de oligonucleotídeos iniciadores FAP, que amplificam um segmento do gene L1, sendo que o amplicon gerado foi submetido ao sequenciamento direto. Entretanto, como as sequências obtidas não apresentaram qualidade aceitável, o amplicon foi clonado e dez clones foram selecionados para um novo sequenciamento. A análise das sequências dos insertos revelou a presença de dois diferentes tipos de BPV. O BPV-1 (gênero Deltapapillomavirus) foi detectado em seis clones, enquanto o BPV-6 (gênero Xipapillomavirus) foi detectado em quatro clones. Esse resultado confirma a ocorrência da co-infecção pelo BPV associada a papilomas cutâneos em bovinos.

Multiple bovine papillomavirus infections associated with cutaneous papillomatosis in brazilian cattle herds

Cutaneous papillomatosis is a pathological condition commonly found in cattle and is characterized by the presence of benign proliferative tumors caused by bovine papillomavirus (BPV) infection. While multiple infections with human papillomavirus (HPV) are common in healthy and immunodeficient humans, studies with the aim of identifying mixed infections are still sporadic in veterinary medicine. The aim of this study is to describe the occurrence of multiple BPV infections in cattle affected by cutaneous papillomatosis. Fifteen skin warts were collected from at least two diverse anatomical regions of six bovines with papillomatosis belonging to three cattle herds from the Paraná state in Brazil. The BPV types present in the skin wart samples were determined by a PCR assay performed with the FAP primer pair for partial L1 gene amplification followed by direct sequencing or by cloning and sequencing of the inserts. Sequence analysis of the obtained amplicons allowed the identification of four characterized BPV types (BPV-1, -2, -6, and -8) and three previously described putative new BPV types (BPV/BR-UEL3, BPV/BR-UEL4, and BPV/BR-UEL5). Double infections were identified in four (A, B, D, and E) of the six animals included in this study. In this work, the strategy adopted to evaluate skin warts from diverse anatomical sites of the same animal allowed the identification of multiple infections with two or three different BPV types. The analysis of four animals belonging to a single cattle herd also showed the presence of six different viral types. These results clearly suggest that both multiple papillomaviral infection and a high viral diversity can be as frequent in cattle as in human beings.

A papilomatose cutânea é comumente observada nos rebanhos bovinos e caracterizada pela presença de tumores proliferativos benignos causados pela infecção pelo papilomavírus bovino (BPV). Enquanto a infecção múltipla pelo papilomavírus humano (HPV) é um achado comum tanto em seres humanos saudáveis quanto em pacientes com imunodeficiência, na medicina veterinária esses relatos ainda são escassos. O objetivo desse estudo foi descrever a ocorrência de infecções múltiplas pelo BPV em rebanhos afetados pela papilomatose cutânea. Quinze papilomas foram obtidos, de pelo menos duas regiões anatômicas diferentes, de seis bovinos com papilomatose e provenientes de três rebanhos de corte localizados no estado do Paraná, Brasil. Os tipos virais presentes nas lesões foram identificados por PCR, utilizando o par de oligonucleotídeos iniciadores FAP, seguidos de sequenciamento direto ou clonagem e novo sequenciamento dos insertos. A análise das sequências obtidas permitiu a identificação do BPV-1, -2, -6 e -8, além de supostos novos tipos (BPV/BR-UEL3, BPV/BR-UEL4, e BPV/BR-UEL5), descritos anteriormente. Infecções por dois tipos diferentes de BPV foram identificadas em quatro animais (A, B, D e E) dos seis incluídos nesse estudo. A estratégia adotada neste estudo permitiu a identificação de infecção múltipla por dois ou três diferentes tipos virais do BPV no mesmo animal. Além disso, a avaliação de quatro animais de um mesmo rebanho demonstrou a presença de seis tipos virais circulantes. Esses resultados sugerem que tanto as infecções múltiplas quanto a grande diversidade viral podem ser frequentes nos bovinos, assim como o observado nos humanos. O reconhecimento da multiplicidade e complexidade das infecções pelo BPV pode colaborar para o entendimento dos aspectos epidemiológicos, clínicos e imunológicos da papilomatose cutânea nos rebanhos bovinos.

Investigation of the possible role of Chlamydophila abortus in reproductive failures in nrazilian herds of domestic ruminants

Chlamydophila abortus (C. abortus) infection is related to reproductive failure in domestic ruminants. Although it has not been well characterized worldwide, this pathogen has already been identified in some European countries and in the USA. In Brazil, preliminary studies have shown serological evidence of C. abortus infection in herds with low antibody prevalence. Until now, the identification of C. abortus in biological samples from females presenting reproductive failures has not been described in Brazilian herds of domestic ruminants. The aim of this study was to evaluate the presence of the C. abortus in a collection of abortions from cattle (n=85), sheep (n=12), and goats (n=8), in samples of vaginal mucus from cows (n=13), sheep (n=90), and goats (n­=20), and in semen from sheep (n=10) and goats (n=5). The specimens (n=243) were evaluated using a PCR assay developed to amplify the 16S-23S rRNA intergenic space of C. abortus. A PCR assay with an internal control, which amplifies a fragment from the ND5 gene of bovine mitochondrial DNA, was used in order to evaluate the efficiency of the DNA extraction and of the PCR reaction. All biological samples (n=243) included in this study were negative for C. abortus in the PCR assay. The internal control enabled the amplification of a product from the bovine mitochondrial ND5 gene in all cattle abortion samples (n=85). Given the serological evidence indicating the presence of C. abortus infection in Brazilian herds of domestic ruminants, and considering the wide sampling evaluated, the failure to identify C. abortus in this survey suggests that the frequency of clinical signs in infected animals may be low or even absent.

A infecção pela Chlamydophila abortus (C. abortus) em ruminantes domésticos está relacionada com distúrbios reprodutivos. Apesar de ainda pouco estudada em todo o mundo, a infecção já foi identificada em alguns países europeus e também nos EUA. No Brasil, estudos preliminares demonstraram evidências sorológicas da infecção em alguns rebanhos com baixa prevalência de anticorpos. Até o momento ainda não foi possível a identificação de C. abortus a partir de material biológico proveniente de fêmeas com problemas reprodutivos em rebanhos brasileiros. O objetivo deste trabalho foi avaliar a presença da C. abortus em uma coleção de abortos bovinos (n=85), ovinos (n=12) e caprinos (n=8), em amostras de muco vaginal bovino (n=13), ovino (n=90) e caprino (n=20), e em sêmen ovino (n=10) e caprino (n=5). As amostras biológicas (n=243) foram avaliadas por meio de técnica de PCR desenvolvida para a amplificação do espaço intergênico 16S-23S RNAr da C. abortus. Um controle interno da reação, que amplifica um fragmento do gene ND5 do DNA mitocondrial de bovino, foi utilizado para a avaliação da eficiência da extração e da amplificação do DNA nas amostras provenientes de abortamento bovino. Todas as amostras biológicas (n=243) incluídas nesse estudo resultaram negativas para a C. abortus na PCR. O controle interno da reação possibilitou a amplificação de um produto do gene ND5 mitocondrial bovino em todas as amostras de aborto bovino (n=85). Apesar de evidências,,sorológicas indicarem a presença da infecção por C. abortus em rebanhos de ruminantes no Brasil, e considerando o número de amostras biológicas avaliadas, a não identificação de C. abortus nesse estudo sugere que a frequência de sinais clínicos nos animais infectados pode ser baixa ou mesmo ausente.