Studies on genetic properties of porcine parvoviruses

Porcine parvovirus (PPV) is considered to be one of the most important causes of reproductive failure in swine. Fetal death, mummification, stillbirths and delayed return to estrus are some of the clinical signs commonly associated with PPV infection in a herd. The virus genome is considered to be conservative, with substitution rates near to that of their host. However, it has been shown that some parvoviruses exhibit a substitution rate close to that commonly determined for RNA viruses. In this scenario, new PPV phenotypes may reduce the effectiveness of the currently used vaccines, recommending the continuous monitoring of the currently prevalent PPV strains. In addition, a number of novel porcine parvoviruses have been described during the last decade, but the importance and characteristics of these viruses remain unknown. In the present dissertation, three studies were performed to address the PPV genetic variability, to monitor the emergence of new PPV strains and the prevalence of novel parvoviruses. In the first study, recent PPV field isolates from Austria, Brazil, Germany and Switzerland were sequenced and analyzed. These samples, together with sequences retrieved from GenBank, were included in three datasets (viral protein complete gene, viral protein partial gene and non-structural protein complete gene). For each dataset, the nucleotide substitution rate was determined and a molecular clock estimated. The analysis revealed that for the new strains, the amino acids substitutions were located mainly in the viral capsid loops. Only the capsid protein datasets present the higher suitability for phylogenetic analysis. In them, a higher divergence was found, with three well defined clusters. By inferring the evolutionary dynamics of the PPV sequences, a nucleotide substitution rate of approximately 10 -4 substitutions per site per year was found for these datasets. An association of the phylogenetic tree with the molecular clock revealed that the main divergence of the PPV strains for the viral protein ocurred in the last 30 years. In the second study, the population dynamic of PPV isolates from swine herds was analyzed using PPV complete protein gene and partial sequences deposited in GenBank. The population dynamic of the virus was calculated using a Bayesian approach with a Bayesian skyline coalescent model. Additionally, an in vitro model was performed by twenty-one consecutives passages of the Challenge strain (a virulent field strain) and NADL2 strain (a vaccine strain) in PK15 cell-line supplemented with polyclonal antibodies raised against the vaccine strain (negative control was not supplemented). The Bayesian analysis indicated a decrease in the population diversity over the years and the predominance of some PPV strains. In agreement, the in vitro study revealed that a lower number of mutations appeared for both viruses in the presence of anti-PPV antibodies in comparison with the control passages without antibodies. In the third study, tonsils and hearts from 100 pigs were collected in a German slaughterhouse in 2010 and tested for PPV, porcine parvovirus 2 (PPV2), porcine parvovirus 3 (PPV3) and porcine parvovirus 4 (PPV4). Positive samples of PPV, PPV2 and PPV3 were sequenced. PPV was observed in 60/100 hearts and 61/100 tonsils and PPV2 in 55/100 hearts and 78/100 tonsils. PPV3 and PPV4 could not be detected in the heart samples but 20/100 and 7/100, respectively, of the tonsils were tested positive. The phylogenetic analysis of the PPV, PPV2 and PPV3 sequences revealed that the German samples could be divided in at least two clusters or clades for each virus. Altogether, it can be concluded that PPV is continuously evolving. Apparently, PPV vaccines largely used in the last 30 years probably have reduced the genetic diversity of the virus and induced the predominance of strains with distinct capsid profile from the original vaccine-based strain. Moreover, the high prevalence of the PPV, PPV2 and PPV3 and their genetic diversity highlight the importance of the continuous monitoring of these viruses

Use of a modified AFLP protocol to discriminate Salmonella enterica subsp.enterica serovar Enteritidis isolates

Salmonella enterica  subsp. enterica (S.) serovar Enteritidis is one of the main pathogens involved in food-borne diseases worldwide. In epidemiological investigations of food-related salmonellosis, subtyping is necessary to improve preventive and control measures. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis is a modified AFLP that uses only one restriction enzyme to produce DNA fragments that are selectively amplified by PCR. In order to assess the applicability of SE-AFLP in S. Enteritidis typing, one hundred and eight strains isolated from poultry, swine and also from human salmonellosis outbreaks in Southern Brazil were analyzed. Strains from other countries and six different S. enterica serovars were also included as controls. SE-AFLP was able to distinguish S. Enteritidis from the other S. enterica serovars analyzed. However, most of S. Enteritidis strains isolated from poultry, salmonellosis outbreaks and most of the strains from other countries shared the same predominant pattern. The low genetic diversity identified in S. Enteritidis suggests that the strains analyzed are clonally related and one predominant SE-AFLP genotype is widely spread in Southern Brazil

Phylogenetic Classification of Feline Immunodeficiency Virus

Background: The feline immunodeficiency virus (FIV) is responsible for a retroviral disease that affects domestic and wild cats worldwide, causing Feline Acquired Immunodeficiency Syndrome (FAIDS). FIV is a lentivirus from the family Retroviridae and its genome has 3 main structural genes: gag, pol and env. Phylogenetic studies have classified FIV into 7 subtypes according to the diversity among strains from the World, mainly in the env gene. Epidemiological analyses have demonstrated the high predominance of FIV-A and FIV-B. This in silico study aimed to perform a phylogenetic analysis to study FIV diversity worldwide. Materials, Methods & Results: A total of 60 whole genome sequences (WGS) and 122 FIV env gene sequences were included in 2 datasets, which were aligned using MAFFT version 7. Recombination among genomes and/or env genes was analyzed with RDP5 software. Phylogenetic analyses with both datasets were performed, after removing the recombinant sequences, by the W-IQ-TREE and constructed and edited by the FigTree. A total of 12 recombination events involving 19 WGS were detected. In addition, 27 recombination events involving 49 sequences were observed in the env gene. A high rate of recombinants was observed inter-subtypes (A/B and B/D) and intra-subtypes (A/A). All recombinants were removed from the subsequent phylogenetic analyses. Phylogenies demonstrated 6 distinct main clades, 5 from domestic cats (A, B, C, E, U) and 1 from wild cat sequences (W) in the WGS, as well as in the specific env gene analyses. Most clustered with subtype B sequences. In the WGS analysis, clade B had a prevalence of 65.9% Brazilian sequences (27/41) and 2.4% Japanese sequences (1/41). In the env gene analyses, clade B showed a prevalence of 43.8% of Brazilian sequences (32/73) and 20.5% of USA sequences (15/73). The results of both analyses also confirm the FIV-wide geographical distribution around the world. In the phylogenetic analyses carried out with WGS, sequences from China (1/41; 2.4%), Colombia (1/41; 2.4%) and the USA (1/41; 2.4%) were identified in clade A; sequence from Canada in clade C (1/41; 2.4%); sequence from Botswana belonged to clade E (1/41; 2.4%); sequences from Brazil clustered into clade U (2/41; 5% - data not yet published); and sequences belonging to the clade W were from Canada (1/41; 2.4%) and the USA (5/41; 12.3%). Specific env gene phylogenetic analyses showed sequences from Colombia (1/73; 1.4%), France (2/73; 2.7%), the Netherlands (3/73; 4.1%), Switzerland (2/73; 2.7%), EUA (6/73; 8.3%), belonging to clade A; sequence from Canada belonging to clade C (1/73; 1.4%); sequences from Brazil belonging to clade U (2/73; 5% - data not yet published); and sequences belonging to clade W from the USA (6/73; 8.3%). Discussion: The results presented here demonstrate that FIV has a rapid viral evolution due to recombination and mutation events, more specifically in the env gene, which is highly variable. Currently, this retrovirus is classified into 7 subtypes (A, B, C, D, E, F and U-NZenv) according to their high genomic diversity. It also highlighted the importance of in silico sequence and phylogeny studies to demonstrate evolutionary processes. This was the first study to address the WGS FIV diversity with a phylogenetic approach. Keywords: FIV, in silico, phylogeny, subtypes, recombination.

Homologous recombination in pestiviruses: Identification of three putative novel events between different subtypes/genogroups

AbstractViruses from the genus Pestivirus of the family Flaviviridae have a non-segmented, single-stranded RNA genome and can cause diseases in animals from the order Artiodactyla. Homologous recombination is rarely reported in this virus family. To detect possible recombination events, all complete pestivirus genomes that are available in GenBank were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Three putative recombinant viruses derived from recombination from different pestivirus subtypes/genogroups were detected: Bovine viral diarrhea virus 1 (BVDV-1) strain 3156, BVDV-2 strain JZ05-1 and Classical swine fever virus (CSFV) strain IND/UK/LAL-290. The present study demonstrated that the pestivirus classification cannot be based only on the analysis of one fragment of the genome because genetic conversions can lead to errors. The designation of the recombinant forms (RF) provides a more informative structure for the nomenclature of the genetic variant. The present work reinforces that homologous recombination occurs in pestivirus populations under natural replication and describes the first evidence of recombination in BVDV-2

Parvovírus canino: uma abordagem evolutiva e clínica

A parvovirose canina é causada pelo parvovírus canino tipo 2 (CPV-2) e consiste em uma enfermidade mundialmente conhecida na medicina de cães, uma vez que é altamente contagiosa, caracterizada principalmente por episódios de hematoquezia, vômitos e desidratação. No Brasil, milhares de animais são infectados todo ano, sendo a via oronasal a principal forma de contágio. A mortalidade é relativamente grande já que a doença possui apenas tratamento sintomático e os animais chegam ao ambulatório em estágio crítico. Sabe-se que a vacinação reduz drasticamente a incidência da doença, porém, a evolução do vírus ainda levanta questões sobre a eficácia de algumas vacinas já que alguns animais, mesmo vacinados, acabam desenvolvendo a virose. A presente revisão aborda aspectos relacionados à parvovirose canina como: seu histórico, grupos de risco, fontes de infecção, sinais clínicos, dados epidemiológicos, métodos de diagnóstico, vacinação e tratamento

Identification of enteric viruses circulating in a dog population with low vaccine coverage

Although the use of vaccines has controlled enteric diseases in dogs in many developedcountries, vaccine coverage is still under optimal situation in Brazil. There is a large popula-tion of nonimmunized dogs and few studies about the identification of the viruses associated with diarrhea. To address this situation, stool samples from 325 dogs were analyzed bypolymerase chain reaction for the detection of common enteric viruses such as Canine ade-novirus (CAdV), Canine coronavirus (CCoV), Canine distemper virus (CDV), Canine rotavirus (CRV)and Carnivorous protoparvovirus 1 (canine parvovirus 2; CPV-2). At least one of these specieswas detected in 56.6% (184/325) of the samples. The viruses detected most frequently ineither diarrheic or nondiarrheic dog feces were CPV-2 (54.3% of the positive samples), CDV(45.1%) and CCoV (30.4%), followed by CRV (8.2%) and CAdV (4.9%). Only one agent wasdetected in the majority of the positive samples (63%), but co-infections were present in 37%of the positive samples and mainly included CDV and CPV-2. The data presented herein canimprove the clinical knowledge in regions with low vaccine coverage and highlight the needto improve the methods used to control these infectious diseases in domestic dogs

Detecção de Salmonella sp. em psitacídeos no cativeiro

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Tumor ósseo multilobular (Condroma rodens) em um cão

Background: The multilobular tumor of bone, also known as chondroma rodens, is a primary tumor of bones with low frequency in dogs. It is considered a slow-growth malignant tumor, locally invasive, able to compress and invade the cerebral tissue. Its occurrence is greater in the flat bones of skull and hard palate. The clinical signs depend on the tumor location and usually are related to the compression of adjacent structures. The aim of this study is present a multilobular tumor of bone clinical case in a dog with has progressive growth on the skull’s frontal part and facial deformation. Clinical, laboratory and therapeutic findings will be discussed in the report.Case: An 8-year-old male crossbred castrated dog, weighing 31 kg, presenting progressive growth in the skull was examined at the University of Caxias do Sul veterinary clinic. According to the owner, the tumor was firstly observed about 3 months ago, and the dog became prostrated since then. In the clinical examination, was noticed an enlarged, symmetric and diffuse volume in the skull’s frontal part, facial deformation, especially around the ocular region, causing visual deficit. It wasn’t detected any other systemic alterations. The radiography of the skull revealed a soft tissue increased volume, suggesting a mass or an encapsulated abscess. Serum biochemistry demonstrated an increase of alcaline phosphatase activity. The other hematological and biochemical parameters were within normal limits. Fine needle aspiration was performed, showing compatible result with bone neoplasm. It was chosen to make a surgical resection, starting with a cross-shaped incision on medial portion of the skull, followed by a skin disclosure to expose the tumor. With the assistance of an orthopedical chisel and metzembaum scissors, the mass was removed. The tumor presented steady and sanded aspect, reddish colored with whitish areas. Due to the anatomic area and evolved structures, it was not possible to make the total removal of the tumor with a safety margin. Fragments were sent to histopathological examination, and the final diagnosis was multilobular tumor of bone. After hospital discharge, antibiotic cefalexin and analgesia was prescribed tramadol hydrochloride and carprofen. Weekly follow-up and reviews were performed, with points with drawn within 15 days. One month after the surgery, the dog came back to the review feeling prostrated and presenting a new mass (in the same location), causing him once more visual deficit. The owner opted to euthanize, not authorizing necropsy.Discussion: The multilobular tumor of bone is considered a slow-growth malignant tumor that usually occurs on the dog’s skull. The clinical signs are related to the compression of the affected structures. In this case, the animal has visual deficit due to the mass growth above ocular region. The multilobular tumor of bone is considered rare in dogs, however it is important to know this disease manifestations, allowing the best intervention to be done. It is important to emphasize that the cytological findings are similar of those on osteosarcoma, so it is considered as a differential diagnosis. The histopathological examination is indispensable to make the final diagnosis. The surgical resection may provide better quality of life and longer survival and must be evaluated according to each case

Ornithobacterium rhinotracheale : genotipificação e sorotipificação de isolados brasileiros

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