Genomic analysis of influenza A virus from captive wild boars in Brazil reveals a human-like H1N2 influenza virus

Influenza is a viral disease that affects human and several animal species. In Brazil, H1N1, H3N2 and 2009 pandemic H1N1 A(H1N1)pdm09 influenza A viruses (IAV) circulate in domestic swine herds. Wild boars are also susceptible to IAV infection but in Brazil until this moment there are no reports of IAV infection in wild boars or in captive wild boars populations. Herein the occurrence of IAV in captive wild boars with the presence of lung consolidation lesions during slaughter was investigated. Lung samples were screened by RT-PCR for IAV detection. IAV positive samples were further analyzed by quantitative real-time PCR (qRRT-PCR), virus isolation, genomic sequencing, histopathology and immunohistochemistry (IHC). Eleven out of 60 lungs (18.3%) were positive for IAV by RT-PCR and seven out of the eleven were also positive for A(H1N1)pdm09 by qRRT-PCR. Chronic diffuse bronchopneumonia was observed in all samples and IHC analysis was negative for influenza A antigen. Full genes segments of H1N2 IAV were sequenced using Illumina's genome analyzer platform (MiSeq). The genomic analysis revealed that the HA and NA genes clustered with IAVs of the human lineage and the six internal genes were derived from the H1N1pdm09 IAV. This is the first report of a reassortant human-like H1N2 influenza virus infection in captive wild boars in Brazil and indicates the need to monitor IAV evolution in Suidae populations.     

Diversity of genome segment B from infectious bursal disease viruses in the United States

Several phylogenetic lineages of the infectious bursal disease virus (IBDV) genome segment B have been identified. Although this genome segment has been shown to contribute to virulence, little is known about the genetic lineages that exist in the United States. The nucleotide genome segment B sequences of 67 IBDV strains collected from 2002 to 2011 in the United States were examined. Although they were from nine different states, a majority (47) of these viruses were from California. A 722-base pair region near the 5' end of genome segment B, starting at nucleotide 168 and ending at 889, was examined and compared to sequences available in GenBank. The nucleotide sequence alignment revealed that mutations were frequently observed and that they were uniformly spaced throughout the region. When the predicted amino acids were aligned, amino acids at positions 145, 146, and 147 were found to change frequently. Six different amino acid triplets were observed and the very virulent (vv) IBDV strains (based on presence of vvIBDV genome segment A sequence) all had the triplet T145, D146, and N147. None of the non-vvIBDV strains had this TDN triplet. Phylogenetic analysis of the 67 nucleotide sequences revealed four significant genome segment B lineages among the U.S. viruses. One of these included the genome segment B typically found in vvIBDV and three contained non-vvIBDV genome segment B sequences. When the available sequences in GenBank were added to the analysis, two additional lineages were observed that did not contain U.S. viruses; one included viruses from China and the other contained viruses from the Ivory Coast. Although the samples tested do not represent all poultry producing regions in the United States, serotype 1 viruses from states outside California all belonged to one genome segment B lineage. The other three lineages observed in the United States were populated with viruses exclusively found in California, except the serotype 2 lineage, where the type strain was a serotype 2 virus from Ohio. The data provide further evidence for the importance of genome segment B identification during routine molecular diagnosis of all IBDV strains.     

Hemorrhagic diathesis and bone marrow aplasia secondary to lomustine overdose in a dog

A 9‐year‐old mixed breed 13 kg spayed female dog was presented for evaluation of two masses in the right abdominal mammary gland region. Surgery was conducted to excise the masses. A grade I complex mammary gland carcinoma and high grade (grade III) mast cell tumor with an inguinal lymph node metastasis were diagnosed. Forty‐seven days after the surgical procedure, the mast cell tumor relapsed, and neoadjuvant treatment with lomustine (81 mg/m²) was prescribed. Thirteen days from initiation of lomustine therapy, the dog was re‐presented to the hospital with bloody diarrhea, hematemesis, epistaxis, an elevated rectal temperature, depression, severe dehydration, and marked dyspnea. The CBC showed severe thrombocytopenia and leukopenia. According to the owner, lomustine (45mg per os [PO]) was mistakenly administered daily for 10 consecutive days (total dose, 810 mg/m²). The dog died and a necropsy was performed. The main gross lesions consisted of severe multifocal hemorrhages in multiple organs, especially in the digestive system. Histopathologic evaluation revealed disseminated hemorrhages, as well as marked bone marrow aplasia. This report describes the clinical, hematologic, gross, and histologic findings in a fatal case of lomustine overdose in a dog.     

The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions

Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.     

Transmission of chronic nociception by spinal neurons expressing the substance P receptor

Substance P receptor (SPR)-expressing spinal neurons were ablated with the selective cytotoxin substance P-saporin. Loss of these neurons resulted in a reduction of thermal hyperalgesia and mechanical allodynia associated with persistent neuropathic and inflammatory pain states. This loss appeared to be permanent. Responses to mildly painful stimuli and morphine analgesia were unaffected by this treatment. These results identify a target for treating persistent pain and suggest that the small population of SPR-expressing neurons in the dorsal horn of the spinal cord plays a pivotal role in the generation and maintenance of chronic neuropathic and inflammatory pain.     

The expression of histamine H4 receptor mRNA in the skin and other tissues of normal dogs

The histamine 4 (H₄) receptor was first cloned and characterized in 2000 using the human H₃ receptor DNA sequence. The H₄ receptor has been shown to participate in various aspects of inflammation, such as chemotaxis, upregulation of adhesion molecule expression and modulation of cytokine secretion. The primary goal of this study was to determine whether H₄ receptor mRNA is expressed in normal canine skin by performing an RT‐PCR. An additional goal was to determine the expression of this receptor in the colon, liver, spleen and kidney. Tissues were collected from five healthy, young‐adult pit bull dogs. Samples were immediately placed in RNAlater^® solution and stored at ?20°C until processed. The amplified products in all skin samples in addition to the colon, liver, spleen and kidney (variable expression) had the expected size of 400–500 bp. The sequenced amplicons matched the National Center for Biotechnology Information published sequence for the canine H₄ receptor. The study results showed that canine normal skin expresses the H₄ receptor mRNA. Further studies using immunohistochemistry should be conducted to demonstrate the expression of the H₄ receptor at the protein level and to localize the expression of this receptor in the skin.     

Effect of oil-based formulations of acaripathogenic fungi to control Rhipicephalus microplus ticks under laboratory conditions

The formulations of acaripathogenic fungi to control ticks have been widely studied. The present study evaluated the efficacy of oil-based formulations of Metarhizium anisopliae sensu lato (s.l.), isolate Ma 959, and Beauveria bassiana, isolate Bb 986, on different Rhipicephalus microplus stages, comparing the efficacy between aqueous suspensions and 10, 15 and 20% mineral oil formulations. Twelve groups were formed: one aqueous control group; three mineral oil control groups, at 10, 15 or 20%; two aqueous fungal suspensions of M. anisopliae s.l. or B. bassiana; and three formulations of M. anisopliae (s.l.) or B. bassiana containing 10, 15, and 20% mineral oil. To prepare aqueous suspensions and oily formulations, fungal isolates were cultivated on rice grains in polypropylene bags. The conidial suspensions and formulations had a concentration of 10(8)conidia/mL. Bioassays were repeated twice. After treatment, the following biological parameters of engorged females were evaluated: hatching percentage, egg production index, nutritional index, and percentage of tick control. The following parameters were evaluated in the bioassays with eggs: period of incubation, period of hatch, and hatching percentage. Mortality was evaluated in bioassays with larvae. M. anisopliae s.l. and B. bassiana oil-based formulations were more effective than aqueous suspensions against R. microplus eggs, larvae and engorged females, however, there was no significant difference between the three oil concentrations used. M. anisopliae s.l. and B. bassiana formulated in mineral oil reached 93.69% and 21.67% efficacy, respectively, while M. anisopliae s.l. and B. bassiana aqueous suspensions attained 18.70% and 1.72% efficacy, respectively. M. anisopliae s.l. oil-based formulations caused significant effects in all biological parameters of engorged females while B. bassiana oil-based formulations modified significantly the nutritional index only. Eggs treated with M. anisopliae s.l. and B. bassiana oil-based formulations showed hatching rates that decreased 102.5 and 3.65 times, respectively. In the bioassay with larvae, M. anisopliae s.l. oil-based formulations caused nearly 100% mortality five days after treatment, while larva treated with B. bassiana oil-based formulations reached 100% mortality at day 20 after treatment. Larva from oil-based control groups showed mortality at day 15 after treatment, which indicated a possible toxic effect of the oil for this R. microplus stage. The results showed that the fungal mineral oil formulations tested were more effective than the aqueous suspension. Oil-based formulations at 10, 15 and 20% enhanced the activity of M. anisopliae s.l. Ma 959, and B. bassiana Bb 986, isolates against R. microplus eggs, larvae, and engorged females tick. Mineral oil was effective as an adjuvant in formulations of M. anisopliae s.l., Ma 959, and B. bassiana, Bb 986, for the control of R. microplus under laboratory conditions.