Emergence of Avian coronavirus Escape Mutants Under Suboptimal Antibody Titers.

To perform a quasispecies assessment of the effect of vaccine combinations and antibody titers on the emergence of Avian coronavirus (AvCoV) escape mutants, 5-week-old males from a commercial chicken breeder lineage were vaccinated intramuscularly with one dose of a monovalent (genotype GI-1) or a bivalent (genotypes GI-1 and GI-11 (n = 40 birds/group) AvCoV vaccine. Seven birds were kept as controls. Six weeks later, pools of sera of each group were prepared and incubated at virus neutralization doses of 10 and 10-1 with the Beaudette strain (GI-1) of AvCoV in VERO cells. Rescued viruses were then submitted to genome-wide deep sequencing for subconsensus variant detection. After treatment with serum from birds vaccinated with the bivalent vaccine at a titer of 10-1, an F307I variant was detected in the spike glycoprotein that mapped to an important neutralizing region, which indicated an escape mutant derived from natural selection. Further variants were detected in nonstructural proteins and non-coding regions that are not targets of neutralizing antibodies and might be indicators of genetic drift. These results indicate that the evolution of AvCoV escape mutants after vaccination depends on the type of vaccine strain and the antibody titer and must be assessed based on quasispecies rather than consensus dominant sequences only because quasispecies may be otherwise undetected.

Molecular diversity of Alphacoronavirus 1 in dogs and cats in Colombia.

Alphacoronavirus 1 (subgenus Tegacovirus, genus Alphacoronavirus, family Coronaviridae), which encompasses transmissible gastroenteritis virus (TGEV), feline coronavirus (FCoV) and canine coronavirus (CCoV), is an important pathogen that can cause severe gastroenteritis and is distributed worldwide. CCoV has two different genotypes: CCoV type I, which has a high identity with FCoV-I, and CCoV type II, which is divided into two subtypes, CCoV IIa (pantropic) and CCoV IIb, which is related to FCoV-II and has been involved in multiple recombination events. Between 2014 and 2018, 43 fecal samples from puppies and young dogs under 1 year of age with hemorrhagic enteritis and from 5 cats under 2 years of age with ascites or thoracic effusion were collected by a private veterinary practice in Bogotá, Colombia. A screening for Coronavirus via RT-PCR (nsp12) and PCR amplification of Canine protoparvovirus (VP1) revealed 27.1% (13/49) and 72.9% (35/49) positive samples, respectively. Positive samples for coronavirus were tested for M, N, S and the sequences grouped in the FCoV, CCoV-I and CCoV-IIb clusters that were distant from the pantropic type (IIa). The N gene formed two clusters, one exclusively with samples from this study in subtype II and another with strains in subtype I. For gene S (subtype I), the samples clustered with the Brazilian samples, while samples positive for S subtype IIb grouped into a cluster distinct from the other reference sequences. The prevalence of coronaviruses identified in this study is within the range reported by different countries worldwide.

Gammacoronavirus and Deltacoronavirus in Quail.

This paper expands on a previous report about coronaviruses in quail. After surveillance carried out in 2009 and 2010, some farmers started vaccinating quail with the Massachusetts avian infectious bronchitis virus serotype. The samples for this study were collected in 2013 from São Paulo state in southeastern Brazil. Pools of trachea, lungs, reproductive tract, kidneys, and enteric contents from quail and laying hens kept in the same farms and from quail-only farms as well as from both healthy birds and those showing infectious bronchitis-like symptoms were sampled in this study. The samples were screened using nested RT-PCR targeting the 3'-untranslated region of the Gammacoronavirus genus. Based on the DNA sequence for the RNA-dependent RNA polymerase (RdRp) gene, the strains isolated from quail clustered within either the Gammacoronavirus or Deltacoronavirus genus, and sequences from both genera were found in one quail sample. The phylogeny based on the partial S1 subunit sequence showed that the gammacoronaviruses detected in quail and layers belonged to the Brazil type. These results suggest that quail are susceptible to Gammacoronavirus and Deltacoronavirus viruses and indicate that the Massachusetts vaccination was not controlling IBV in quail or chickens.

Feline Coronavirus 3c Protein: A Candidate for a Virulence Marker?

Feline infectious peritonitis virus (FIPV) is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP), whereas feline enteric coronavirus (FECV) is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus) have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a-c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account.

Ocorrência do subtipo B do vírus da imunodeficiência felina em gatos domésticos da região sul do estado do Rio Grande do Sul, Brasil / Occurrence of subtype B of the feline immunodeficiency virus in domestic cats from the south region of Rio Grande do Sul state, Brazil

No Brasil existem poucos estudos sobre a ocorrência da infecção pelo vírus da imunodeficiência felina (FIV), assim como a determinação dos subtipos circulantes, o que é indispensável para o desenvolvimento de vacinas e novos testes diagnósticos. O presente trabalho investigou a ocorrência da infecção pelo FIV entre os anos de 2010 e 2011 em gatos domésticos submetidos a atendimento clínico na cidade de Pelotas e região. Amostras de sangue total de 70 animais, incluindo suspeitos (28) ou não suspeitos (42) da infecção pelo FIV, foram submetidas à reação de PCR nested. Os resultados indicaram uma frequência de infecção de 15,7% (11/70) e a análise dos fatores associados (sexo, idade e condição clínica) evidenciou uma maior ocorrência em gatos com idade superior a 10 anos e acometidos por infecções crônicas e recidivantes. Oito amostras positivas na PCR nested foram submetidas a sequenciamento genômico e somente o subtipo B foi detectado na região estudada.

In Brazil there are few studies on the occurrence of the feline immunodeficiency virus (FIV) infection and its subtypes, which are essential for the development of vaccines and new diagnostic tests. The present study investigated the occurrence of the FIV infection between 2010 and 2011 in domestic cats submitted to medical attendance in the city of Pelotas and nearby area. Total blood samples of seventy cats, suspected (28) or not (42) of infection by FIV were analyzed by nested PCR in order to perform a diagnosis. The results pointed to a FIV infection frequency of 15.7% (11/70) and the analysis of the risk factors related to infection (sex, age and clinical condition) evidenced a greater occurrence in cats up to 10 years of age with chronic and recurrent infections. Eight samples found positive by nested PCR were submitted to DNA sequencing indicating that only the subtype B was detected in the studied region.

Mutagenic fate of insecticide fenitrothion in the environment-mutagenicity increases both by anaerobic biodegradation and photodegradation.

In the present study, our objectives were (1) using the Ames assay, to evaluate the change in mutagenicity of a fenitrothion-containing solution during aerobic biodegradation, anaerobic biodegradation, and photodegradation, and (2) to identify possible mutagenic transformed products (TPs) that contributed substantially to any increase in mutagenicity. Mutagenicity of the fenitrothion-containing solution did not increase during aerobic biodegradation with any of the tested bacterial strains. In contrast, the mutagenicity increased for strain YG1029 during anaerobic biodegradation because of the generation of a strongly mutagenic TP, amino-fenitrothion. During photodegradation, mutagenicities increased slightly for YG1021 and YG1024, possibly owing to the production of a previously unreported mutagenic TP.

Multiple ulcers of the ileum due to Cytomegalovirus infection in a patient who showed no evidence of an immunocompromised state.

68-year-old woman presented with abdominal pain and vomiting. After initial conservative therapy, laparotomy showed multiple ulcers of the ileum, one of which had perforated and adhered to the uterus. The affected segment of the ileum was resected. Numerous cytomegalic cells, corresponding to endothelia and macrophages, with intranuclear inclusion bodies, were found in microscopic sections of the ulcerated lesions. These findings were consistent with cytomegalic vasculitis and enteritis. Cytomegalovirus infections of the alimentary tract have been reported mainly in severely immunocompromised patients or those with predisposing disorders such as ulcerative colitis; their prognosis is usually poor. In our patient, there was no obvious immunocompromised state or other gastrointestinal disorders. The postoperative course has been uneventful for 2 years after surgery. The prognosis of Cytomegalovirus-associated lesions in the alimentary tract may be quite good in the immunocompetent patient.

[Thymic cyst resembling a bronchogenic cyst].

Bronchogenic cysts are common cystic lesions in the mediastinum. Most are located in the middle or posterior ares of the mediastinum, especially around the carina. We encountered a patient with a mass in the superior and anterior areas of the mediastinum, where bronchogenic cysts are not usually found. The mass was therefore, thought to be a thymic cyst. The cyst was located just behind the sternum and in front of the left brachio-cephalic vein, and was 5.5 x 3.5 x 1.2 cm. It originated in the upper and median part of the thymus, and was not attached to the respiratory tract. Histological examination showed ciliacted epithelium and a layer of smooth muscle in the wall of the cyst. The cyst contained viscid, yellowish, turbid fluid. Preoperative computed tomography and magnetic resonance imaging showed findings characteristic of bronchogenic cysts, except for the location. We know of no previous reported case of a thymic cyst resembling a bronchogenic cyst.

[Investigation of microwave coagulo-necrotic therapy for 21 patients with small hepatocellular carcinoma less than 5 cm in diameter].

Microwave coagulo-necrotic therapy (MCN) was performed in 21 patients with small hepatocellular carcinoma less than 5cm in diameter and this therapeutic method was determined to be effective from the following results. 1) A marked low density area was seen in the region receiving the therapy and no enhanced findings were observed by enhanced CT one month postoperatively. 2) Recurrence appeared in 5 patients (23.8%) and only one patient died 1 year and 6 months after the operation. However, the other 20 patients survived for a maximum of 3 years and 2 months postoperatively. 3) The levels of total bilirubin and GOT in these patients were similar to those of patients who underwent hepatectomy. However, the levels of prothrombin time and hepaplatin test in patients with MCN changed less than in those who received hepatectomy. 4) Tendency of the renal failure was seen in 2 patients, and the other 19 patients did not have any complication postoperatively.

Transfusion-associated graft versus host disease following hepatectomy for hepatocellular carcinoma--a case report.

A case of transfusion-associated graft versus host disease (TA-GVHD) following hepatectomy for hepatocellular carcinoma is described in a 53 year-old male patient. The intraoperative bleeding was estimated to be 1220 ml, and he was transfused with 4 units of fresh whole blood. On postoperative day (POD) 12, a fever of 38 degrees C developed, followed by a systemic erythema on POD 14, and a marked progressive leukopenia starting from POD 19. The patient died of multiple organ failure (MOF) on POD 29. Just before death, the results of skin, bone marrow, and liver biopsies had no physical evidence of GVHD. TA-GVHD was found in the HLA typing of the patient's family. This TA-GVHD case was considered to be a reduced immunity due to severe surgical stress or preoperative transcatheter arterial embolization (TAE), in view of the fact that he was transfused with fresh whole blood during the operation. TA-GVHD has frequently been reported in patients after open heart surgery, but also after hepatectomy. It is therefore necessary to take all available means to prevent it by restricting the use of blood preparations as much as possible, and if hetero blood transfusions are performed, blood should be irradiated prior to transfusion.

A new technique of hepatectomy using an occlusion balloon catheter for the hepatic vein.

To minimize intraoperative bleeding and allow more accurate resection, we have devised a new technique for hepatectomy. In addition to occlusion of the afferent vessels, we occlude the hepatic vein at the hepatocaval junction using a balloon catheter inserted transhepatically under intraoperative ultrasonic guidance. We have performed eight hepatectomies using this method. A sequence of 15 minutes of vascular occlusion followed by 5 minutes reperfusion was repeated throughout the operation, and the total ischemic time ranged from 45 to 90 minutes. This method greatly decreased intraoperative bleeding without causing significant elevation of the postoperative transaminase or lactic dehydrogenase levels when compared with occlusion of only the afferent vessels or nonischemic resection using a microwave tissue coagulator in patients undergoing equivalent resections. Also, the postoperative prothrombin time recovered to a significantly higher level, and there were no fatal postoperative complications using this method. Our method is useful for systematic hepatic resection along the hepatic vein or for resection of tumor sited at the confluence of the hepatic vein.

Replacement of donor lymphoid tissue in small-bowel transplants.

The presence of recipient lymphocytes in grafts is thought to equate with rejection. Thus, we wished to follow the fate of lymphocytes after transplant of the small bowel. Three complete small-bowel transplants, two with the liver from the same donor also transplanted, were done successfully. Patients were immunosuppressed with FK 506. 5 to 11% of lymphocytes in the recipients' peripheral blood were of donor origin during the early postoperative period when there were no clinical signs of graft-versus-host disease. However, donor cells were no longer detectable after 12 to 54 days. Serial biopsy specimens of the grafted small bowel showed progressive replacement of lymphocytes in the lamina propria by those of the recipient's HLA phenotype. Lymphoid repopulation was complete after 10 to 12 weeks but the epithelial cells of the intestine remained those of the donor. The patients are on enteral alimentation after 5, 6, and 8 months with histopathologically normal or nearly normal intestines. Re-examination of assumptions about the rejection of intestinal grafts and strategies for its prevention are required following these observations.

A rare type of intramural esophageal diverticulosis.

Benign stenosis of the mid-thoracic esophagus developed in a 28-yr-old man. At operation, the esophageal wall was adherent to the surrounding tissues at the level of the stricture, and the esophageal wall appeared scarred and felt hard. Pathologic examination of the resected specimen showed many submucosal cystic spaces associated with mucosal depressions. Based on these findings, a diagnosis of esophageal intramural diverticulosis was made. However, some cystic spaces ran parallel to the long axis of the esophagus, and one had penetrated the muscular layers and led into the inflammatory mass. Muscularis mucosa surrounded the cystic spaces, and inflammation was slight. These findings suggest that the etiology of the esophageal diverticula of this case was a congenital malformation.

[Effect of peri-operatively administrated OK-432 on immunopotency in primary liver cancer--immunological and histological study in cases receiving intrahepatic arterial administration].

OK-432 purified from streptococcal preparations was administrated via either hepatic artery or peripheral vein to the patients with primary liver cancer to prevent the decrease of immunopotency in the peri-operative periods. In "OK-432 administration" group, the immunological parameters such Su-PS skin reactions, number of lymphocytes, NK activity, and Leu 11 positive cells of peripheral blood showed more increased levels than those in "untreated" group. Histologically, the resected specimens showed varying degrees of lymphocyte infiltration throughout the cancerous tissue in five out of nine patients who received OK-432 via hepatic artery. In patients received OK-432 via the hepatic artery which was feeding cancer lesion, there were formation of lymph follicles in the cancerous tissues and destruction of the cancer nests by infiltrating lymphocytes. Together with the immunological and histological findings, the intraarterial administration of OK-432 seemed to be useful as a multidisciplinary treatment.

[An unresectable gastric cancer radically resectable following UFT, ADM, MMC therapy].

A 58-year-old woman who was inoperable because of S3 (pancreas and colon) and P1 at the initial operation was treated with UFT, ADM and MMC at a dosage of 242.7 g, 418 mg and 166 mg, respectively. After the chemotherapy (2 years and 6 months from initial operation), a second look operation revealed that a tumor was localized in the submucosal layer and there were no lymph node metastases. Thus, it could be resected radically. Until now, various methods of chemotherapy for unresectable gastric cancer have been reported, and many cases of CR contained fluoropyrimidine derivates. In the case under study, combination chemotherapy containing UFT was performed with good results.