N-glycosylation of infectious bronchitis virus M41 spike determines receptor specificity.

Infection of chicken coronavirus infectious bronchitis virus (IBV) is initiated by binding of the viral heavily N-glycosylated attachment protein spike to the alpha-2,3-linked sialic acid receptor Neu5Ac. Previously, we have shown that N-glycosylation of recombinantly expressed receptor binding domain (RBD) of the spike of IBV-M41 is of critical importance for binding to chicken trachea tissue. Here we investigated the role of N-glycosylation of the RBD on receptor specificity and virus replication in the context of the virus particle. Using our reverse genetics system we were able to generate recombinant IBVs for nine-out-of-ten individual N-glycosylation mutants. In vitro growth kinetics of these viruses were comparable to the virus containing the wild-type M41-S1. Furthermore, Neu5Ac binding by the recombinant viruses containing single N-glycosylation site knock-out mutations matched the Neu5Ac binding observed with the recombinant RBDs. Five N-glycosylation mutants lost the ability to bind Neu5Ac and gained binding to a different, yet unknown, sialylated glycan receptor on host cells. These results demonstrate that N-glycosylation of IBV is a determinant for receptor specificity.

Rapid detection and quantification of plasmid-mediated colistin resistance genes (mcr-1 to mcr-5) by real-time PCR in bacterial and environmental samples.

AIM: The aim of the study was to validate a rapid method to detect and quantify colistin resistance genes (mcr-1 to mcr-5) by real-time polymerase chain reaction (RT-PCR) in diverse matrices. METHODS AND RESULTS: The detection limit of two newly designed SYBR Green real-time PCR assays for mcr-4 and mcr-5 and of previously published protocols for mcr-1 to mcr-3 was assessed using serial dilutions of reference strains. The assays could detect all five mcr genes with the lower limit of 102 copy numbers. Escherichia coli isolates (n = 1062) and environmental samples (n = 93) were tested for the presence of mcr genes. The assays enabled the detection of colistin resistance genes both in bacterial isolates and in complex environmental samples. CONCLUSIONS: This method represents a set of sensitive, rapid and effective assays for the screening of colistin resistance directly from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Colistin is an antimicrobial commonly used in animals and has recently emerged as a last-resort treatment in humans. Plasmid-mediated mcr genes confer resistance to colistin and represent a major threat for public health since they can be easily disseminated through horizontal gene transfer. The rapid and sensitive detection of mcr genes is of utmost necessity.

A sensitive, reproducible, and economic real-time reverse transcription PCR detecting avian metapneumovirus subtypes A and B.

Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

Attenuated live infectious bronchitis virus QX vaccine disseminates slowly to target organs distant from the site of inoculation.

Infectious bronchitis (IB) is a highly contagious respiratory disease of poultry, caused by the avian coronavirus infectious bronchitis virus (IBV). Currently, one of the most relevant genotypes circulating worldwide is IBV-QX (GI-19), for which vaccines have been developed by passaging virulent QX strains in embryonated chicken eggs. Here we explored the attenuated phenotype of a commercially available QX live vaccine, IB Primo QX, in specific pathogens free broilers. At hatch, birds were inoculated with QX vaccine or its virulent progenitor IBV-D388, and postmortem swabs and tissues were collected each day up to eight days post infection to assess viral replication and morphological changes. In the trachea, viral RNA replication and protein expression were comparable in both groups. Both viruses induced morphologically comparable lesions in the trachea, albeit with a short delay in the vaccinated birds. In contrast, in the kidney, QX vaccine viral RNA was nearly absent, which coincided with the lack of any morphological changes in this organ. This was in contrast to high viral RNA titers and abundant lesions in the kidney after IBV D388 infection. Furthermore, QX vaccine showed reduced ability to reach and replicate in conjunctivae and intestines including cloaca, resulting in significantly lower titers and delayed protein expression, respectively. Nephropathogenic IBVs might reach the kidney also via an ascending route from the cloaca, based on our observation that viral RNA was detected in the cloaca one day before detection in the kidney. In the kidney distal tubular segments, collecting ducts and ureter were positive for viral antigen. Taken together, the attenuated phenotype of QX vaccine seems to rely on slower dissemination and lower replication in target tissues other than the site of inoculation.

Unrevealed genetic diversity of GII Norovirus in the swine population of North East Italy.

Noroviruses (NoVs) are one of the major causative agents of non-bacterial gastroenteritis in humans worldwide. NoVs, belonging to Caliciviridae, are classified into ten genogroups (G) and eight P-groups based on major capsid protein (VP1) and of the RNA-dependent-RNA-polymerase (RdRp), respectively. In swine, the main genogroup and P-group identified are GII and GII.P; which can infect humans too. To date, only one case of GIIP.11 have been identified in swine in Italy while the circulation of other P-types is currently unknown. In the present study, 225 swine faecal samples were collected from 74 swine herds in Veneto region through on-farm monitoring. NoV circulation was particularly high in older pigs. The phylogenetic analysis showed the co-circulation of NoVs belonging to two different P-types: GII.P11 and GII.P18, here described for the first time in Italy, presenting an extensive genetic diversity, never described before worldwide. Distinct NoV genetic subgroups and unique amino acid mutations were identified for each P-type for the first time. This study demonstrated the co-circulation of diverse swine NoVs subgroups in Italy, raising questions on the origin of such diversity and suggesting that continuous monitoring of swine NoVs is needed to track the emergence of potentially zoonotic viruses by recombination events.

Author Correction: Unrevealed genetic diversity of GII Norovirus in the swine population of North East Italy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Subpopulations in aMPV vaccines are unlikely to be the only cause of reversion to virulence.

Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine (strain VCO3/50) and progenitor (strain VCO3/60616) consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence was not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, can be involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release.

[Initial cancer of the breast. Radiotherapy and radical surgical treatment]. / Il cancro iniziale della mammella. Radioterapia e terapia chirurgica radicale.

The controversy over the treatment of initial cancer of the breast is well known. An account is given of the factors that determine the prognosis, and the clinical and instrumental examinations on which staging is based. Lastly, radiotherapeutical approaches founded on precise classification dependent on systematic investigations of a radiological, radio-isotopic biochemical and immunological order, and, of course, on accurate information of surgical relevance, are presented.

[Metabolic endoradiotherapy with P32 in skeletal metastases from breast neoplasms. Note I]. / La endoradiotherapia metabolica con 32P nelle metastasi scheletriche da neoplasia del seno. Nota 1

22 patients suffering from breast neoplasia with particularly painful bone metastasis were treated with radiophosphorus. Only occasionally was an evident recalcification condition encountered and survival, although exceptional in some cases, did not deviate from normal. On the basis, also, of clinical and experimental observations reported in the literature, it is held that the use of 32P in metabolic radiotherapy of bone metastases is worthwhile and is justified because of the encouraging successes obtained, especially in pain remission.

[Possibilities of metabolic endoradiotherapy with P32 in the prevention of osseous metastases from breast neoplasms. Note II].

To reach a definitive conclusion on the value of using 32P in the prophylaxis of bone metastases in association with surgical and traditional radiotherapeutic treatment, observations should be extended to a much greater number of cases. This notwithstanding, the results obtained justify this methodology based on continuous sustained, internal irradiation which would seem to make possible a diminution in the frequency of bone metastases in the early years of a delay in their onset.

[Radioisotope methods and technics in obstetrical and gynecological diagnosis]. / Tecniche e metodiche radioisotopiche nella diagnostica ostetrica e ginecologica

Certain applications of radioisotopes to obstetric and gynaecological diagnosis are illustrated. Radioisotopes have been widely employed since the improvement in instrumental methods and the availability of short-life radioisotopes has removed the basic limitation which once prevented their use during the entire fecund period of the woman owing to the risk of radiation damage to the reproductive system. Particular attention is paid to the advances made in placental localization, in the study of thyroid and renal function in pregnancy, in the study of the diffusion of female genital tumours and relapses, and in early diagnosis of tumours of the uterus and ovaries. Dosimetric problems arising when using diagnostic techniques with radioisotopes in obstetrics and gynaecology are analysed.

[Radiological picture Turner's syndrome]. / L'indagine radiologica nella sindrome di Turner.

A review of the relevant literature is followed by presentation of the radiological picture, particularly that of the skeleton, in 8 patients with the clinical and endocrinological features and the chromosome pattern of Turner's syndrome. An indication is given of the weight to be attached to the main and lesser radiological signs, especially in cases where the absence of somatic features and detectable hypogonadism impede early diagnosis.

[Radiothermolumiscent dosimetry in plesioroentgenotherapy]. / Dosimetria a radiotermoluminescenza in plesioroentgenterapia

The employment of thermoluminescence dosimeters in the evaluation of X-rays emitted by a plesiotherapy apparatus was investigated. It was found that lithium fluoride microdosimeters gave good linearity of response in function of the energy involved. This response was also repeatable as required.

[Histoenzymatic changes and dosimetric studies of some organs and areas, using diagnostic radioisotopes. Critical review of a 10-year experiment]. / Modificazioni istoenzimatiche e rilievi dosimetrici su alcuni organi e distretti per impiego di radioisotopi a scopo diagnostico. Resoconto critico di una esperienza decennale.

Ten years experience of dosimetry and study of the histochemical changes brought on by internal irradiation are reviewed. The research is broken down into three stages: 1) identification of enzymatic damage in small laboratory animals; 2) study of the dose really absorbed at various points of critical organs of large animals, 3) the collection of histoenzymatic data resulting from research on large animals treated internally with isotopes commonly used in diagnosis.

[The general practitioner and clinical records: a survey of the knowledge of users of a software package]. / Medico di base e cartelle cliniche: indagine conoscitiva su utenti in possesso di un pacchetto software.

The FATMA project--subproject 6, Line of Research 2--aims at implementing a new computerized information network for general practitioners using the same kind of software and homogeneously located throughout Italy. Thus, patients' case form data can be collected for public health purposes. After a study of literature in order to check whether other countries had implemented similar projects and what they consisted of, a survey based on a computerized questionnaire was carried out on a group of g.p.s. who had already received a software for the management of their clinical and professional activities from Janssen Informedica. This survey aimed at judging some aspects of users in order to select potential monitors of the information network who would test a new software. The analysis regards both g.p.s' social and professional data and the use of a computer package with reference to the quality and quantity of the clinical data they input. 2,010 questionnaires were filled in all their parts and mailed back to Informedica. It came out that g.p.s. believe more and more they can improve the organization and management of their work by using a software equipped with routines and easy access procedures so that clinical data, diagnostic reports and treatment can be collected, placed on files and updated. Thus, a new software should be developed. While meeting g.p.s' needs, it must overcome the limits of packages now available concerning the possibility of carrying out epidemiological studies. The software can become a tool to collect clinical data of patients, diseases and diagnostic procedures through standard protocols directly from g.p.s records.