Emiliana Falcone

First Pandemic H1N1 Outbreak from a Pig Farm in Italy

The first outbreak of the pandemic H1N1 virus in a swine breeder farm in Italy in November 2009 was reported. Clinical signs observed in sows included fever, depression, anorexia and agalactia, while in piglets diarrhoea and weight loss. The morbidity in sows was approximately 30% and the accumulated mortality rate was similar with those usually reported in piggeries (<10%). Virus was isolated from piglets (A/Sw/It/290271/09) and the sequencing of the whole genome was then performed. Comparison with all (H1N1)v sequences available in GenBank shows A/Sw/It/290271/09 three unique amino-acid (aa) changes in PB2 (S405T), PB1 (K386R) and PA (K256Q), not yet associated to any well characterized phenotype markers of Influenza viruses. All eight aa at positions representing the so-called species specific swine-human signatures, found in both swine and in the pandemic H1N1v, are also present. The M2 protein displays the C55F and the PA protein the S409N substitutions, both corresponding to enhanced transmission phenotype markers. Phylogenetic analysis showed that the virus was genetically related to the pandemic H1N1 virus. In addition, serological samples were collected from 40 sows, of which 20 resulted positive to the pandemic H1N1 virus by HI test proving a virus circulation in the farm.

Molecular characterization of bovine rotavirus strains circulating in northern Italy, 2003–2005

A total of 232 stools collected from calves with rotavirus infection in herds located in northern Italy from 2003 to 2005 was investigated. Determination of the rotavirus G and P types was carried out using nested RT-PCR. G6 was the most prevalent genotype, accounting for 78.5% of samples, G10 accounted for 9.9% of samples and viruses of G8 type were found in 4.7% of samples. In 3% of samples, viruses were not classified due to concomitant infection with more G type strains, whereas viruses in 3.9% of samples could not be characterized with any of the G-specific primers used in this study. Most common P types were P[11] and P[5], accounting for 65.1% and 25%, respectively. In 2.6% of cases, samples reacted with multiple P-specific primers; no P[1] serotype was identified. The G6P[11] combination was predominant throughout the study period, i.e. 52.5% in 2003, 50% in 2004 and 40% in 2005. The incidence of G6P[5] increased from 13.1% in 2003 to 27% in 2004 and 25.5% in 2005. The G10P[11] combination decreased markedly from 18% in 2003 to 2.6% in 2004, rising again to 7.3% in 2005. G8P[11] viruses were similarly present in 2003 (5%) and 2004 (4.3%), declining slightly in 2005 (1.8%).

Experimental Infection of Calves with Bovine Viral Diarrhoea Virus Type-2 (BVDV-2) Isolated from a Contaminated Vaccine

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100% homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.

Molecular adaptation of an H7N3 wild duck influenza virus following experimental multiple passages in quail and turkey

To investigate the molecular adaptation of influenza viruses during natural interspecies transmission, we performed a phenotypic and genotypic analysis of a low-pathogenic duck H7N3 influenza virus after experimental passages in turkey and quail. Results obtained showed differences in the HA receptor-binding and in NA enzyme activities in viruses recovered after passages in quail, compared to those obtained from passages in turkey. Sequencing of the HA, NA and genes of internal proteins of the viruses obtained from quail and turkey, identified several amino acid substitutions in comparison with the progenitor virus. Of note, in the quail-adapted viruses the emergence of a 23-amino acid deletion in the stalk of the NA and the introduction of a glycosylation site in the HA were a reminiscence of changes typically observed in nature confirming a potential role of the quail in the adaptation of wild birds viruses to domestic poultry.

Rapid diagnosis of avian infectious bronchitis virus by the polymerase chain reaction

Abstract A simple, sensitive and specific polymerase chain reaction (PCR) procedure was developed in order to detect infectious bronchitis virus (IBV) directly in tissue samples. Viral RNA was extracted from allantoic fluids and cell cultures infected experimentally with different strains of IBV and from tissues of naturally infected birds. Viral RNA was then amplified and identified by a nested RT-PCR assay using two sets of primers flanking a well-conserved region of the nucleocapsid gene. The selected IBV nucleocapsid sequence was detected successfully by simple direct electrophoresis of amplified material.

Urinary and faecal mutagenicity in Sprague-Dawley rats dosed with the food mutagens quercetin and rutin

The natural flavonoid quercetin was administered to Sprague-Dawley rats by ip injection or gastric intubation of a single dose of 500, 1000 or 2000 mg/kg body weight. Mutagenicity assays with Salmonella typhimurium strain TA98 showed moderate mutagenic activity in the urines and faecal extracts but not in plasma samples from the treated animals. The mutagenic activity detected in the urines accounted for about 0.5% of the administered dose, irrespective of the route of administration and the dose level. Higher mutagenicity was demonstrated in faecal extracts. Rutin (quercetin-3-O-rutinoside) was administered by gavage and ip injection at 2000 mg/kg. Although the chemical was inactive as a mutagen in vitro, significant mutagenicity was detected in the urines and faecal extracts of the treated rats. Such activity was similar to that detected after administration of free quercetin in a dose some four times lower (by weight).

Genetic heterogeneity of bovine viral diarrhoea virus in Italy

The genetic characteristics, of 38 field isolates of bovine viral diarrhoea virus (BVDV) collected in 1999 from sick or healthy and persistently infected cattle of dairy farms situated in northern Italy, were investigated. A partial 5′-untranslated region (5′-UTR) sequence of each isolate was determined and a phylogenetic analysis was performed. All the isolates were classified as belonging to the BVDV-1 genotype and could be assigned to different BVDV-1 groups, namely BVDV-1b (n = 20), BVDV-1d (n = 6) and BVDV-1e (n = 10). Two remaining isolates could be classified as BVDV-1f and BVDV-1h, respectively. These results provided evidence for genetic heterogeneity of BVDV in Italy, and contribute to a better knowledge of the circulation of BVDV strains, and to their classification.

Standardization of an Inactivated H7N1 Avian Influenza Vaccine and Efficacy Against A/Chicken/Italy/13474/99 High-Pathogenicity Virus Infection

Abstract The minimum requirements for assessing the immunogenicity of an experimental avian influenza (AI) vaccine prepared from inactivated A/Turkey/Italy/2676/99 (H7N1) low-pathogenicity (LP) AI (LPAI) virus were determined in chickens of different ages. A correlation between the amount of hemagglutinin (HA) per dose of vaccine and the protection against clinical signs of disease and infection by A/Chicken/Italy/13474/99 highly pathogenic (HP) AI (HPAI) virus was established. Depending on the vaccination schedule, one or two administrations of 0.5 μg of hemagglutinin protected chickens against clinical signs and death and completely prevented virus shedding from birds challenged at different times after vaccination.

West Nile Virus: Characteristics of an African Virus Adapting to the Third Millennium World

The emergence and spread of West Nile Virus (WNV) from North through South America during the last decade, and the recent outbreaks of disease in both humans and horses in Europe suggest that the epidemiology of this infection is evolving. WNV is now considered among the emerging threats for both human and veterinary public health in areas like Europe where it was previously regarded to as an exotic agent. Further knowledge has built up from studies investigating the characteristics of the virus and its genome evolution capacity, the adaptation to new avian host species, the changes in vector competence and biology, and the host-pathogen interactions, including the immune response. Also, the new needs for preparedness to future major outbursts of disease have stimulated research on virus detection and characterization, filling the gaps in both specialized diagnostic technology and the need for field rapid assays. This review will present an overview of WNV virology, remarking the impact of virus diversity and evolution on theoretical and practical aspects involved in both risk definition, detection and control of infection.

Genotyping of Bovine Viral Diarrhoea Viruses Isolated from Cattle in Northern Italy

Following the first official report of a clinically severe outbreak of bovine viral diarrhoea disease occurring in a farm in northern Italy, which had originated from the use of a live vaccine contaminated with a strain of BVD genotype II virus, a retrospective study on the prevalence of BVDV genotypes in Italy became highly relevant. For this purpose, the genotype of 78 BVDV-positive specimens, obtained in 1998–1999 from dairy cattle in an area near to where the outbreak occurred, was characterized by PCR technology. Two sets of primers, spanning the 5′ UTR of BVDV genome, were used sequentially in a first round of RT-PCR, performed on viral RNA extracted directly from 15 clinical samples and 63 BVDV-infected cell-culture fluids; a second PCR assay followed to selectively amplify only BVDV genotype II. All the viruses under study were characterized as BVDV genotype I. As well as contributing to a better understanding of the prevalence of BVDV genotypes in the field, the results of the present study illustrate the possibility that novel BVDV strains can emerge in susceptible animals through the use of contaminated immunobiological products for bovine use.