M. Tollis

Immunization of monkeys with rabies ribonucleoprotein (RNP) confers protective immunity against rabies

The ability of rabies virus ribonucleoprotein (RNP) to induce protective immunity against rabies and to prime for production of virus-neutralizing antibodies (VNA) was studied in monkeys. Following two immunizations with RNP, monkeys developed a strong anti-RNP response and were protected against a challenge infection with a lethal dose of street rabies virus. Monkeys that were primed with RNP and then immunized with a single dose of human diploid cell vaccine (HDCV) developed VNA titres comparable to the VNA titres in non-primed monkeys after a second HDCV immunization. The utility of rabies RNP for the pre-exposure prophylaxis of human rabies is discussed.

ELISA Test for the Detection of Influenza H7 Antibodies in Avian Sera

Abstract Using a monoclonal antibody (MAb) specific for the H7 influenza surface glycoproteins, a serological enzyme-linked immunosorbent assay (ELISA) test has been developed. This MAb was made using the low-pathogenicity (LP) avian influenza (AI) strain (BS2676/99) isolated in Italy during a recent outbreak. The test is able to detect H7 antibodies in avian sera. The H7 ELISA has a 99% concordance of results with the classical hemagglutination inhibition (HI) test.

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.

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.

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.

Molecular characterization of low pathogenicity H7N3 avian influenza viruses isolated in Italy

Abstract The complete coding regions of the surface glycoproteins, nucleoprotein (NP), polymerase 2 (PB2), and matrix (M) of A/turkey/214845/02 and A/turkey/220158/99 (H7N3) low pathogenicity avian influenza (LPAI) viruses isolated in October 2002 in Italy were amplified and sequenced to determine the epidemiologic relationships with an A/turkey/Italy/4603/99 (H7N1/4603/99) LPAI virus isolated during the 1999–2001 epizootic in Italy. The hemagglutinin (HA) of H7N3 viruses showed 97.8% nucleotide similarity with A/turkey/Italy/4603/99 (H7N1), and NP, M, and PB2 gene similarities were 93.6%, 98.2%, and 96.2%, respectively. Phylogenetic analyses of HA, PB2, and M genes showed that H7N3 and H7N1 viruses were closely related. Sequence analysis revealed a 23 amino acid deletion in the stalk of the neuraminidase of H7N3 viruses and a unique deletion of amino acid glycine in position 17 in the NP gene of H7N1 virus.

A preliminary vaccine potency trial of a Newcastle disease virus inactivated with binary ethylenimine.

Inactivation of Newcastle Disease Virus (NDV) by binary ethylenimine (BEI) is reported. The activity of an oil vaccine prepared with BEI-inactivated NDV was compared to a vaccine prepared with formalin-inactivated NDV.The BEI inactivated vaccine had almost twice the efficacy.

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.