L. Gallina

Inhibition of viral RNA synthesis in canine distemper virus infection by proanthocyanidin A2

Canine distemper virus (CDV) is a contagious and multisystemic viral disease that affects domestic and wild canines as well as other terrestrial and aquatic carnivores. The disease in dogs is often fatal and no specific antiviral therapy is currently available. In this study, we evaluated the in vitro antiviral activity against CDV of proanthocyanidin A2 (PA2), a phenolic dimer belonging to the class of condensed tannins present in plants. Our results showed that PA2 exerted in vitro antiviral activity against CDV with a higher selectivity index compared to ribavirin, included in our study for the previously tested anti-CDV activity. The time of addition assay led us to observe that PA2 was able to decrease the viral RNA synthesis and to reduce progeny virus liberation, at different times post infection suggesting multiple mechanisms of action including inhibition of viral replicative complex and modulation of the redox milieu. These data suggest that PA2, isolated from the bark of Aesculus hippocastanum, has potential usefulness as an anti-CDV compound inhibiting viral replication.

TaqMan Based Real Time PCR for the Quantification of Canine Distemper Virus

Scagliarini, A., Dal Pozzo, F., Gallina, L., Vaccari, F. and Morganti, L., 2007. TaqMan based real time PCR for the quantification of canine distemper virus. Veterinary Research Communications, 31(Suppl. 1), 261–263

Original Findings Associated with Two Cases of Bovine Papular Stomatitis

ABSTRACT Bovine papular stomatitis virus was isolated from two calves in an animal house with biosafety level 3 confinement. The hypotheses on the origin of the infection, the interesting features of the partial amino acid sequences of the major envelope viral protein, and the importance of diagnostic tools available for animal diseases that are not listed by the World Organization for Animal Health (OIE) are discussed.

Antiviral efficacy of EICAR against canine distemper virus (CDV) in vitro.

Canine distemper virus (CDV) is a highly contagious pathogen of carnivores. In dogs, the disease is characterized by high lethality rates and no specific antiviral therapy is available. The aim of this study was to verify the in vitro antiviral activity of the 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR) and to compare it with the 1-(beta-d-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin, RBV). EICAR was more active than RBV against CDV replication, while both molecules exhibited low selectivity indexes. A reversal of their antiviral activity was observed after addition of guanosine, suggesting their involvement in the inhibition of the inosine monophosphate dehydrogenase enzyme (IMPDH). RBV and EICAR had a time- and concentration-dependent anti-CDV activity, mainly displayed during the first 10h post-infection. The involvement of the inhibition of the viral RNA-dependent RNA polymerase (vRdRp) is discussed, as well as the role of CDV as a model to study more potent and selective antiviral molecules active against other Paramyxoviridae.

Therapeutic paint of cidofovir/sucralfate gel combination topically administered by spraying for treatment of orf virus infections.

The aim of the research was to study a new cidofovir/sucralfate drug product to be used as a spray for treating the mucosal and/or skin lesions. The product, i.e., a water suspension of sucralfate (15% w/w) and cidofovir (1% w/w), combines the potent antiviral activity of the acyclic nucleoside phosphonate cidofovir ((S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine) and the wound healing properties of sucralfate gel (sucrose octasulphate basic aluminum salt). The product was characterized in vitro with respect to compatibility between drug and carrier, spray particle size, spray deposition, drying kinetics, and drug content and release. An interaction between the two active substances was found. The interaction between sucralfate and cidofovir was counteracted by introducing sodium dihydrogen phosphate (16% w/w) in the preparation. The spray formulation containing cidofovir/sucralfate gel painted the skin and dried quickly to a scab, remaining firmly adhered to the lesions. The therapeutic paint was tested in vivo on lambs infected with orf virus by treating the animals with different cidofovir/sucralfate formulations (0.5% or 1% cidofovir + sucralfate 15% + NaH2PO4 16% w/w) and with sucralfate gel suspension alone as control. The treatment with formulations containing cidofovir and phosphate salt for four consecutive days resulted in a rapid resolution of the lesions, with scabs containing significantly lower amounts of viable virus when compared with untreated lesions and lesions treated with sucralfate suspension alone.

Co-infection with multiple variants of canine parvovirus type 2 (CPV-2).

Battilani, M., Gallina, L., Vaccari, F. and Morganti, L., 2007. Co-infection with multiple variants of canine parvovirus type 2 (CPV-2). Veterinary Research Communications, 31(Suppl. 1), 209–212

Cervus elaphus papillomavirus (CePV1): New insights on viral evolution in deer

We identified a novel papillomavirus (CePV1) in a fibropapilloma of a 1.5 year old male red deer (Cervus elaphus) shot in the Italian Alps in Brescia province. PV particles were first observed by electron microscopy and PV DNA was then identified by PCR using degenerate primers. Subsequently we cloned the entire genome and determined its complete sequence. CePV1 genome is 8009 bp long and contains all 9 ORFs and the long untranslated regulatory region characteristic for Delta-papillomaviruses. Pairwise nucleotide alignments and phylogenetic analyses based on concatenated E1-E2-L1 ORFs allowed to determine the highest similarity with the Capreolus caprelus papillomavirus CcaPV1. The analysis of the host-parasite phylogenetic tree interactions suggest the co-divergence of CePV1 and C. elaphus while the identified topological incongruences leading us to speculate that CcaPV1 could eventually be the result of an earlier host switch event.

In Vitro Evaluation of Antiviral Activity of Ribavirin Against Canine Distemper Virus

Canine distemper virus (CDV) a member of the Morbillivirus genus of the family Paramyxoviridae, is a highly infectious and an acknowledged lethal pathogen of many carnivores. The disease is controlled by vaccination but an increasing number of distemper cases has been recorded even in vaccinated dogs all over the world and the affected animals can display catharral and or neurological symptoms. At present, there is no specific therapy, even if there is an increasing demand for one from the dog owners. For this reason the research on antiviral compounds against distemper is aimed at finding active molecules to treat the infection. Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carbox-amide, RBV) is a broad spectrum antiviral agent (Garcia et al., 2001; Crance et al., 2003; Poynard et al., 1998) that has been licensed for clinical use (as an aerosol) in the treatment of respiratory syncytial virus and for the therapy of measles pneumonia (Forni et al., 1994) and subacute slerosing panencephalitis (Hara et.al, 2003; Hosoya et al., 2004). The aim of the present study was to evaluate the in vitro antiviral activity of ribavirin against CDV.

Detection and quantification of Betanodavirus by real time PCR

Betanodavirus infection is now widespread in several fish species worldwide and it causes severe economic losses in European sea bass (Dicentrarchus labrax) farming in Italy. Betanodavirus are small ssRNA non-envelope viruses. Based on phylogenetic analysis of the coat protein gene, Betanodavirus can be classified into four genotypes: SJNNV, TPNNV, BFNNV, RGNNV (Nishizawa et al., 1997). The virus is widespread both in farmed and wild animals (Gagné et al., 2004; Gomez et al., 2004) and it has high resistance to environmental and chemico-physical disinfectant treatment, so control by direct prophylaxis is particularly difficult. Furthermore, fattening-farm is often conducted in sea cages or in extensive brackish ponds with no clear-cut separation between wild and farmed fish (Munday et al., 2002). Despite a recent work (Thiéry et al., 2004) reporting the presence of a SJNNV-like virus in a sole farmed in the Mediterranean, the main genotype widespread in this region and responsible for the outbreaks on sea bass farms is the RGNNV. Unfortunately, there is no vaccine available for this disease, due to a limited knowledge about the pathogenesis of Betanodavirus infection, so control is based on early diagnosis and broodstock control. In this study we set up a new Real Time PCR diagnostic method. This technique was found to be rapid, practical and sensitive. Several Real Time PCR methods have recently been set up to detect numerous pathogens significantly contributing to studies on pathogenesis (Gilad et al., 2004), diagnosis and antiviral activity (Yun et al., 2003).

A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.