High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies.

Factor V is an essential clotting factor that plays a key role in the blood coagulation cascade on account of its procoagulant and anticoagulant activity. Eighty percent of circulating factor V is produced in the liver and the remaining 20% originates in the α-granules of platelets. In humans, the factor V gene is about 80 kb in size; it is located on chromosome 1q24.2, and its cDNA is 6914 bp in length. Furthermore, nearly 190 mutations have been reported in the gene. Factor V deficiency is an autosomal recessive coagulation disorder associated with mutations in the factor V gene. This hereditary coagulation disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft-tissue bleeds to potentially fatal hemorrhages. Current treatment of this condition consists in the administration of fresh frozen plasma and platelet concentrates. This article describes the cases of two patients with severe factor V deficiency, and of their parents. A high level of mutational heterogeneity of factor V gene was identified, nonsense mutations, frameshift mutations, missense changes, synonymous sequence variants and intronic changes. These findings prompted the identification of a new mutation in the human factor V gene, designated as Jaén-1, which is capable of altering the procoagulant function of factor V. In addition, an update is provided on the prospects for the treatment of factor V deficiency on the basis of yet-to-be-developed recombinant products or advanced gene and cell therapies that could potentially correct this hereditary disorder.

Modified haemagglutination inhibition assay for the detection of canine parvovirus type 2 antibodies in dog sera.

Canine parvovirus type 2 (CPV-2) infection is associated with severe gastroenteritis in puppies. Quantification of CPV-2 specific antibodies before vaccination can reveal the presence of interfering maternal-derived immunity and facilitate timing of effective immunisation. Inhibition of haemagglutination (HI) is commonly used to measure CPV-2-specific antibody levels in serum. However, the presence of nonspecific agglutinins in canine serum and artefactual precipitation of red blood cells (RBC) are both limitations of the assay. In this study, we compared the standard HI protocol with a refined HI protocol, in which canine serum was pre-incubated with porcine RBC for 12 h to remove nonspecific agglutinins and a lower concentration (0.1% vs. 0.8%) of porcine RBC suspensions was used to limit artefactual precipitation of RBC. A panel of canine sera, collected from 80 dogs of different ages and with different neutralising antibody titres, was analysed. Nonspecific agglutinins were identified in most (97%) serum samples from puppies <4 months of age and in only 7% dogs 6 months old. Pre-treatment of serum samples was effective in removing nonspecific agglutinins from all samples and artefactual precipitation of RBCs was not noted when 0.1% RBC suspensions were used. Refinement of the HI protocol has increased the accuracy of interpretation and reduced the interference of nonspecific agglutinins, primarily seen in puppies. This reduces the likelihood of incorrect assessment of passive or active immunity in puppies when deciding whether to administer or defer vaccination, which could potentially leave them susceptible to CPV-2 infection.

Oral administration of modified live canine parvovirus type 2b induces systemic immune response.

Different strategies have been proposed to overcome maternally derived antibody (MDA) interference with canine parvovirus type 2 (CPV-2) immunisation, including intranasal vaccination, which presents some practical limitations. In the present study, the results of the oral administration of a commercial CPV-2b modified live virus (MLV) vaccine in pups with MDA are reported. The CPV-2b vaccine was orally administered to 14 6-week-old pups with a bait. Blood samples and rectal swabs were collected at different days post-vaccination (dpv) to determine CPV-2 antibody titres and DNA loads. Thirteen pups were positive to serological and virological tests after the first vaccination and one pup became positive after the second vaccine administration. The findings of this study suggest that systemic immunity against CPV-2 may be achieved by the use of an MLV CPV-2b vaccine administered orally even in the presence of MDA titres that usually interfere with vaccination.

Bubaline alphaherpesvirus 1 induces a latent/reactivable infection in goats.

Latent infection is a common mechanism used by several alphaherpesviruses to persist in their host but it is not clear whether this mechanism is also triggered in heterologous infections. Cross-species infections have been documented repeatedly for alphaherpesviruses of ruminants, a group of closely related viruses. Herewith we report latent infection with bubaline alphaherpesvirus 1 (BuHV-1) in experimentally infected goats and subsequent virus reactivation after treatment with dexamethasone (DMS) at 10 months after infection. After DMS treatment, the virus was isolated in one such animal in the nasal swabs from day 3 to 9 post treatment and in the ocular swabs at day 6. The goat was euthanized 48 days after DMS treatment and viral DNA was detected by PCR in the trigeminal ganglia and in two cervical ganglia. Additionally, BuHV-1 DNA was detected by PCR in the trigeminal ganglia of the other 3 goats.

In vitro virucidal activity of sodium hypochlorite against canine parvovirus type 2.

Canine parvovirosis is a very contagious, severe and often lethal infectious disease of dogs caused by canine parvovirus type 2 (CPV-2). Parvoviruses are very resistant to several disinfectants while are sensitive to halogens such as sodium hypochlorite which is often used for decontamination of veterinary clinics and animal housing facilities due to its broad spectrum of activity. If compliance with vaccination programmes and with proper disinfection plans is ensured, there should be no continuous, nor frequent, CPV-2 outbreaks in kennels and veterinary clinics. However, a continuous spread of CPV-2 infections is observed, even in kennels where an appropriate vaccination programme is applied, and this imposes a re-evaluation of disinfection protocols using sodium hypochlorite. The aim of the present study was to determine the effect of concentration, contact time and presence of organic matter on the virucidal activity of sodium hypochlorite against several CPV-2 strains. A sensitive in vitro assay capable of measuring the infectivity of CPV-2 was employed to determine the efficacy of three different concentrations of sodium hypochlorite. The data indicate that using a 0.75% sodium hypochlorite solution for a short contact time (1 min) can reduce significantly the CPV-2 titres and that even lower concentrations, i.e. 0.37%, can efficiently inactivate the viruses provided that the contact time is extended to 15 min. Results also confirm the importance of cleaning before disinfection since the presence of organic matter totally abrogated the virucidal activity of sodium hypochlorite solutions against the three CPV-2 strains.

Goats are susceptible to Bubaline alphaherpesvirus 1 infection: Results of an experimental study.

Herpesvirus infections are generally subjected to strong host species restriction, although virological and serological investigations have revealed the possibility of cross-species infections in closely related animal species. In this study we evaluated susceptibility of goats to infection by Bubaline alphaherpesvirus 1 (BuHV-1). Four goats were inoculated intra-nasally with BuHV-1 and monitored clinically, virologically and serologically for 42days. None of the goats displayed clinical signs although all the animals variably shed the virus by the nasal route during the first 12days after infection. BuHV-1 was also detected in the white blood cells of two animals in the first week post infection. The results suggest that goats are susceptible to BuHV-1 infection and that they could play an epidemiological role in the circulation/transmission of the virus among domestic and wild ruminants and impact to some extent on the control plans for herpesviruses in cattle.

In vitro inhibition of caprine herpesvirus 1 by acyclovir and mizoribine.

Caprine herpesvirus 1 (CpHV-1) infection in goats induces genital vesicular-ulcerative lesions that strictly resemble the lesions induced by herpesvirus 2 in the human host. The immunosuppressive drug Mizoribine (MIZ) was found to increase the antiviral activity of Acyclovir (ACV) against herpesvirus infections, raising interesting perspectives on new combined therapeutic strategies. In this study the anti-CpHV-1 activity in vitro of ACV alone or in combination with MIZ was characterized. When applied alone at non-toxic concentrations, ACV had a slight effect on CpHV-1 replication while in combination with MIZ a dose-dependent inhibition of the virus yield was observed with an IC50 of ACV of 28.5 µM. These findings suggest that combined therapy of ACV and MIZ is potentially exploitable in the treatment of genital infection by herpesviruses.

Assessing the efficacy of cidofovir against herpesvirus-induced genital lesions in goats using different therapeutic regimens.

Caprine herpesvirus 1 (CpHV-1) infection in goats induces genital vesicular-ulcerative lesions that strictly resemble those produced by human herpesvirus 2 in humans. In previous studies, the potent inhibition of CpHV-1 by cidofovir was demonstrated. Cidofovir antiherpetic activity was evaluated in goats infected experimentally by the vaginal route with CpHV-1 and then treated locally at different times after infection. The administration of 1% cidofovir cream onto vaginal mucosa was able to prevent the onset of genital lesions and to decrease significantly the titers of the virus shed by the infected animals, notably in the groups treated shortly after infection (24 and 48 h). The efficacy of cidofovir against caprine herpesvirus infection was higher when the treatment was started shortly after infection than when lesions were already present and advanced. Herpesvirus genital infection of goats is a useful animal model to study the activity of antiviral drugs against human herpesvirus infections.

Caprine herpesvirus 1 vaccine with the LTK63 mutant as a mucosal adjuvant induces strong protection against genital infection in goats.

Caprine herpesvirus 1 provides a unique virus-animal model to investigate potential tools applicable for the therapy and prophylaxis of genital herpesvirus infections of humans. In order to evaluate the efficacy of mucosal immunization in the goat model, an inactivated CpHV-1 vaccine was adjuvated with the enzymatically inactive mutant of the heat-labile enterotoxin of Escherichia coli, LTK63, and used to immunize goats by the vaginal route, by administering two doses at a 3-week interval. The mucosal vaccine was safe, as neither local nor systemic reactions were associated with the vaccine administration. The vaccinated animals displayed high levels of secretory IgA and were significantly protected after challenge with the virulent CpHV-1 strain, with marked decrease in virus shedding, while the unvaccinated goats were not. These findings suggest that mucosal immunization is potentially exploitable in the control of genital infection by herpesviruses.

Identification of group A porcine rotavirus strains bearing a novel VP4 (P) Genotype in Italian swine herds.

The VP4 gene of a G5 Italian porcine rotavirus strain, 344/04-1, was nontypeable by PCR genotyping. The amino acid sequence of the full-length VP4 protein had low identity (

First detection of canine parvovirus type 2c in pups with haemorrhagic enteritis in Spain.

Canine parvovirus type 2 (CPV-2), the aetiological agent of haemorrhagic enteritis in dogs, includes three antigenic variants, types 2a, 2b and 2c. CPV-2c has been detected initially in Italy and subsequently in Vietnam. We report the first identification of this novel antigenic variant in Spain, where it caused an outbreak of fatal enteritis in basset hound pups in association with canine coronavirus type I and type II. We suggest that this new antigenic variant of CPV-2 could spread throughout Europe and that there is a subsequent need to update current CPV vaccines.

Hemocompatibility of low-friction boron-carbon-nitrogen containing coatings.

Mechanical heart valves are exposed to extreme mechanical demands, which require a surface showing not only nonhaemostatic properties, but also wear resistance and low friction. As alternative to different forms of amorphous carbon (a-C), so-called diamond-like carbon (DLC), the suitability of boron carbonitride (BCN) coatings is tested here for hemocompatible coatings. They have similar mechanical properties like a-C surfaces, but superior chemical stability at ferrous substrates or counterparts. BCN films with different nitrogen content were compared with hydrogenated a-C films regarding their mechanical properties, surface energy, adsorption of albumin and fibrinogen, blood platelet adherence, and activation of the contact system of the clotting cascade and kinin system. Similar mechanical properties and biological response have been found in the BCN films with respect to a-C, indicating the potential of these coatings for biomedical applications. The increase in the crystallinity and tribological properties of the BCN samples with a higher incorporation of N was also followed by a lower protein adsorption and low activation of the contact system, but an increased adherence of thrombocytes.

Identification of a novel VP4 genotype carried by a serotype G5 porcine rotavirus strain.

Rotavirus genome segment 4, encoding the spike outer capsid VP4 protein, of a porcine rotavirus (PoRV) strain, 134/04-15, identified in Italy was sequenced, and the predicted amino acid (aa) sequence was compared to those of all known VP4 (P) genotypes. The aa sequence of the full-length VP4 protein of the PoRV strain 134/04-15 showed aa identity values ranging from 59.7% (bovine strain KK3, P8[11]) to 86.09% (porcine strain A46, P[13]) with those of the remaining 25 P genotypes. Moreover, aa sequence analysis of the corresponding VP8* trypsin cleavage fragment revealed that the PoRV strain 134/04-15 shared low identity, ranging from 37.52% (bovine strain 993/83, P[17]) to 73.6% (porcine strain MDR-13, P[13]), with those of the remaining 25 P genotypes. Phylogenetic relationships showed that the VP4 of the PoRV strain 134/04-15 shares a common evolutionary origin with porcine P[13] and lapine P[22] rotavirus strains. Additional sequence analyses of the VP7, VP6, and NSP4 genes of the PoRV strain 134/04-15 revealed the highest VP7 aa identity (95.9%) to G5 porcine strains, a porcine-like VP6 within VP6 genogroup I, and a Wa-like (genotype B) NSP4, respectively. Altogether, these results indicate that the PoRV strain 134/04-15 should be considered as prototype of a new VP4 genotype, P[26], and provide further evidence for the vast genetic and antigenic diversity of group A rotaviruses.

Antibody levels and protection to canine parvovirus type 2.

The relationship between maternally derived antibody (MDA) levels and protection to canine parvovirus (CPV) infection in pups is reported. Twelve pups with a wide range of haemagglutination inhibiting (HI) titres of MDA to CPV were divided into four groups, with each group balanced for antibody titres. The dogs were inoculated with a field CPV-2b strain and clinical signs, virus shedding and antibody response were assessed. The CPV was not detected in the faeces of dogs with HI titres of 320 at any time. In dogs with HI titres up to 160, active CPV replication after challenge was demonstrated by real-time polymerase chain reaction. The successful infection of dogs with HI titres of 80 and 160 was confirmed by seroconversion, evaluated at day 14 post-infection. These findings demonstrated that CPV infection could also occur in the presence of MDA HI titres (> or =80) usually considered fully protective.

Lapine rotaviruses of the genotype P[22] are widespread in Italian rabbitries.

An epidemiological survey was carried out to investigate the distribution of the VP7 and VP4 specificities of lapine rotaviruses (LRVs) in rabbitries from different geographical regions of Italy. Almost all the strains were characterized as P[22],G3, confirming the presence of the newly-recognized rotavirus P[22] VP4 allele in Italian rabbits. Only one P[14],G3 LRV strain was identified and two samples contained a mixed (P[14] + [22],G3) rotavirus infection. All the LRV strains analyzed exhibited a genogroup I VP6 specificity and a long dsRNA electropherotype. However, one of the P[14],G3 strains possessed a super-short pattern. Altogether, these data highlight the epidemiological relevance of the P[22] LRVs in Italian rabbitries.

Sequence analysis of the VP7 and VP4 genes identifies a novel VP7 gene allele of porcine rotaviruses, sharing a common evolutionary origin with human G2 rotaviruses.

During an epidemiological survey encompassing several porcine herds in Saragoza, Spain, the VP7 and VP4 of a rotavirus-positive sample, 34461-4, could not be predicted by using multiple sets of G- and P-type-specific primers. Sequence analysis of the VP7 gene revealed a low amino acid (aa) identity with those of well-established G serotypes, ranging between 58.33% and 88.88%, with the highest identity being to human G2 rotaviruses. Analysis of the VP4 gene revealed a P[23] VP4 specificity, as its VP8* aa sequence was 95.9% identical to that of the P14[23],G5 porcine strain A34, while analysis of the VP6 indicated a genogroup I, that is predictive of subgroup I specificity. Analysis of the 10th and 11th RNA segments revealed close identity to strains of porcine and human origin, respectively. The relatively low overall aa sequence conservation (<89% aa) to G2 human rotaviruses, the lack of N-glycosylation sites that are usually highly conserved in G2 rotaviruses, and the presence of several amino acid substitutions in the major antigenic hypervariable regions hampered an unambiguous classification of the porcine strain 34461-4 as G2 serotype on the basis of sequence analysis alone. The identification of a borderline, G2-like, VP7 gene allele in pigs, while reinforcing the hypotheses of a tight relationship in the evolution of human and animal rotaviruses, provides additional evidence for the wide genetic/antigenic diversity of group A rotaviruses.

Canine distemper and related diseases: report of a severe outbreak in a kennel.

An outbreak of canine distemper in a kennel of German shepherds in the province of Bari is reported. Six 42-day-old pups developed typical signs of canine distemper (fever, conjunctivitis, respiratory distress and enteritis) and died within 7-10 days. Neurological symptoms were observed only in one pup. Four additional pups, which had shown no sign of illness, were separated and vaccinated, but two of these developed a severe, fatal nervous form 15 days later. Post-mortem examination, carried out on two pups which died without neurological signs, showed pneumonia and enteritis, more severe in one of the two examined pups. Smears from the brain and the conjunctiva of both dogs tested positive for canine distemper virus (CDV) by an immunofluorescent assay, confirmed by the identification of viral RNA using RT-PCR. Bordetella bronchiseptica and a canine adenovirus strain, characterized as canine adenovirus type 2 by a differential PCR assay, were isolated from the lungs of the pup showing the most pronounced lesions. Furthermore, canine coronavirus was detected by PCR in the intestinal content of this pup, suggesting a multifactorial aetiology of the outbreak.

Safety and efficacy of a modified-live canine coronavirus vaccine in dogs.

The safety and the efficacy of a modified-live (ML) canine coronavirus (CCoV) vaccine strain 257/98-3c was evaluated in 14 dogs seronegative and virus negative for CCoV. For the safety test, four dogs were inoculated, two by intramuscular and two by oronasal route, with 10 times the vaccinal dose. During the observation period (28 days) all dogs did not display any local or systemic reaction. For the efficacy test, eight dogs were vaccinated by intramuscular (four dogs-group A) or by oronasal route (four dogs-group B). Two dogs were maintained as non-vaccinated controls. In the dogs of group A, vaccinal virus was not detected in faecal samples by virus isolation (VI) and by PCR assay, while in the dogs of group B, the virus was revealed for six median days only by PCR. Twenty-eight days later, the vaccinated and control dogs were challenged with a field CCoV strain. After the challenge, the vaccinated dogs did not display clinical signs and the dogs of group A shed virus for 5.5 median days, evaluated by VI, and for 10 median days evaluated by PCR. Virus shedding was not observed, both by VI and PCR assay, in the dogs of group B. The two control dogs displayed moderate clinical signs and the virus was detected by VI for 14.5 median days starting from day 3 post-challenge (dpc 3) and by PCR assay for 23 median days starting from dpc 1.

A severe dual infection by feline panleukopenia virus and feline calicivirus in an adult cat.

A dual infection by feline panleukopenia virus (FPV) and feline calicivirus (FCV) in a 7 month-old cat is described. The animal developed a severe illness with depression, anorexia, fever, leucopoenia, nasal and ocular discharge and oral ulcers. Both FPV and FCV were isolated in cell cultures from a rectal swab and the presence of FCV was confimed by polymerase chain reaction. Antibodies to both the viruses were detected in the serum. The severity of the disease induced by the mixed viral infection highlights the need for intensifying FPV vaccination in cats.

Experimental infection of goats at different stages of pregnancy with caprine herpesvirus 1.

Three goats from a group of five caprine herpesvirus 1 (CpHV.1) seronegative pregnant goats were inoculated intranasally with a virulent BA.1 strain of CpHV.1. Goat n.1 was infected on day 45 of pregnancy, goat n.2 on day 92 and goat n.3 on day 127. Each of the three goats produced a single foetus 10-60 days after infection. Foetus n.1 was never found and so it could not be examined for virological findings. Goat n.2 delivered at term of gestation and CpHV.1 was detected by PCR and isolated from most of the foetal organs. Foetus n.3 was partially autolysed and the virus was only detected by PCR but not isolated from foetal organs. The results confirm the damaging effect of CpHV.1 infection on pregnancy, the difficulty in diagnosing the CpHV.1 induced abortion, and the importance developing appropriate prophylactic programmes.