Mutation analysis of the spike protein in Italian feline infectious peritonitis virus and feline enteric coronavirus sequences.

Feline coronavirus (FCoV) exists as two different genotypes, FCoV type I and II, each including two biotypes, feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV), the latter being a virulent variant originating from the former virus. Recently, two amino acid substitutions, M1058L and S1060A, within the spike protein have been associated to the FECV/FIPV virulence change. In this study, we have analysed the frequency of detection of such mutations in FIPV and FECV strains circulating in Italian cats and obtained information about their evolutionary relationships with reference isolates. A total of 40 FCoV strains, including 19 strains from effusions or tissue samples of FIP cats and 21 strains from faecal samples of non-FIP cats, were analysed. Mutation M1058L was detected in 16/18 FCoV-I and 1/1 FCoV-II strains associated with FIP, while change S1060A was presented by two FIPV strains. By phylogenetic analysis, FCoV sequences clustered according to the genotype but not according to the biotype, with FECV/FIPV strains recovered from the same animal being closely related. Further studies are needed to better define the genetic signatures associated with the FECV/FIPV virulence shift.

Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.

SARS-CoV-2 emerged from animals and is now easily transmitted between people. Sporadic detection of natural cases in animals alongside successful experimental infections of pets, such as cats, ferrets and dogs, raises questions about the susceptibility of animals under natural conditions of pet ownership. Here, we report a large-scale study to assess SARS-CoV-2 infection in 919 companion animals living in northern Italy, sampled at a time of frequent human infection. No animals tested PCR positive. However, 3.3% of dogs and 5.8% of cats had measurable SARS-CoV-2 neutralizing antibody titers, with dogs from COVID-19 positive households being significantly more likely to test positive than those from COVID-19 negative households. Understanding risk factors associated with this and their potential to infect other species requires urgent investigation.

Dilated cardiomyopathy in mucolipidosis type 2.

Mucolipidosis II and III are lysosomal storage diseases caused by pathogenetic mutations in GNPTAB and GNPTG genes which cause an impaired activity of the lysosomal hydrolase N-acetylglucosamine- 1-phosphotransferase, a key enzyme in the synthesis of the mannose-6-phosphate targeting signals on lysosomal enzymes. Patients with MLII alpha/beta present coarse facial features, cessation of statural growth, important skeletal manifestations, impaired neuromotor development and cardiorespiratory involvement. All children appear to have cardiac involvement, but severe dilated cardiomyopathy is uncommon. In this report we describe the case of an 11-month-old girl who is affected by a MLII. Analysis of the GNPTAB gene identified at a heterozygous level the previously described gene variants c. 2693delA p(Lys898Serfs*13) and c. 2956C>T p(Arg986Cys). Her main clinical features were coarse face with gingival hypertrophy, dysostosis multiplex, recurrent respiratory infection and an early onset of dilated cardiomyopathy, an uncommon feature for MLII. To our knowledge, dilated cardiomyopathy has been previously described in literature in only two cases of MLII and in one patient affected by MLIII.

Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy.

SARS-CoV-2 originated in animals and is now easily transmitted between people. Sporadic detection of natural cases in animals alongside successful experimental infections of pets, such as cats, ferrets and dogs, raises questions about the susceptibility of animals under natural conditions of pet ownership. Here we report a large-scale study to assess SARS-CoV-2 infection in 817 companion animals living in northern Italy, sampled at a time of frequent human infection. No animals tested PCR positive. However, 3.4% of dogs and 3.9% of cats had measurable SARS-CoV-2 neutralizing antibody titers, with dogs from COVID-19 positive households being significantly more likely to test positive than those from COVID-19 negative households. Understanding risk factors associated with this and their potential to infect other species requires urgent investigation. ONE SENTENCE SUMMARY: SARS-CoV-2 antibodies in pets from Italy.

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.

Circulation of multiple subtypes of bovine viral diarrhoea virus type 1 with no evidence for HoBi-like pestivirus in cattle herds of southern Italy.

Pestiviruses of cattle include bovine viral diarrhoea 1 (BVDV-1) and 2 (BVDV-2) plus an emerging group, named HoBi-like pestivirus. In the present paper, the results of an epidemiological survey for pestiviruses circulating in cattle in southern Italy are presented. Molecular assays carried out on a total of 924 bovine samples detected 74 BVDV strains, including 73 BVDV-1 and 1 BVDV-2 viruses. Phylogenetic analysis carried out on partial 5'UTR and Npro sequences revealed the presence of 6 different subtypes of BVDV-1 and a single BVDV-2c strain. BVDV-1 displayed a high level of genetic heterogeneity, which can have both prophylactic and diagnostic implications. In addition, the detection of BVDV-2c highlights the need for a continuous surveillance for the emergence of new pestivirus strains in cattle farms in southern Italy.

Evidence for Circulation of Bovine Viral Diarrhoea Virus Type 2c in Ruminants in Southern Italy.

Recently, bovine viral diarrhoea virus type 2c (BVDV-2c) was responsible for a severe outbreak in cattle in northern Europe. Here, we present the results of an epidemiological survey for pestiviruses in ruminants in southern Italy. Pooled serum samples were obtained from 997 bovine, 800 ovine, 431 caprine and eight bubaline farms, and pestiviral RNA was detected by molecular methods in 44 farms consisting of 16 cattle and one buffalo herds and of 21 sheep and six goat flocks. Twenty-nine and 15 farms were infected by BVDV-1 and BVDV-2 strains, respectively. BVDV-1 strains were recovered mainly from cattle and were heterogeneous, belonging to the subtypes 1b, 1u, 1e, 1g and 1h. In contrast, all BVDV-2 viruses but two were detected in sheep or goats and were characterized as BVDV-2c by sequence analysis of 5'UTR. These strains displayed high genetic identity to BVDV-2c circulating in cattle in northern Europe and were more distantly related to a BVDV-2c isolate recovered from a cattle herd in southern Italy more than 10 years before. The circulation of a BVDV-2c in small ruminants suggests the need for a continuous surveillance for the emergence of pestivirus-induced clinical signs in southern Italian farms.

HoBi-Like Pestivirus and Its Impact on Cattle Productivity.

The clinical features and economic impact of the infection caused by an emerging group of pestiviruses, namely HoBi-like pestivirus, in a cattle herd of southern Italy are reported. In 2011, the virus was first associated with respiratory disease, causing an abortion storm after 1 year and apparently disappearing for the following 3 years after persistently infected calves were slaughtered. However, in 2014, reproductive failures and acute gastroenteritis were observed in the same herd, leading to a marked decrease of productivity. A HoBi-like strain closely related to that responsible for previous outbreaks was detected in several animals. Application of an intensive eradication programme, based on the detection and slaughtering of HoBi-like pestivirus persistently infected animals, resulted in a marked improvement of the productive performances.

Measurement of the quality factor of a new low-frequency differential accelerometer for testing the equivalence principle.

A cryogenic differential accelerometer has been developed to test the weak equivalence principle to a few parts in 10(15) within the framework of the general relativity accuracy test in an Einstein elevator experiment. The prototype sensor was designed to identify, address, and solve the major issues associated with various aspects of the experiment. This paper illustrates the measurements conducted on this prototype sensor to attain a high quality factor (Q ∼ 10(5)) at low frequencies (<20 Hz). Such a value is necessary for reducing the Brownian noise to match the target acceleration noise of 10(-14) g/√Hz, hence providing the desired experimental accuracy.

Lights and shades on an historical vaccine canine distemper virus, the Rockborn strain.

Both egg- and cell-adapted canine distemper virus (CDV) vaccines are suspected to retain residual virulence, especially if administered to immuno-suppressed animals, very young pups or to highly susceptible animal species. In the early 1980s, post-vaccine encephalitis was reported in dogs from various parts of Britain after administration of a particular batch of combined CDV Rockborn strain/canine adenovirus type-1 vaccine, although incrimination of the Rockborn strain was subsequently retracted. Notwithstanding, this, and other reports, led to the view that the Rockborn strain is less attenuated and less safe than other CDV vaccines, and the Rockborn strain was officially withdrawn from the markets in the mid 1990s. By sequencing the H gene of the strain Rockborn from the 46th laboratory passage, and a commercial vaccine (Candur(®) SH+P, Hoechst Rousell Vet GmbH), the virus was found to differ from the commonly used vaccine strain, Onderstepoort (93.0% nt and 91.7% aa), and to resemble more closely (99.6% nt and 99.3% aa) a CDV strain detected in China from a Lesser Panda (Ailurus fulgens). An additional four CDV strains matching (>99% nt identity) the Rockborn virus were identified in the sequence databases. Also, Rockborn-like strains were identified in two vaccines currently in the market. These findings indicate that Rockborn-like viruses may be recovered from dogs or other carnivores with distemper, suggesting cases of residual virulence of vaccines, or circulation of vaccine-derived Rockborn-like viruses in the field.

Genetic analysis of feline panleukopenia viruses from cats with gastroenteritis.

Thirty-nine parvovirus strains contained in faecal samples collected in Italy (n=34) and UK (n=5) from cats with feline panleukopenia were characterized at the molecular level. All viruses were proven to be true feline panleukopenia virus (FPLV) strains by a minor groove binder probe assay, which is able to discriminate between FPLV and the closely related canine parvovirus type 2. By using sequence analysis of the VP2 gene, it was found that the FPLV strains detected in Italy and UK were highly related to each other, with a nucleotide identity of 99.1-100 and 99.4-99.8% among Italian and British strains, respectively, whereas the similarities between all the sequences analysed were 98.6-100%. Eighty-eight variable positions were detected in the VP2 gene of the field and reference FPLV strains, most of which were singletons. Synonymous substitutions (n=57) predominated over non-synonymous substitutions (n=31), and the ratio between synonymous and non-synonymous substitutions (dN/dS) was 0.10, thus confirming that evolution of FPLV is driven by random genetic drift rather than by positive selection pressure. Some amino acid mutations in the VP2 protein affected sites that are thought to be responsible for antigenic and biological properties of the virus, but no clear patterns of segregation and genetic markers, were identified, confirming that FPLV is in evolutionary stasis.

Migraine detection through spontaneous EEG analysis.

Spontaneous EEG patterns are studied to detect migraine patients both during the attack and in headache-free periods. The EEG signals are analyzed through the wavelets and both scale-dependent and scale-independent features are computed to characterize the patterns. The classification is carried out by a supervised neural network. The efficiency of the method is evaluated through the Receiver Operating Characteristic (ROC) analysis and the Wilcoxon-Mann-Whitney (WMW) test. Although a high discrimination is observed with one single neural output, a complete separation among MwA patients and healthy subjects is obtained when a scatter plot is drawn in the plane of two suitable neural outputs.

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 (

Heterogeneity within the hemagglutinin genes of canine distemper virus (CDV) strains detected in Italy.

Canine distemper virus (CDV) is a highly contagious viral pathogen causing lethal disease in dogs and other mammalians. A high degree of genetic variation is found between recent CDV strains and the old CDV isolates used in the vaccines and such genetic variation is regarded as a possible cause of the increasing number of CDV-related diseases in dogs. The H gene shows the greatest extent of genetic variation that allows for distinction of various lineages, according to a geographical pattern of distribution and irrespective of the species of identification. In the present study, hemagglutinin (H) genes obtained from field strains detected from clinical specimens of Italian dogs were analyzed genetically. Phylogenetic analysis revealed that a homogeneous group of CDV strains is widespread in Italian dogs, all which are included into the European lineage. Unexpectedly, strains 179/04 and 48/05 clustered along with CDVs of the Arctic lineage, the highest identity being to strain GR88 (98.0 and 98.4%aa, respectively). The full-length sequence of a red fox CDV strain, 207/00 was also determined and analyzed. The H protein of the fox CDV strain was unrelated to strains within the major European lineage. These results suggest that at least three different CDV lineages are present in Italy.

New approaches for the molecular characterization of canine parvovirus type 2 strains.

Characterization of the canine parvovirus type 2 (CPV-2) is sometimes ambiguous, frequently requiring more than one technique for definitive prediction of the viral type. Taking into account the single-nucleotide polymorphisms encountered in the VP2-protein gene between types 2a and 2b and between type 2b and Glu-426 mutant (type 2c), two different minor groove binder (MGB) probe assays were developed for rapid identification of the CPV-2 variants. A total of 315 samples collected from dogs with diarrhoea were screened for CPV-2 by a real-time polymerase chain reaction (PCR) assay capable of detecting all CPV-2 types. In order to compare the type-specific assays with the traditional techniques [haemagglutination inhibition with monoclonal antibodies, PCR-restriction fragment-length polymorphism (RFLP), sequence analysis] for prediction of CPV-2 antigen specificity, the 203 samples tested CPV-2 positive were analysed using the different methods. The results showed a 100% concordance between the MGB probe assays and the combined conventional methods, with 116 samples characterized as type 2a, 32 as type 2b and 55 as type 2c. Therefore, the MGB probe assays represent a quick, reliable tool for prediction of CPV-2 antigen specificity, with regard to the more time-consuming assays currently used.

Additional predictive value of heart rate variability in high-risk patients surviving an acute myocardial infarction.

The aim of this study was to investigate the usefulness of heart rate variability (HRV) in predicting cardiac mortality in patients with a recent acute myocardial infarction at high risk of events. Time- and frequency-domain HRV was assessed on 24-hour pre-discharge Holter recording of 81 patients (mean age 65 +/- 10 years, 76.5% males) with 1) a recent acute myocardial infarction, and 2) left ventricular ejection fraction < 40% and/or a number of premature ventricular beats > or = 10/hour. There were 15 total cardiac deaths (18.5%) in a follow-up time of 29 +/- 15 months (range 6 to 48 months), with sudden death occurring in 11 patients (13.6%). Median values of low frequency (LF) were lower in patients with, compared to those without, total (p = 0.04) and sudden (p = 0.02) cardiac death. Similarly the low frequency/high frequency (LF/HF) ratio was lower in patients with fatal events (p = 0.03 and p = 0.02, respectively). Furthermore, mean of the standard deviations of all RR intervals for all 5-min segments < 20 ms, among time-domain variables, and very-low frequency (VLF) < 18 ms, among frequency-domain variables, significantly predicted cardiac death (relative risk-RR 2.94, p = 0.03; and RR 3.85, p < 0.005, respectively). Furthermore, VLF < 18 ms and LF/HF ratio < 1.05 significantly predicted the occurrence of sudden death (RR 3.52, p = 0.04; and RR 3.49, p = 0.04, respectively). Thus, our data show that HRV analysis is a useful tool for identifying patients with an actual increased risk of fatal cardiac events among patients who are basically considered at high risk according to the presence of an impaired left ventricular function and/or frequent ventricular arrhythmias.

Circadian distribution of ischemic attacks and ischemia-related ventricular arrhythmias in patients with variant angina.

BACKGROUND: There are limited data about the circadian distribution of ischemic episodes in patients with variant angina. Furthermore, no previous study investigated whether ischemia-related ventricular arrhythmias follow a circadian variation in these patients. METHODS: The circadian variation of transient ischemia and ischemia-induced ventricular arrhythmias was assessed by cosinor methodology on 24-hour Holter recordings of 26 patients with variant angina. RESULTS: On the whole, 301 ischemic episodes were detected in the population, with premature ventricular complexes occurring in 49 of them (16%). Ischemic episodes followed a typical circadian variation (acrophase hr 02:36, p < 0.01) in the total sample. However, a significant circadian variation of ischemic episodes was detectable in the 14 patients without (n = 167, acrophase hr 04:00, p < 0.0001), but not in the 12 patients with (n = 134, p = 0.14) hemodynamically significant coronary stenoses, independently of the location (anterior/inferior) of ischemia. There was no significant circadian variation of ischemia-related ventricular arrhythmias. CONCLUSIONS: Among patients with variant angina, a clearcut circadian variation of ischemia is present in those without, but not in those with, hemodynamically significant coronary artery stenoses, thus suggesting that different pathophysiologic mechanisms may operate, at least in part, in triggering coronary spasm in these two subgroups of patients. There was no significant circadian variation of ischemia-related ventricular arrhythmias in these patients.

The "warm-up" phenomenon occurs in patients with chronic stable angina but not in patients with syndrome X.

Most patients with chronic stable angina show an improvement in ischemic threshold when a second exercise test is performed a few minutes after a first positive test. In this study we evaluated whether this "warm-up" phenomenon also occurs in patients with syndrome X. We performed 2 consecutive exercise tests in 14 patients with chronic stable angina and 11 patients with syndrome X. The second exercise test was performed after 10 minutes from the end of the first one, always after complete recovery to baseline of ST segment. In patients with stable angina, heart rate (108+/-18 vs 99+/-16 beats/min, p = 0.005), rate-pressure product (17,020+/-4,541 vs 15,215+/-3,734 beats/min x mm Hg, p = 0.028), and exercise time (587+/-297 vs 444+/-244 seconds, p = 0.002) at 1-mm ST depression were higher in the second test than in the first one and a significant improvement in these parameters during the second test was also observed at peak exercise. Conversely, in patients with syndrome X, there were no significant differences between the 2 tests in heart rate (128+/-18 vs 131+/-23 beats/min), rate-pressure product (19,922+/-5,153 vs 19,390+/-5,654 beats/min x mm Hg), and exercise time (592+/-243 vs 566+/-228 seconds) at 1-mm ST-segment depression. Similarly, in this group of patients, no significant differences in exercise variables between the 2 tests were observed at peak exercise. Thus, unlike patients with chronic stable angina, patients with syndrome X have no evidence of warm-up in response to repeated exercise testing.

Autonomic changes associated with spontaneous coronary spasm in patients with variant angina.

OBJECTIVES: This study sought to investigate whether changes in nervous autonomic tone may have a role in the mechanisms triggering spontaneous coronary spasm in variant angina. BACKGROUND: Previous studies have suggested that both sympathetic and vagal activation may act as a trigger of epicardial artery spasm in patients with variant angina, but the actual role of autonomic changes in spontaneous coronary spasm remains unknown. METHODS: We analyzed the changes in heart rate variability associated with episodes of ST segment elevation detected on Holter monitoring in 23 patients with variant angina (18 men, 5 women; mean [+/-SD] age 59 +/- 12 years). For study purposes, episodes of transmural ischemia lasting > or = 3 min and without any ST segment changes in the previous 40 min were selected for analysis. Heart rate variability indexes were calculated at 2-min intervals, at 30,15,5 and 1 min before ST elevation and at peak ST segment elevation. Ninety-three of 239 total ischemic episodes (39%) fulfilled the inclusion criteria. RESULTS: The results showed that 1) high frequency (HF) (0.04 to 0.15 Hz), a heart rate variability index specific for vagal activity, decreased in the 2 min preceding ST segment elevation (p < 0.001) and returned to basal levels at peak ST segment elevation; 2) heart rate and low frequency (0.04 to 0.15 Hz), which are partially correlated with sympathetic activity, showed a significant increase at peak ST segment elevation (p < 0.001 for both); 3) the pattern of the HF reduction before ST segment elevation was consistently confirmed in several subgroups of ischemic episodes, including those of patients with or without coronary stenoses, those of patients with anterior or inferior ST segment elevation, those occurring during daily or nightly hours and silent episodes. There were no significant variations in heart rate variability in control periods selected from Holter tapes of patients and before ST segment elevation induced by balloon inflation in 20 patients undergoing coronary angioplasty. CONCLUSIONS: Our data show that changes in autonomic tone are likely to contribute to trigger or predispose to epicardial spasm. In particular, although not excluding an active role for adrenergic mechanisms, our data suggest that a vagal withdrawal may often be a component of the mechanisms leading to spontaneous coronary vasospasm.