Search

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.

Canine coronavirus (CCoV) strains with the ability to spread to internal organs, also known as pantropic CCoVs (pCCoVs), have been detected in domestic dogs and wild carnivores. Our study focused on the detection and molecular characterization of pCCoV strains circulating in Italy during the period 2014–2017 in autochthonous dogs, in dogs imported from eastern Europe or illegally imported from an unknown country. Samples from the gut and internal organs of 352 dogs were screened for CCoV; putative pCCoV strains, belonging to subtype CCoV‐IIa, were identified in the internal organs of 35 of the examined dogs. Fifteen pCCoV strains were subjected to sequence and phylogenetic analyses, showing that three strains (98960‐1/2016, 98960‐3/2016, 98960‐4/2016) did not cluster either with Italian or European CCoVs, being more closely related to alphacoronaviruses circulating in Asia with which they displayed a 94%–96% nucleotide identity in partial spike protein gene sequences. The pCCoV‐positive samples were also tested for other canine viruses, showing co‐infections mainly with canine parvovirus.

Saliva is an appropriate specimen for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) diagnosis. The possibility of pooling samples of saliva, using non-invasive bibula strips for sampling, was explored employing Bovine coronavirus (BCoV) spiked saliva. In laboratory, up to 30 saliva-soaked strips were pooled in a single tube with 2 mL of medium. After quick adsorption with the medium and vortexing, the liquid was collected and tested with a quantitative molecular assay to quantify viral RNA genome copies. On testing of single and pooled strips, the difference between the median threshold cycles (Ct) value of test performed on the single positive saliva sample and the median Ct value obtained on the pool of 30 strips, was 3.21 cycles. Saliva pooling with bibula strips could allow monitoring of COVID-19 on a large scale, reducing costs for the health bodies in terms of medical material and skilled personnel. Finally, saliva sampling is noninvasive and less traumatic than nasopharyngeal swabs and can be self-collected.

Highlights • A molecular survey for selected viral enteropathogens was conducted in dogs with and without acute gastroenteritis. • Canine parvovirus and coronavirus were strongly associated with occurrence of enteric disease. • Canine circovirus infections correlated with acute gastroenteritis only when associated with other enteric viruses., Canine circovirus (CanineCV) is a canine virus, whose pathogenetic role is still uncertain. Based on recent data suggesting its role as entheropathogen, a case-control study was conducted between 2013 and 2016 to investigate the association of CanineCV with gastroenteritis in dogs, alone or in combination with other viral pathogens, including canine parvovirus (CPV), canine coronavirus (CCoV) and canine distemper virus (CDV). A total of 219 dogs suffering from acute gastroenteritis disorders and 67 controls randomly recruited among healthy dogs or patients presenting without enteric signs were screened by a panel of real-time (RT-)PCR assays for CanineCV, CPV, CCoV and CDV. A high prevalence of viral infections was detected in dogs with gastroenteritis (77.16%), with CPV representing the most frequently detected enteropathogen, followed by CanineCV and CCoV. While CPV and CCoV infections displayed a strong association with occurrence of acute gastroenteritis (p

Canine adenoviruses are a major cause of disease in dogs, coyotes, red foxes and wolves, as well as in other carnivores and marine mammals. Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB), respectively. In this study, a duplex real-time PCR assay for simultaneous detection and characterisation of CAdV-1 and CAdV-2 was developed by using a single primer pair and virus-specific probes. The assay was validated testing standard DNAs produced on purpose and clinical samples of various matrices known to be positive for CAdV-1, CAdV-2 or both viruses. Precise calculation of DNA loads in samples containing a wide range of viral amounts was allowed by generating a standard curve for absolute quantification. The assay was proven to be highly specific, since no cross-reactions with the different CAdV type was observed, and sensitive, being able to detect less than 10 copies of CAdV-1/CAdV-2 DNA. The low intra-assay and interassay coefficient of variations demonstrated a high repeatability, thus confirming the potential use of this assay for quantitative detection of CAdV-1 and CAdV-2 for rapid diagnosis and epidemiological investigations.

Highlights • A molecular survey for CIRD agents was conducted during years 2011–2013. • Canine parainfluenza virus was the most frequently detected pathogens. • The emerging pathogens canine respiratory coronavirus, Mycoplasma cynos and canine pneumovirus were sporadically detected. • Most severe forms of respiratory disease were observed in dogs infected with M. cynos alone or in combination with either CRCoV or M. canis. • CIRD vaccines should be updated to include emerging pathogens., A molecular survey for traditional and emerging pathogens associated with canine infectious respiratory disease (CIRD) was conducted in Italy between 2011 and 2013 on a total of 138 dogs, including 78 early acute clinically ill CIRD animals, 22 non-clinical but exposed to clinically ill CIRD dogs and 38 CIRD convalescent dogs. The results showed that canine parainfluenza virus (CPIV) was the most commonly detected CIRD pathogen, followed by canine respiratory coronavirus (CRCoV), Bordetella bronchiseptica, Mycoplasma cynos, Mycoplasma canis and canine pneumovirus (CnPnV). Some classical CIRD agents, such as canine adenoviruses, canine distemper virus and canid herpesvirus 1, were not detected at all, as were not other emerging respiratory viruses (canine influenza virus, canine hepacivirus) and bacteria (Streptococcus equi subsp. zooepidemicus). Most severe forms of respiratory disease were observed in the presence of CPIV, CRCoV and M. cynos alone or in combination with other pathogens, whereas single CnPnV or M. canis infections were detected in dogs with no or very mild respiratory signs. Interestingly, only the association of M. cynos (alone or in combination with either CRCoV or M. canis) with severe clinical forms was statistically significant. The study, while confirming CPIV as the main responsible for CIRD occurrence, highlights the increasing role of recently discovered viruses, such as CRCoV and CnPnV, for which effective vaccines are not available in the market.

Highlights • A multiplex TaqMan assay for differentiation of bovine pestiviruses was developed. • The assay was proven to be specific allowing to discriminate among pestiviruses. • The emerging HoBi-like strains were correctly typed by the new assay., HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 100–101 viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples.

Highlights • A real-time RT-PCR assay was developed for detection of bovine pestiviruses. • The assay was proven to be rapid, sensitive, and reproducible. • The emerging HoBi-like strains were correctly detected by the new assay., A real-time RT-PCR assay based on the TaqMan technology was developed for rapid and sensitive detection of pestiviruses infecting cattle, i.e., bovine viral diarrhea virus (BVDV) 1, BVDV-2, and the emerging HoBi-like pestiviruses. The assay was linear and reproducible, being able to detect as few as 10 copies of viral RNA. By real-time RT-PCR analysis of 986 biological samples collected from cattle herd with clinical signs suggestive of pestivirus infection and from animals recruited in a pestivirus surveillance programme, 165 pestivirus positive samples were detected, including 6 specimens, 2 nasal swabs, and 4 EDTA–blood samples, that tested negative by a gel-based RT-PCR assay targeting the 5′UTR. The developed TaqMan assay represents a new reliable and effective tool for rapid and sensitive diagnosis of infections caused by all pestiviruses circulating in cattle, thus being useful for extensive surveillance programs in geographic areas where HoBi-like pestiviruses are co-circulating with BVDV-1 and BVDV-2.

Highlights • The full-length genome of canine coronavirus type I was determined. • Sequence analysis showed unique features with respect to canine coronavirus type II. • By phylogeny, canine coronavirus type I formed a separate cluster. • The results may contribute to the understanding of the Alphacoronavirus-1 evolution., Canine coronavirus types I (CCoV-I) and II (CCoV-II) are usually responsible for mild enteritis in dogs. While the CCoV-II genome has been completely sequenced, to date there are no complete genomic sequence data available publicly for CCoV-I. Thus, the aim of the present study was to analyze the full-length genome of a CCoV-I prototype strain that had been recovered from a dog with diarrhea in Italy. CCoV-I strain 23/03 has a genome of 30,000 nucleotides, excluding the 3′ poly(A) tail, displaying the typical Alphacoronavirus-1 organization and the highest genetic relatedness to CCoV-II. However, two distinct features were observed in the CCoV-I genome: (i) the presence of an additional ORF between the spike (S) protein gene and ORF3a; (ii) the diversity of the S protein, which is more closely related to that of feline coronavirus type I and presents a furin cleavage site. The present study may contribute to a better understanding of the Alphacoronavirus-1 evolutionary pattern and may be paradigmatic of how coronaviruses evolve through gene losses, acquisition and exchanges among different members.

Intra-vitam diagnosis of feline infectious peritonitis (FIP) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. With the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive FIP were collected intra-vitam for detection of feline coronavirus (FCoV) antibodies and RNA by means of indirect immunofluorescence (IIF) assay and real-time RT-PCR (qRT-PCR), respectively. In 5 effusions there was no evidence for either FCoV RNA or antibodies, 51 and 52 specimens tested positive by IIF and qRT-PCR, respectively, although antibody titres ≥ 1:1600, which are considered highly suggestive of FIP, were detected only in 37 effusions. Three samples with high antibody levels tested negative by qRT-PCR, whereas 18 qRT-PCR positive effusions contained no or low-titre antibodies. qRT-PCR positive samples with low antibody titres mostly contained low FCoV RNA loads, although the highest antibody titres were detected in effusions with CT values > 30. In conclusion, combining the two methods, i.e., antibody and RNA detection would help improving the intra-vitam diagnosis of effusive FIP., Highlights • Effusions from cats with suspected FIP were analysed for detection of feline coronavirus (FCoV) antibodies and RNA. • Using a cut-off of 1:1600 for FCoV antibodies, only 40/61 samples were in agreement between the two tests. • Most effusions with low FCoV loads were found to contain low specific antibody titres. • Combining serological and molecular methods results in an increase of the diagnostic efficiency.

A calf persistently infected with Hobi-like pestivirus displayed severe clinical signs and subsequently died. Gross lesions and histopathological changes were suggestive of hemorrhagic and necrotic inflammation involving several tissues. A Hobi-like pestivirus pair was isolated from the dead calf, i.e., cytopathogenic (CP) and noncytopathogenic (NCP) strains strictly related to each other and to Italian prototype isolates at the genetic level. Two biotype-specific real-time reverse transcription-PCR assays determined the time of the emergence of the CP virus as 1 month before the calf's death. This highest RNA titers were reached in lymphoid and nervous system tissues, whereas only traces of CP viral RNA were found in blood. In contrast, great NCP virus loads were present in all tissues and biological fluids. The present report provides new insights into the pathogenesis and molecular mechanisms of this emerging group of pestiviruses.

Highlights ► Mixed infections prevail among CCoV infections. ► CCoV triple (CCoV-I/CCoV-IIa/CCoV-IIb) infections are reported for the first time. ► The identification of a divergent strain is reported. ► CCoV-II is more conserved in the first part of the S gene compared to CCoV type I., Canine coronavirus (CCoV) is an etiologic agent of diarrhea in dogs and is known to have spread worldwide. Mild disease or asymptomatic carriage are probably in many cases common outcomes of infection. To date, two different genotypes of CCoV are known, CCoV type I (CCoV-I) and CCoV type II (CCoV-II). CCoV type II is divided in two subtypes, CCoV-IIa (classical strains) and CCoV-IIb, with CCoV-IIb emerging as a result of a putative recombination between CCoV-IIa and transmissible gastroenteritis virus (TGEV). The aim of the present study was to investigate the presence of CCoV in Greece and to genetically analyze the circulating strains. Between December 2007 and December 2009, 206 fecal samples were collected from dogs with diarrhea from kennels, pet shops and veterinary clinics of different country regions. RT-PCR and real time RT-PCR assays were used for CCoV detection and characterization. CCoV was identified in 65.1% of the dogs presenting diarrhea, being more frequently detected in animals younger than 3 months old and in animals housed in groups. In 47% of the positive samples more than one CCoV genotype/subtype were detected, with triple CCoV-I/CCoV-IIa/CCoV-IIb infections being identified for the first time. Molecular and phylogenetic analysis revealed that CCoV-I Greek strains share low genetic relatedness to each other and to the prototype CCoV-I strains in the 5’ end of the S gene. Moreover, a divergent CCoV-IIa strain was identified. The circulation of highly variable CCoV-I and CCoV-IIb emerging strains, as well as the detection of the divergent strain, raise concerns on the importance of these new strains as primary pathogens of diarrhoeic syndromes diagnosed in dogs.

Infection with pantropic canine coronavirus was detected during outbreaks in France and Belgium. This was concurrent in most cases with canine parvovirus 2c. One outbreak was a deadly acute systemic disease with a single pantropic canine coronavirus infection. This is the first report of a fatality associated with pantropic canine coronavirus alone outside Italy.

Recently, canine coronavirus (CCoV) strains with putative recombinant origin with porcine transmissible gastroenteritis virus (TGEV) were shown to be widespread in Europe. In this study, a killed vaccine against TGEV-like CCoV strains, included in the new subtype CCoV-IIb, was developed through inactivation with betapropiolactone and emulsification with MF59™ adjuvant. Safety, immunogenicity and efficacy of the developed vaccine were evaluated in vivo. Five 10-week-old beagle pups were administered (three weeks apart) two vaccine doses, whereas two animals served as unvaccinated controls. The vaccine was shown to be safe as no local neither systemic reactions were observed after first and second dose administration. Serum antibodies against CCoV were detected in vaccinates starting from study day 14 (by enzyme-linked immunosorbent assay) or 28 (by virus neutralisation test). Subsequent challenge with virulent CCoV-IIb resulted in the development of mild gastroenteric disease in control pups, whereas vaccinates did not display clinical signs. Faecal shedding of the challenge virus occurred in both treatment groups, but vaccinated dogs were found to shed very low viral titres in comparison to controls. The developed vaccine may help control the CCoV-IIb-induced disease (and active virus circulation) in environments, such as kennels and shelters, where the pathogenic potential of this virus is greater as a consequence of predisposing factors and concurrent infections.

A hypervirulent strain (CB/05) of canine coronavirus was employed to infect oronasally 11-week-old pups. Peripheral blood monocytes (CD14 + ), T lymphocytes (CD4 + and CD8 + ) and B lymphocytes (CD21 + ) were studied by flow cytometry within 5 days post-infection (p.i.) and at later time points. Infection with CB/05 resulted in a profound depletion of T cells and a slight loss of B cells in the first week p.i. In particular, while the CD8 + and the B lymphocytes returned to baseline levels by day 7 p.i., the CD4 + T cells remained significantly low until day 30 p.i. and recovered completely only at day 60 p.i. Monocytosis was also observed after CB/05 infection with a peak at day 5 p.i. The prolonged depletion of peripheral CD4 + T cells did not alter the levels of serum IgG or IgM. The impact of CB/05 infection on the immune performance of infected pups is discussed.

Recently, a coronavirus strain (179/07-11) was isolated from water buffalo (Bubalus bubalis) and the virus which displayed a strict genetic and biological relatedness with bovine coronavirus (BCoV) was referred to as bubaline coronavirus (BuCoV). Here, we report the characterisation of four BuCoVs strains identified in the faeces or intestinal contents of water buffalo calves with acute gastroenteritis. Single BuCoV infections were detected in all but one cases from which two clostridia species were also isolated. Sequence and phylogenetic analyses of the 5' end of the spike-protein gene showed that three BuCoVs were closely related to the prototype strain 179/07-11, whereas the fourth isolate (339/08-C) displayed a higher genetic identity to recent BCoV reference strains. Three strains adapted to the in vitro grow on human rectal tumour cells were also evaluated for their ability to replicate in a bovine cell line (Madin Darby bovine kidney) and to cause haemagglutination of chicken erythrocytes and all displayed biological properties similar to those already described for the prototype BuCoV. The present report shows that albeit genetically heterogeneous, the different BuCoV strains possess a common biological pattern which is different from most BCoV and BCoV-like isolates.

Recently, an outbreak of fatal infection caused by a pantropic variant (strain CB/05) of canine coronavirus (CCoV) has been reported. In this study, evidence is provided that immunity induced by natural exposure to enteric CCoV is not fully protective against strain CB/05. Twenty-two, 10-week-old beagles with a recent natural infection by enteric CCoV were randomly distributed in two experimental groups of eight (groups A and B) and one control group of six (group C) dogs. Dogs in groups A and B were inoculated oronasally with different doses (4 x 10(5) or 4 x 10(3)TCID(50)) of the pantropic strain CB/05, whereas dogs in group C were used as negative controls. Clinical, post-mortem and virological investigations showed that, despite the high serum antibody titres induced by the prior natural infection with enteric CCoV, dogs were susceptible to experimental infection with strain CB/05. This was shown by the occurrence of faecal shedding, and dogs displaying moderate clinical signs, mainly vomiting and diarrhoea. Involvement of the lymphoid tissues was evident as demonstrated by the acute lymphopenia (below 70% of the initial counts), gross lesions in spleen and lymph nodes and detection of CB/05 RNA in thymus, spleen and lymph nodes of some infected dogs. The presence of viral RNA in lymphoid tissues was observed only in dogs euthanised in the early stages of infection and the clinical course of the infection was unrelated to the viral dose administered. The present study demonstrates that strain CB/05 is able to induce infection and disease in dogs seropositive to enteric CCoV, thus highlighting the need for extensive epidemiological investigation and for the possible development of novel antigenically relevant vaccines.

Recombinant canine coronaviruses, resembling the transmissible gastroenteritis virus of swine (TGEV) in a 5' fragment of the S glycoprotein, have been detected recently and showed to be present in canine populations. The 5' fragment of the S protein (S') of a TGEV-like canine coronavirus (CCoV), strain 174/06, was expressed in an Escherichia coli cell-free system. The purified recombinant polypeptide was employed to develop an ELISA test for the detection of TGEV-like CCoV-specific antibodies in dog sera. Four canine sera positive for TGEV-like CCoV, six sera positive to classical CCoV-II strains and 10 negative control sera were examined. The recombinant S' was not recognized by antibodies to classical CCoV-II, as only sera from dogs infected experimentally with TGEV-like CCoV reacted strongly with the recombinant S' polypeptide whereas dog sera with antibodies to classical CCoV-II did not react. As classical CCoV-II and TEGV-like CCoVs are related antigenically, the recombinant S' ELISA is a useful method to investigate serologically the prevalence of TGEV-like CCoVs in dogs.

Coronaviruses (CoVs) are positive-stranded, non-segmented RNA viruses generally responsible for the emergence of respiratory and enteric disease in humans, companion animals and livestock. Their aptitude to evolve by genetic recombination and/or point mutation is recognized, thus giving rise to new viral genotypes and mutants with different tissues or host tropism. In particular, a probable origin from the strictly related bovine coronavirus (BCoV) or, alternatively, from a common ancestor has been suggested for some group 2a CoVs, including canine respiratory coronavirus (CRCoV). In this study, we report the sequence analysis of the viral RNA 3'-end of an Italian CRCoV, strain 240/05, together with the sequence comparison with extant bovine-like viruses including the sole CRCoV strain 4182 previously described. Interestingly, although the structural proteins show the same features of CRCoV 4182, the genomic region between the spike and the envelope protein genes of CRCoV 240/05 encodes for three distinct products, including the equivalent bovine 4.9 kDa non-structural protein and a truncated form of the 4.8 kDa protein, whereas CRCoV 4182 has a unique 8.8 kDa protein.

The sequence of the full-length gene encoding for the main capsid protein VP2 of 58 canine parvovirus (CPV) type 2c strains, along with recent CPV-2a/2b strains, was determined and analysed in comparison with reference CPV isolates. The CPV-2c strains displayed a low genetic variability and shared amino acid changes already detected in recent CPV-2a/2b isolates, with a phylogenetic clustering accounting for their geographical distribution. Analysis of the selection pressure driving CPV evolution confirmed that the VP2 gene is under purifying selection. The emergence and global spread of the new CPV variant provides an interesting model to better understand virus evolution.

Four canine coronavirus type II (CCoV-II) strains were identified in the guts and internal organs of pups which had died of acute gastroenteritis. The CCoV-II strains were strictly related to porcine transmissible gastroenteritis virus (TGEV) in the N-terminal domain of the spike protein, whereas in the other parts of the genome, a higher genetic relatedness to recent CCoV-II isolates was observed. Experimental infection of dogs with a TGEV-like isolate induced mild gastroenteritis without any systemic involvement. By virus neutralization tests, antigenic differences between reference and TGEV-like CCoVs were found. Our data support the potential recombinant origin of the TGEV-like CCoVs. Copyright © 2009, American Society for Microbiology. All Rights Reserved., This work was supported by grants from the Italian Ministry of Health, Ricerca finalizzata 2008, project Mammalian Coronaviruses: Molecular Epidemiology, Vaccine Development and Implications for Animal and Human Health (Nicola Decaro), and from the Italian Ministry of Education, University and Research, PRIN 2006, project Infezione del Cane da Coronavirus Pantropico: Aspetti Epidemiologici, Patogenetici e Molecolari (Canio Buonavoglia).

Malignant catarrhal fever (MCF) is a fatal, systemic disease of cattle and other domestic and wild ruminants that, in Europe, is caused by Ovine herpesvirus 2 (OvHV-2). American bison ( Bison bison) are highly susceptible to the disease. An adult American bison, housed in a zoo in southern Italy in close cohabitation with a group of domestic sheep ( Ovis aries aries) displayed clinical signs that resembled the acute form of MCF. By real-time polymerase chain reaction, OvHV-2 DNA was detected intravitam in blood, in nasal and ocular swabs, and postmortem in tissue samples of the bison. By indirect fluorescent antibody test, high MCF antibody titers were found in the bison serum. Ovine herpesvirus 2 DNA and antibodies were also found in blood samples from the domestic sheep, thus suggesting a potential role of these animals as a source of the infection. To the authors' knowledge, this is the first MCF case in captive ruminants in Italy and the second confirmed case in captive bison of European zoos.

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of bovine coronavirus (BCoV) RNA in clinical samples is described. The assay is based on TaqMan technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the transmembrane-protein gene of BCoV. The BCoV real-time RT-PCR assay was able to detect the tested BCoV and BCoV-like viruses (canine respiratory coronavirus and bubaline coronavirus), whereas other common viral pathogens of cattle were not recognised by the established oligonucleotide set, thus showing that the test was specific for bovine-like CoVs. The detection limit of the assay was 20 BCoV RNA copies (1-log higher with respect to traditional gel-based RT-PCR) and the reproducibility was satisfactory, thus allowing for a sensitive and accurate measurement of the viral RNA load in clinical samples. Two hundred and twenty clinical specimens (92 rectal, 82 nasal and 46 ocular swabs) were subjected to gel-based and real-time RT-PCR. By conventional amplification, 43 rectal, 54 nasal and 34 ocular samples tested positive, whereas the TaqMan assay was able to detect the BCoV nucleic acid in 49 rectal, 60 nasal and 37 ocular swabs. The rapidity and high throughput of the BCoV TaqMan assay makes this method a powerful tool for a sensitive and specific diagnosis of BCoV infection in cattle.

A pantropic canine coronavirus (CCoV) strain (CB/05) has been recently associated to a fatal outbreak of systemic disease in young dogs. We report the clinical, virological and serological findings in dogs experimentally infected with strain CB/05. The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. Leucopenia (acute lymphopenia) occurred in all infected dogs, with values dropping below 60% of the initial counts. Considering the severity of the CB/05-induced disease, two of the youngest pups were euthanized for ethical reasons at days 8–9 postinfection, whereas the other pups underwent a slow but progressive improvement of their clinical status with complete recovery. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. All pups seroconverted for CCoV, as shown by the high optical density values and antibody titres detected by ELISA and virusneutralisation tests, respectively. The present study confirms that strain CB/05 is highly pathogenic for dogs, being able to induce a severe disease (and in some cases the death) even in experimental conditions.

Four outbreaks of bovine respiratory disease (BRD) associated with bovine Coronavirus (BCoV) infection in Italian cattle herds were reported. In 3 outbreaks, BRD was observed only in 2–3-month-old feedlot calves, whereas in the remaining outbreak, lactating cows, heifers, and calves were simultaneously affected. By using reverse transcription polymerase chain reaction (RT-PCR), BCoV RNA was detected in all outbreaks without evidence of concurrent viral pathogens (i.e., bovine respiratory syncytial virus, bovine herpesvirus type 1, bovine viral diarrhea virus, bovine parainfluenza virus). Common bacteria of cattle were recovered only from 2 outbreaks of BRD: Staphylococcus spp. and Proteus mirabilis (outbreak 1) and Mannheimia haemolytica (outbreak 4). A recently established real-time RT-PCR assay showed that viral RNA loads in nasal secretions ranged between 3.10 × 10 2 and 7.50 × 10 7 RNA copies/μl of template. Bovine Coronavirus was isolated from respiratory specimens from all outbreaks except outbreak 1, in which real-time RT-PCR found very low viral titers in nasal swabs.

Canine respiratory coronavirus (CRCoV) is a group II coronavirus that was firstly identified in lung samples of dogs with canine infectious respiratory disease (CIRD) in UK in 2003. We report for the first time the identification of CRCoV in Italy, together with serological evidence that the virus has been circulating in the Italian dog population as from 2005. Serological investigations on 216 dog sera, carried out by an ELISA test using the strictly related bovine coronavirus (BCoV) as antigen, revealed an overall CRCoV seroprevalence of 32.06% in the last 2 years. RT-PCR targeting the S-gene of CRCoV was carried out on 109 lung samples collected from carcasses of dogs submitted for diagnostic investigations. Positive results were obtained from the lungs of a dog of the Apulia region that was co-infected with canine parvovirus type 2. Sequence analysis of the S-gene fragment amplified by RT-PCR (595 bp) showed similarity to group II coronaviruses, with the highest nucleotide identity (98%) to the only CRCoV strain currently available in the GenBank database (strain T101). The results of the present study show that CRCoV is present also in continental Europe, although further studies are required to determine the real pathogenic potential of the virus.

Pantropic canine coronavirus (CCoV) was first detected in young dogs in Italy in 2005, but the complete genome sequence of this virus had not yet been determined. Here, we report the full-length genome sequence of the prototype strain CB/05, which showed that this virus is genetically similar to CCoV-IIa viruses.

Highlights • HoBi-like pestivirus was administered to pregnant sheep. • Infected animals displayed reproductive disorders. • Persistently infected lambs were born that shed the virus through all body fluids. • Sheep may serve as an alternative model for HoBi-like pestivirus vaccine studies., In order to evaluate sheep as experimental model to test the efficacy of HoBi-like pestivirus vaccines for cattle, 10 sheep at different stages of pregnancy (30 or 50 days) were experimentally infected with the Italian prototype isolate Italy-1/10-1. Irrespective of the stage of pregnancy, virus inoculation resulted in reproductive failures, consisting of abortion, stillbirths or birth of weak or persistently infected (PI) lambs. Aborted fetuses, stillborn and dead lambs displayed extensive histopathological changes, consisting of hemorrhages, congestion and mononuclear infiltration in major organs. Pestiviral antigens were detected by immunohistochemistry in most tissues with remarkable signals in lungs and kidneys. PI lambs were constantly viremic, shed the virus through the nasal secretions and feces and, in all cases but one, did not have detectable HoBi-like pestivirus antibodies before the assumption of colostrum. The single seropositive infected lamb showed low-titer viremia and viral shedding that ceased only several weeks after the 3-month observation period. The study proves that sheep are susceptible to the reproduction failures caused by HoBi-like pestivirus infection and can serve as a suitable model for the evaluation of the fetal protection induced by homologous experimental vaccines.

Hobi-like pestivirus, a new tentative species within genus Pestivirus, was firstly detected in foetal bovine serum batches and later associated to respiratory distress and reproductive failures in cattle. In the present study, the cross-antibody response between bovine viral diarrhoea virus 1 (BVDV-1) and the emerging pestivirus was evaluated in the sheep model. Ten sheep were immunised against BVDV-1 or Hobi-like pestivirus using inactivated preparations and the induced antibody responses were evaluated against the homologous and heterologous viruses. The results showed that heterologous antibody titres were significantly lower than the homologous ones, thus suggesting the need to develop specific vaccines against the emerging pestiviral species.

An epidemiological survey for Canine parvovirus 2 (CPV-2) and Canine coronavirus (CCoV) was conducted in Albania. A total of 57 fecal samples were collected from diarrheic dogs in the District of Tirana during 2011–2013. The molecular assays detected 53 and 31 CPV- and CCoV-positive specimens, respectively, with mixed CPV–CCoV infections diagnosed in 28 dogs. The most frequently detected CPV type was 2a, whereas IIa was the predominant CCoV subtype. A better comprehension of the CPV–CCoV epidemiology in eastern European countries will help to assess the most appropriate vaccination strategies to prevent disease due to infections with these widespread agents of acute gastroenteritis in the dog.

An outbreak of canine infectious respiratory disease (CIRD) associated to canine pneumovirus (CnPnV) infection is reported. The outbreak occurred in a shelter of the Apulia region and involved 37 out of 350 dogs that displayed cough and/or nasal discharge with no evidence of fever. The full-genomic characterisation showed that the causative agent (strain Bari/100-12) was closely related to CnPnVs that have been recently isolated in the USA, as well as to murine pneumovirus, which is responsible for respiratory disease in mice. The present study represents a useful contribution to the knowledge of the pathogenic potential of CnPnV and its association with CIRD in dogs. Further studies will elucidate the pathogenicity and epidemiology of this novel pneumovirus, thus addressing the eventual need for specific vaccines.

The first reported incidence of Hobi-like viruses in Europe dates to a 2010 outbreak of respiratory disease in cattle in Italy. In this study, a Hobi-like virus was detected in archival samples, collected in 2007 in Italy from a cattle herd displaying respiratory disease, during the validation of a nested PCR protocol for rapid characterization of bovine pestiviruses. Phylogeny conducted with full-length pestivirus genomes and three informative genomic sequences, placed the strain detected in the samples, Italy-129/07, into the Hobi-like virus branch. Italy-129/07, similar to other Hobi-like viruses isolated in Italy, was more closely related to viruses of South American origin, than Hobi-like viruses of Southeast Asian origin. This suggests a possible introduction of this emerging group of pestiviruses into Italy as a consequence of using contaminated biological products such as fetal bovine serum originating in South America. This report of a Hobi-like virus associated with respiratory disease along with the full-genomic characterization of the virus detected provides new data that contributes to the body of knowledge regarding the epidemiology, pathobiology and genetic diversity of this emerging group of pestiviruses. Importantly, it dates the circulation of Hobi-like viruses in Italy to 2007, at least three years before previous reports.

A calf persistently infected by Hobi-like pestivirus was monitored for about 6 months, displaying clinical signs typical of bovine viral diarrhea virus persistent infection and shedding the virus through all body secretions, with maximal titers detected in urine. This report provides new insights into the pathogenesis of the emerging pestivirus.

A Hobi-like pestivirus pair consisting of cytopathogenic (cp) and non-cytopathogenic (noncp) strains, Italy 83/10cp and Italy 83/10ncp, was isolated from the lung of a heifer that died of respiratory disease. The noncp and cp viruses were isolated on Madin–Darby bovine kidney cells and separated by plaque purification and end point dilution. Analysis of the nearly full-length genomes revealed that the two viruses were very closely related to each other and to the noncp Hobi-like strain Italy 1/10-1, which had been isolated a few weeks earlier from the same herd. One major difference between noncp and cp viruses concerned the presence of a cellular Jiv sequence in the 3′ domain of the NS2-encoding region of the cp strain. This is the first study, to our knowledge, reporting the isolation and molecular characterization of a Hobi-like virus pair.