ERDRP-0519 inhibits feline coronavirus in vitro.

BACKGROUND: Coronaviruses (CoVs) are major human and animal pathogens and antiviral drugs are pursued as a complementary strategy, chiefly if vaccines are not available. Feline infectious peritonitis (FIP) is a fatal systemic disease of felids caused by FIP virus (FIPV), a virulent pathotype of feline enteric coronavirus (FeCoV). Some antiviral drugs active on FIPV have been identified, but they are not available in veterinary medicine. ERDRP-0519 (ERDRP) is a non-nucleoside inhibitor, targeting viral RNA polymerase, effective against morbilliviruses in vitro and in vivo. RESULTS: The antiviral efficacy of ERDRP against a type II FIPV was evaluated in vitro in Crandell Reese Feline Kidney (CRFK) cells. ERDRP significantly inhibited replication of FIPV in a dose-dependent manner. Viral infectivity was decreased by up to 3.00 logarithms in cell cultures whilst viral load, estimated by quantification of nucleic acids, was reduced by nearly 3.11 logaritms. CONCLUSIONS: These findings confirm that ERDRP is highly effective against a CoV. Experiments will be necessary to assess whether ERDRP is suitable for treatment of FIPV in vivo.

Mucosal disease-like syndrome in a calf persistently infected by Hobi-like pestivirus.

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.

Persistent infection caused by Hobi-like pestivirus.

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.

European surveillance for pantropic canine coronavirus.

Highly virulent pantropic canine coronavirus (CCoV) strains belonging to subtype IIa were recently identified in dogs. To assess the distribution of such strains in Europe, tissue samples were collected from 354 dogs that had died after displaying systemic disease in France (n = 92), Hungary (n = 75), Italy (n = 69), Greece (n = 87), The Netherlands (n = 27), Belgium (n = 4), and Bulgaria (n = 1). A total of 124 animals tested positive for CCoV, with 33 of them displaying the virus in extraintestinal tissues. Twenty-four CCoV strains (19.35% of the CCoV-positive dogs) detected in internal organs were characterized as subtype IIa and consequently assumed to be pantropic CCoVs. Sequence and phylogenetic analyses of the 5' end of the spike protein gene showed that pantropic CCoV strains are closely related to each other, with the exception of two divergent French viruses that clustered with enteric strains.

Hobi-like pestivirus: both biotypes isolated from a diseased animal.

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.

Hobi-like pestivirus in aborted bovine fetuses.

An outbreak of abortion affecting multiparous cows was associated with Hobi-like pestivirus infection. Viral RNA and antigens were detected in the tissues of two aborted fetuses. Molecular assays for other common abortogenic agents tested negative. At the genetic level, the Hobi-like pestivirus displayed the closest relatedness to Italian, Australian, and South American viruses, whereas it diverged from the prototype Thai isolate. These findings may have important implications for the pestivirus control/eradication programs in cattle herds.

A nested PCR approach for unambiguous typing of pestiviruses infecting cattle.

An atypical pestivirus ('Hobi'-like pestivirus, putative bovine viral diarrhoea 3, BVDV-3) was identified firstly in contaminated foetal calf serum batches and isolated subsequently from an outbreak of respiratory disease in a cattle herd in Italy. The isolation of the novel pestivirus from animals affected clinically posed concerns about the validity of BVDV eradication programs, considering that 'Hobi'-like pestivirus (BVDV-3) is undetected or mistyped by the molecular diagnostic tools currently employed. In this paper, the development of a nested PCR (nPCR) assay for unambiguous typing of all bovine pestiviruses is reported. The assay consisted of a first-round amplification using an oligonucleotide pair which binds to conserved sequences located in the 5' untranslated region and capsid gene, followed by a heminested PCR using virus-specific forward primers. The assay performances were evaluated analytically, showing good sensitivity and specificity. By analysis of 100 BVDV-positive samples typed using a nPCR assay discriminating ruminant pestiviruses, five samples recognised previously as BVDV-2 were not typed when submitted to the new assay (n=2) or reacted as 'Hobi'-like pestivirus BVDV-3 (n=3). Sequence analysis of the first-round amplification products showed that the untyped strains were border disease viruses, whereas the other three strains were true 'Hobi'-like viruses. The development of a molecular assay able to identify simultaneously all bovine pestiviruses known currently will help warrant biosafety of live vaccines and other biological products and assess the molecular epidemiology of 'Hobi'-like pestivirus, thus leading to the improvement of the eradication programs through unambiguous typing of pestiviruses infecting cattle.

Molecular characterization of a canine coronavirus NA/09 strain detected in a dog's organs.

In the present study, the detection of a pantropic canine coronavirus (CCoV) strain in a dog with lethal diarrhoea is reported. RT-PCR and real-time RT-PCR assays were used for the detection, characterization and quantitation of CCoV. Sequence and phylogenetic analysis of the CCoV NA/09 revealed a high degree of sequence identity with the pantropic strain CB/05, indicating the presence of CB/05-like pantropic strains in Greece. The absence of the 38-nucleotide deletion in ORF3b, which is characteristic of CB/05, indicates the need to identify new genetic markers for pantropic variants of CCoV, probably in the spike-protein gene region.

Atypical pestivirus and severe respiratory disease in calves, Europe.

In 2010, a HoBi-like pestivirus was isolated from clinically affected calves in Italy. This European virus reproduced a milder form of disease under experimental conditions and was genetically related to previously reported HoBi-like strains. Isolation of this novel virus from a clinical outbreak may have implications for cattle health and prophylactic programs.

Detection and characterization of canine astroviruses.

Astroviruses (AstVs) have been identified only occasionally in dogs. A canine AstV, strain Bari/08/ITA, was detected from a pup with gastroenteric signs and the virus was isolated in cell culture and characterized molecularly. In the full-length capsid protein, the virus displayed genetic similarities (83.5 % aa identity) to another canine AstV strain, although a high rate of variation occurred in the hypervariable domain, which is related to AstV antigenic specificity. Specific antibodies were detected in the convalescent dog, indicating seroconversion, and in 59 % of a collection of dog serum samples. Using primers specific for canine AstV, designed to detect a conserved region of ORF1b, canine AstVs were detected in 24.5 % of young pups with gastroenteritis, either alone or in mixed infections with other canine pathogens. In contrast, AstVs were detected in only 9.3 % of asymptomatic pups. These findings indicate that canine AstVs are common in dogs and may suggest a possible role as canine enteric pathogens.

A simple pooling salivary test for SARS-CoV-2 diagnosis: A Columbus' egg?

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.

Natural Bovine Coronavirus Infection in a Calf Persistently Infected with Bovine Viral Diarrhea Virus: Viral Shedding, Immunological Features and S Gene Variations.

The evolution of a bovine coronavirus (BCoV) natural infection in a calf persistently infected with bovine viral diarrhea virus (BVDV) was described. The infected calf developed intermittent nasal discharge, diarrhea and hyperthermia. The total number of leukocytes/mL and the absolute differential number of neutrophils and lymphocytes resulted within the normal range, but monocytes increased at T28 (time 28 post-infection). Flow-cytometry analysis evidenced that the CD8+ subpopulation increased at T7 and between T28 and T35. BCoV shedding in nasal discharges and feces was detected up to three weeks post infection and high antibody titers persisted up to T56. The RNA BCoV load increased until T14, contrary to what was observed in a previous study where the fecal excretion of BCoV was significantly lower in the co-infected (BCoV/BVDV) calves than in the calves infected with BCoV only. We can suppose that BVDV may have modulated the BCoV infection exacerbating the long viral excretion, as well as favoring the onset of mutations in the genome of BCoV detected in fecal samples at T21. An extensive study was performed to verify if the selective pressure in the S gene could be a natural mode of variation of BCoV, providing data for the identification of new epidemic strains, genotypes or recombinant betacoronaviruses.

Recombinant canine coronaviruses in dogs, Europe.

Coronaviruses of potential recombinant origin with porcine transmissible gastroenteritis virus (TGEV), referred to as a new subtype (IIb) of canine coronavirus (CCoV), were recently identified in dogs in Europe. To assess the distribution of the TGEV-like CCoV subtype, during 2001-2008 we tested fecal samples from dogs with gastroenteritis. Of 1,172 samples, 493 (42.06%) were positive for CCoV. CCoV-II was found in 218 samples, and CCoV-I and CCoV-II genotypes were found in 182. Approximately 20% of the samples with CCoV-II had the TGEV-like subtype; detection rates varied according to geographic origin. The highest and lowest rates of prevalence for CCoV-II infection were found in samples from Hungary and Greece (96.87% and 3.45%, respectively). Sequence and phylogenetic analyses showed that the CCoV-IIb strains were related to prototype TGEV-like strains in the 5' and the 3' ends of the spike protein gene.

Recombinant canine coronaviruses related to transmissible gastroenteritis virus of Swine are circulating in dogs.

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.

Genetic heterogeneity and recombination in canine noroviruses.

Alphatronlike (genogroup IV [GIV]) noroviruses (NoVs) have been recently identified in carnivores. By screening a collection of 183 fecal samples collected during 2007 from dogs with enteric signs, the overall NoV prevalence was found to be 2.2% (4/183). A unique strain, Bari/91/07/ITA, resembled GIV.2 NoVs in its ORF1 (polymerase complex), while it was genetically unrelated in its full-length ORF2 (capsid gene) to GIV animal and human NoVs (54.0 to 54.4% amino acid identity) and to any other NoV genogroup (<54.7% amino acid identity). It displayed the highest identity (58.1% amino acid identity) to unclassified human strain Chiba/040502/04/Jp. Interestingly, the very 5' end of ORF2 of the canine virus matched short noroviral sequences (88.9% nucleotide identity and 98.9% amino acid identity) identified from oysters in Japan, indicating that similar viruses may be common environmental contaminants.

An outbreak of canine coronavirus in puppies in a Greek kennel.

Canine coronavirus (CCoV) is usually the cause of mild gastroenteritis in dogs and is known to have spread worldwide. However, to date, no CCoV cases have been confirmed in Greece. In the present work, the authors investigated an outbreak of enteritis in puppies from a Greek kennel for the presence of CCoV. Dogs were presented with clinical signs of diarrhea, anorexia, weakness, depression, dehydration, and 1 death. Canine coronavirus type II was detected by reverse transcription nested polymerase chain reaction in all 11 puppies, whereas 1 puppy presented dual infection with CCoV type II and canine parvovirus 2. Surprisingly, sequence analysis of the samples revealed higher similarity to the pantropic CCoV II strain CB/05 than to other reference strains, in the most variable region of the S gene.

Characterization of Canine parvovirus 2 variants circulating in Greece.

The aim of the present study was to characterize Canine parvovirus 2 (CPV-2) variants currently circulating in Greece. Between March 2008 and March 2009, 167 fecal samples were collected from diarrheic dogs from different regions of Greece. Canine parvovirus 2 was detected by standard polymerase chain reaction, whereas minor groove binder probe assays were used to distinguish genetic variants and discriminate between vaccine and field strains. Of 84 CPV-2-positive samples, 81 CPV-2a, 1 CPV-2b, and 2 CPV-2c were detected. Vaccine strains were not detected in any sample. Sequence analysis of the VP2 gene of the 2 CPV-2c viruses revealed up to 100% amino acid identity with the CPV-2c strains previously detected in Europe. The results indicated that, unlike other European countries, CPV-2a remains the most common variant in Greece, and that the CPV-2c variant found in Europe is also present in Greece.

Circulation of pantropic canine coronavirus in autochthonous and imported dogs, Italy.

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.

Molecular characterization of a canine respiratory coronavirus strain detected in Italy.

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.

A candidate modified-live bovine coronavirus vaccine: safety and immunogenicity evaluation.

A modified-live vaccine against the respiratory form of bovine coronavirus (BCoV) infection was developed by progressive attenuation of a respiratory strain (438/06-TN). The vaccine was found to be safe as four colostrum-deprived newborn calves remained healthy after oronasal administration of ten doses of the vaccine. The immunogenicity of the vaccine was assessed by intramuscular injection of one vaccine dose to 30 BCoV-antibody negative 2-3-month-old calves. At 30 days post-vaccination, all vaccinated calves displayed high antibody titres against BCoV. Sequence analysis of the S gene of wild-type and cell-adapted 438/06-TN strain detected 10 nucleotide changes, 9 of which were non-synonymous.