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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 • 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.

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.

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.