Fecal Shedding of Bovine Astrovirus CH13/NeuroS1 in Veal Calves

LETTER

Specific astrovirus genotype species are associated with neurological disease and encephalitis in humans, mink, pigs, sheep, and cattle (1). In humans (2) and pigs (3), these encephalitis-associated astroviruses were also detected in fecal and/or nasopharyngeal samples, suggesting that the gastrointestinal and respiratory tracts may serve as viral reservoirs. Two encephalitis-associated astroviruses have been identified in the central nervous system of neurologically diseased cattle, namely, bovine astrovirus (BoAstV)-CH13/NeuroS1 (4, 5) and BoAstV-CH15 (6). However, transmission routes and nonnervous target tissues for these astroviruses are mostly unknown. Here, we investigated whether BoAstV-CH13/NeuroS1 and BoAstV-CH15 are part of the respiratory and intestinal virome of veal calves.

Retrospectively, we analyzed 273 rectal swabs and 666 nasal swabs from veal calves of unknown health status as well as nasal swabs of 96 calves with signs of respiratory disease that had been collected in 63 Swiss farmyards in 2016 and 2017 (see supplemental material). We tested all samples for BoAstV-CH13/NeuroS1 and -CH15 using specific reverse transcriptase quantitative PCR (RT-qPCR) protocols (7, 8). All respiratory samples were RT-qPCR negative for both viruses. All rectal swabs were negative for BoAstV-CH15, but two samples (VC34 and VC65) from calves in different farms scored positive for BoAstV-CH13/NeuroS1, with quantification cycle (C_q) values of 25.8 and 27.3, respectively (see Fig. S1 in the supplemental material). Next-generation sequencing of RNA extracts of these two samples yielded three scaffolds with sequence similarities to astroviruses in VC34 and two in VC65. These scaffolds had almost a full-genome length and showed the typical molecular features of astrovirus genomes. Two scaffolds, one in each animal (VC34/346 and VC65/698), showed a high similarity of >94% to sequences of BoAstV-CH13/NeuroS1 strains on the nucleotide level. In addition, two scaffolds (VC34/375 and VC65/693) were closely related (>85%) to previously described astroviruses in fecal samples from cattle. Finally, one scaffold in VC34 (VC34/338) matched best (∼86%) to sequences of porcine astrovirus 4 (PAstV-4) strains (see Table S1 in the supplemental material). These correlations were supported by phylogenetic analysis (Fig. 1; see Table S2 in the supplemental material).

FIG 1.

Phylogenetic comparison of astrovirus sequences identified in fecal samples from veal calves. Amino acid sequences of the nonstructural protein Nsp1ab and the capsid precursor protein were analyzed by the maximum likelihood method using the MEGA X software and 1,000 bootstraps. Branch length corresponds to the numbers of substitutions per site. Bold triangles indicate sequences that cluster with bovine astroviruses identified previously in fecal samples. Bold circles indicate sequences that cluster with ruminant encephalitis-associated strains (open circles). BoAstV, bovine astrovirus; OvAstV, ovine astrovirus; PAstV, porcine astrovirus. Human Astrovirus (HuAstV-1) serves as the outgroup. GenBank accession numbers are indicated in brackets.

We tested additional fecal swab samples, which had been collected during the study period on the two farms where the two animals that were RT-qPCR positive for BoAstV-CH13/NeuroS1 originated (n = 27 for VC34; n = 46 for VC65). One additional animal (VC301) which had been kept in the same herd with VC65 scored positive, with a C_q value of 28.4. We Sanger sequenced ∼80% of the genome of the VC301 strain and found the sequence to be >99% identical to VC65/698. All other animals from these two farms remained BoAstV-CH13/NeuroS1 negative. Fecal and nasal swabs taken from the same three RT-qPCR-positive animals but collected at different time points during their lives were BoAstV-CH13/NeuroS1 negative (Fig. S1), suggesting that viral shedding was temporary.

Taken together, this study represents the first identification of BoAstV-CH13/NeuroS1 fecal excretion in ruminants. Thus, enteric BoAstV-CH13/NeuroS1 infection in calves may represent a viral reservoir, and fecal shedding likely constitutes a route of virus transmission. Moreover, the identification of an astrovirus genome with high similarity to PAstV-4 in fecal samples of calves further questions the concept of a strict species specificity of mammalian astroviruses in livestock.