First Isolation, Molecular Characterization, and Serological Survey of Bovine Adenovirus Type 2 in Japan

Bovine adenovirus type 2 (BAdV-2), a member of the Mastadenovirus genus of the Adenoviridae family, is involved in mild respiratory disease and is rarely isolated from cattle in the world. Here, we isolated BAdV-2 for the first time in Japan, from the feces of cattle with severe respiratory symptoms. The isolate, designated strain KY19-1, was identified morphologically and genetically. Electron microscopic observation of infected cells showed intranuclear, non-enveloped virus particles, approximately 70 nm in diameter and with typical adenoviral morphology. Whole-genome sequencing revealed that the viral genome is 33,175 bp long, with 105 bp inverted terminal repeats and encoding 32 predicted genes. KY19-1 has a similar genomic organization to the prototype BAdV-2 strain No. 19 with overall 99.1% nucleotide identity. Notable differences were found in the E3 region of the genome, which may affect various biological properties, including pathogenicity. The seroepidemiology of BAdV-2 was also investigated, using a virus-neutralization test, and 92.8 % of the Japanese cattle (n=1,325) were seropositive, with the positivity rate increased by age. Further experimental infections with KY19-1 may elucidate the pathogenic properties of BAdV-2 and the importance of this virus as a causal agent of bovine respiratory disease.

Complete Genome Sequence of Bovine Adenovirus Type 7 Strain Fukuroi, Isolated from a Cow with Respiratory Disease

ABSTRACT We determined the complete genome sequence of the bovine adenovirus type 7 prototype strain Fukuroi using next-generation sequencing technology. We found that the viral genome is 30,034 bp long and has the shortest inverted terminal repeats among known adenoviruses.

Whole genome sequencing of live attenuated bovine adenovirus type 7 vaccine strain TS-GT reveals biomarkers for virulence attenuation

Bovine adenovirus type 7 (BAdV-7) is one of the most important respiratory and enteric pathogens in the cattle industry. Although live attenuated vaccines are used to control the virus in Japan, limited information is available on the genomic regions that determine viral pathogenicity. Here we analyzed the attenuated strain TS-GT after isolation of the virus from the hexavalent bovine respiratory disease vaccine Cattlewin-6 lot #40-1. The complete genome sequence of TS-GT was determined using next-generation sequencing. The genome is 30,052 bp long and contains 45-bp inverted terminal repeats (ITR) and 30 predicted genes. A genome sequence comparison showed that 99.9% of the TS-GT genome is identical to the prototypic and pathogenic BAdV-7 strain Fukuroi; however, the TS-GT genome contains a novel mutation and four indels. A mutation in TS-GT of the minor capsid protein pVI causes an amino acid change, and possibly induces an alteration of its membrane-lytic function; this may underlie the temperature-specific growth characteristic of this strain. A 19-bp insertion in the 3’ ITR may affect its promoter activity and change the expression of downstream coding genes. In this way, the insertion may play important roles in the attenuation of the virus. Although further mutational and functional analyses are required, our results provide insights into the molecular basis of BAdV-7 attenuation and open new avenues for future analyses.

Nuclear and Nucleolar Localization of Bovine Adenovirus-3 Protein V

The L2 region of bovine adenovirus-3 (BAdV-3) encodes a Mastadenovirus genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81–120, 190–210, and 380–389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21–50 and 380–389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380–389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.

Bovine Adenovirus-3 Tropism for Bovine Leukocyte Sub-Populations

A number of characteristics including lack of virulence and the ability to grow to high titers, have made bovine adenovirus-3 (BAdV-3) a vector of choice for further development as a vaccine-delivery vehicle for cattle. Despite the importance of blood leukocytes, including dendritic cells (DC), in the induction of protective immune responses, little is known about the interaction between BAdV-3 and bovine blood leukocytes. Here, we demonstrate that compared to other leukocytes, bovine blood monocytes and neutrophils are significantly transduced by BAdV404a (BAdV-3, expressing enhanced yellow green fluorescent protein [EYFP]) at a MOI of 1–5 without a significant difference in the mean fluorescence of EYFP expression. Moreover, though expression of some BAdV-3-specific proteins was observed, no progeny virions were detected in the transduced monocytes or neutrophils. Interestingly, addition of the “RGD” motif at the C-terminus of BAdV-3 minor capsid protein pIX (BAV888) enhanced the ability of the virus to enter the monocytes without altering the tropism of BAdV-3. The increased uptake of BAV888 by monocytes was associated with a significant increase in viral genome copies and the abundance of EYFP and BAdV-3 19K transcripts compared to BAdV404a-transduced monocytes. Our results suggest that BAdV-3 efficiently transduces monocytes and neutrophils in the absence of viral replication. Moreover, RGD-modified capsid significantly increases vector uptake without affecting the initial interaction with monocytes.