Alterations of the receptor-binding properties of swine influenza A viruses (SIVs) during their isolation in embryonated chicken eggs have not been well studied. In this study, the receptorbinding properties of classical H1 SIVs isolated solely in eggs or Madin-Darby canine kidney (MDCK) cells were examined. Sequencing analysis revealed substitutions of D190V/N or D225G in the haemagglutinin (HA) proteins in egg isolates, whereas MDCK isolates retained HA genes identical to those of the original viruses present in the clinical samples. Egg isolates with substitution of either D190V/N or D225G had increased haemagglutinating activity for mouse and sheep erythrocytes, but reduced activity for rabbit erythrocytes. Additionally, egg isolates with D225G had increased haemagglutination activity for chicken erythrocytes. A direct binding assay using a sialyl glycopolymer that possessed either a 5-N-acetylneuraminic acid (Neu5Ac) a2,6galactose (Gal) or a Neu5Aca2,3Gal linkage revealed that the egg isolates used in this study showed higher binding activity to the Neu5Aca2,3Gal receptor than MDCK isolates. Increased binding activity of the egg isolates to the Neu5Aca2,3Gal receptor was also confirmed by haemagglutination assay with resialylated chicken erythrocytes by Galb1,3/4GlcNAca2,3-sialyltransferase. These observations were reinforced by flow-cytometric and N-glycan analyses of the erythrocytes. The a2,3-linked sialic acids were expressed predominantly on the surface of mouse and sheep erythrocytes. Chicken erythrocytes expressed Neu5Aca2,3Gal more abundantly than Neu5Aca2,6Gal, and rabbit erythrocytes expressed both 5-N-glycolylneuraminic acid (Neu5Gc) a2,6Gal and Neu5Aca2,6Gal. Our results demonstrate clearly that classical H1 SIVs undergo alterations in receptor-binding activity associated with an amino acid substitution in the HA protein during isolation and propagation in embryonated chicken eggs.

Perinatal transmission plays a critical role in the spread of bovine leukemia virus (BLV) infection in cattle herds. In the Holstein breed, we previously identified BLV resistant and susceptible bovine leukocyte antigen (BoLA)-DRB3 alleles, including BoLA-DRB3*009:02 and *014:01:01 with a low BLV proviral load (PVL), and *015:01 and *012:01 with a high PVL. Here, we evaluated the perinatal BLV transmission risk in dams with different BoLA-DRB3 alleles. BoLA-DRB3 alleles of 120 dam-calf pairs from five dairy farms in Japan were identified; their PVL was quantified using the BLV-Coordination of Common Motifs (CoCoMo)-qPCR-2 assay. Ninety-six dams were BLV-positive, and 29 gave birth to BLV-infected calves. Perinatal transmission frequency was 19% in dams with resistant alleles suppressed to a low PVL level, and 38% and 25% in dams with susceptible and neutral alleles that maintained high PVL levels, respectively. Notably, all calves with resistant alleles were BLV free, whereas 30% of calves with susceptible genes were infected. Thus, vertical transmission risk was extremely lower for dams and calves with resistant alleles compared to those with susceptible alleles. Our results can inform the development of effective BLV eradication programs under field conditions by providing necessary data to allow for optimal selection of dams for breeding.

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Eleven swine influenza viruses (SIVs) isolated from pigs in

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For broad detection of pestivirus A (bovine viral diarrhea virus 1: BVDV1) and pestivirus B (BVDV2) by a reverse transcription loop-mediated isothermal amplification (RT-LAMP) test, the P25 primer set was designed using nucleotide sequences of 5'-untranslated region of 1454 BVDVs.The base coverage of each primer against diverse BVDVs were more than 99% in each base position.The one step LAMP test with the P25 primer set could detect both BVDV1 (TK) and BVDV2 (KZ), but did not amplify 5 other bovine viruses. Detection limit of the LAMP test was 10 3 copies of synthesized DNAs, and 10 -3 and 10 -4 dilutions of viral RNAs of TK and KZ strains, respectively, whereas that with current Aebischer's primer set was 10 -2 dilution and negative of these RNAs, respectively. All of the 63 viral RNA samples of persistently infected (PI) cattle, consisting of the 1a (12), 1b (31), 1c (11), and 2a (9) subgenotypes, were broadly detected with the P25, while only 65% of them were positive with Aebischer's primer set. The validation study showed that the RT-LAMP test with the P25 had 100 % sensitivity and 100 % specificity against that with updated Vilcek's PCR primers. Also, by using the P26 primer set which contained 3 species-specific primers, all 63 RNA samples were clearly distinguished from BVDV1 or BVDV2 by the typing RT-LAMP test. These results indicate that the one step RT-LAMP test using P25 or P26 primer sets would be useful for broad detection and rapid differentiation of BVDV1 and BVDV2.