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Abstract Practice of inoculating porcine epidemic diarrhea virus (PEDV) in piglets generating feedback material might influence the genetic evolution and attenuation of PEDV. The study was conducted to evaluate evolutionary rate and attenuation following serial in vitro and in vivo propagation. In the study, PED-JPFP0-PJ, Passage 0 (P0), was isolated from infected pigs and serially passaged in Vero cells for 5 consecutive times, P1-P5. P0, P2 and P5 were then subjected to orally inoculate 3-day-old piglets. At 24 h post inoculation, intestines of each passage (F1), were collected, and subsequently sub-passaged in piglets for 2 additional passages (F2-F3). Virus titration, PEDV genomic copies number, VH:CD ratios, and immunohistochemistry were evaluated. S and ORF3 genes were characterized. The results of the study demonstrated that virus titer and virulence were negatively correlated with increased passages, both in vitro and in vivo. Increased substitution rate was observed in higher passages. The evolutionary rate of S gene was higher than that of ORF3. Seven aa changes at positions 223, 291, 317, 607, 694, 1114 and 1199, with reduced N-linked glycan were observed in P5F3. In conclusion, serial passage of PEDV, both in vitro and in vivo, influence the genetic development and the attenuation of PEDV.

Abstract Shared needles are a possible iatrogenic and hematogenous inanimate vector of African Swine Fever virus (ASFV) in farm conditions. To evaluate that possible transmission, sixty, 4-week-old pigs were procured from an ASF free herd free. Upon arrival, pigs were randomly divided into two sets. Set 1 served as seeder pigs, and were randomly allocated to 4 groups. The other pigs were divided into 8 groups, and served as sentinels. Seeder pigs were oronasally challenged with ASFV at high (108 copy numbers/mL), moderate (106 copy numbers/mL) or low (101 copy numbers/mL) challenge titer, except a subgroup that remained unchallenged (negative control). At 7 days post challenge (peak viremia), all four seeder groups were intradermally and intramuscularly (IM) injected with a vaccine adjuvant (Diluvac Forte, MSD Animal Health, The Netherlands) using a needle-free device (IDAL 3G, MSD Animal Health, The Netherlands) and conventional needles, respectively. The same needle or needle-free device was then used to inject the same volume of adjuvant into set 2 (n = 48) pigs. All pigs were observed for clinical disease daily and assayed for the presence of ASFV DNA by quantitative PCR. All seeder groups developed viremia (except the control pigs). ASFV viremia was detected in all sentinel groups injected via the intramuscular route. Transmission rate from the IM route via conventional needles was positively correlated with virus titer in blood circulation of seeders. Sentinels intramuscularly exposed to needles from high titer challenged seeders displayed more severe and acute clinical disease compared to that of exposed to low titer challenged seeders. No viremia nor clinical signs were observed in the sentinel groups injected via the intradermal route. This study confirmed the hematogenous transmission of ASFV between pigs through needle-sharing.