Analysis of Fetal bovine Serum reveals the presence of emerging Hobi-like viruses in Argentinean livestock.

PECORA ANDREA; PEREZ AGUIRREBURUALDE MARIA SOL; DUS SANTOS MARIA JOSE

SAPPORO

Congreso; XXX World Buiatrics Congress; 2018

Fetal bovine serum (FBS) is widely used as a growth supplement in cell culture media and as a component of biological products used in animal and human health. HoBi-like viruses are a group of emerging pestiviruses that affect cattle, buffaloes and goats. Since its first identification in Brazilian FBS in 2004, HoBi-like viruses have been isolated in other European and Asian countries. HoBi-like virus infection seems to be clinically indistinguishable from that caused by BVDV, which includes respiratory disease, reproductive failures and persistently infected animals in herds. Though BVDV is endemic in Argentina, HoBi-like viruses had been neither detected nor isolated locally. In this sense, we have recently reported serological evidence of this agent in water buffalo herds (Pecora et al, 2017).The first aim of this work was to evaluate the presence of traces of HoBi-like viruses in local FBS samples. Moreover, this work sought to determine which BVDV genotype prevails in Buenos Aires region. Semi-elaborated FBS samples (n=112) were obtained prior to the irradiation stage, belonging to a laboratory that commercializes blood products obtained from slaughterhouses located in Buenos Aires province. The samples were subjected to RNA extraction, followed by a reverse transcriptase reaction. Two polymerase chain reaction (PCR) were carried out to amplify 5´pestiviral untranslated region (5´UTR): one with the 324-326 primers (Vilcek et al, 1994) and another with the N2-R5 primers, which detect BVDV or HoBi-like viruses, respectively (Bauermann et al, 2014b). In the PCR-positive samples, amplification products were direct sequenced. Then, a phylogenetic study was performed, by the Kimura and Neighbor Joining methods. Since the FBS had not been irradiated, HoBi-like virus positive samples were subjected to passages in Marbin Darbin Bovine Kidney (MDBK) cells in order to isolate the viruses. Lastly, guinea pigs were vaccinated with oily formulations containing the isolated HoBi-like viruses to evaluate their immunogenicity. In this way, the animals´ antibody profile against Pestiviruses was determined by cross-virus neutralization assays.The PCR performed with 324-326 primers amplified 42 FBS samples which were grouped in BVDV-1a (n=6), BVDV-1b (n=34) and BVDV-2 (n=2). The PCR carried out with N2-R5 primers amplified other 4 FBS samples, which belonged to the Hobi-like virus clade. After several cellular passages, the 4 HoBi-like viruses were successfully isolated. When using these viruses as immunogens, all the vaccinated guinea pigs developed neutralizing antibodies against a HoBi-like virus reference strain (Italy 83/10) ranging from 1/128 to 1/512. Antibody titers against BVDV-1a, BVDV-1b and BVDV-2 were lower in all the cases.In conclusion, HoBi-like virus genome was detected in 4 non-irradiated FBS, which indicates that the virus is circulating in bovine cattle in the Buenos Aires region. The isolated viruses evoked a specific antibody response in guinea pigs. Future work will be performed to characterize these agents.On the other hand, the high percentage of BVDV-1b strains found in this work (81% of the BVDV positive samples) is in agreement with previous data, which reported the high frequency of BVDV genotype 1b in Argentina (Pecora et al, 2014). Finally, we want to highlight the importance that the FBS be irradiated for commercializing, to avoid contamination with endemic or emerging pestiviruses.