Activation of the PI3K/Akt Pathway Early during Vaccinia and Cowpox Virus Infections Is Required for both Host Survival and Viral Replication▿

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to ≥90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers

Evaluation of the genetic variability found in Brazilian commercial vaccines for infectious bronchitis virus

Infectious bronchitis virus (IBV) is currently one of the most important pathogens in the poultry industry. The H120 and Ma5 are the only viral strains approved by the Brazilian government as the constituent of vaccines. Despite the systematic vaccination in Brazil, IBV has not yet been controlled and diseases associated with this virus have been reported in vaccinated chickens. Here, we investigated the genetic variability of H120 and Ma5 strains present in the IBV vaccines from different Brazilian manufacturers. We performed DNA sequencing analyses of the S1 spike glycoprotein gene to investigate its genetic variability and the presence of viral subpopulations among vaccines, between batches, and also in each vaccine after a single passage was performed in chicken embryonated eggs. Our results revealed up to 13 amino acid substitutions among vaccines and some of them were localized in regions of the S1 glycoprotein that play a role in virus–host interaction. Secondary nucleotide peaks identified in the chromatogram for the S1 gene sequence revealed that all original vaccines (H120 and Ma5) were composed by different subpopulations of IBV. Moreover, new viral subpopulations were also found in vaccines after a single passage in chicken embryonated eggs. These findings indicate that H120 and Ma5 viral strains used in vaccines market in Brazil can still mutate very rapidly during replication, leading to amino acid substitutions in proteins involved in the stimulation of the immune response, such as the S1 glycoprotein. Therefore, our data suggest that the genetic variability of these viral strains should be taken into consideration to ensure an effective immune response against IBV

Differential role played by the MEK/ERK/EGR-1 pathway in orthopoxviruses vaccinia and cowpox biology

Appropriation of signalling pathways facilitates poxvirus replication. Poxviruses, as do most viruses, try to modify the host cell environment to achieve favourable replication conditions. In the present study, we show that the early growth response 1 gene (egr-1) is one of the host cell factors intensely modulated by the orthopoxviruses VV (vaccinia virus) and CPV (cowpox virus). These viruses stimulated the generation of both egr-1 mRNA and its gene product, throughout their entire replication cycles, via the requirement of MEK [mitogen-activated protein kinase/ERK (extracellular-signal-regulated kinase) kinase]/ERK pathway. We showed that, upon VV infection, EGR-1 translocates into the nucleus where it binds to the EBS (egr-1-binding site) positioned at the 5′ region of EGR-1-regulated genes. In spite of both viruses belonging to the same genus, several lines of evidence, however, revealed a remarkable contrast between them as far as the roles played by the MEK/ERK/EGR-1 pathway in their biological cycles are concerned. Hence (i) the knocking-down of egr-1 by siRNA (small interfering RNA) proved that this transcription factor is of critical relevance for VV biology, since a decrease of about one log cycle in virus yield was verified, along with a small virus plaque phenotype, whereas the gene silencing did not have a detrimental effect on either CPV multiplication or viral plaque size; (ii) while both pharmacological and genetic inhibition of MEK/ERK resulted in a significant decrease in VV yield, both approaches had no impact on CPV multiplication; and (iii) CPV DNA replication was unaffected by pharmacological inhibition of MEK/ERK, but phosphorylation of MEK/ERK was dependent on CPV DNA replication, contrasting with a significant VV DNA inhibition and VV DNA replication-independence to maintain ERK1/2 phosphorylation, observed under the same conditions