No abstract

The seroprevalence of Neospora caninum infection was estimated from a sample of 889 cattle from 43 dairy herds in three counties in the Asturias region of Spain. The true prevalence of infection was estimated to be 30.6 per cent (95 per cent confidence interval (CI) 27.6 to 33.6). Seropositivity was associated with abortion during the previous year (odds ratio (OR)=3.31, P<0.001) and was slightly higher among purchased cattle (37.6 per cent), than among cattle raised on the farm (29.1 per cent) (P=0.078). Seropositive cows were more likely than seronegative cows to have had a seropositive dam (OR=2.3, P=0.011), suggesting that congenital transmission contributed to about 56 per cent of the infections. Herds with a true seroprevalence above 10 per cent had more dogs on the farm, than herds with a lower prevalence (P=0.032). The ORS relating abortion to seropositivity in individual herds ranged from 0.7 to 19, indicating that some herds experienced few abortions caused by N. caninum, while others experienced more abortions due to the organism. Overall, 38.7 per cent of the abortions were estimated to have been attributable to N. caninum.

Viruses rely on the cellular machinery of host cells to synthesize their proteins, and have developed different mechanisms enabling them to compete with cellular mRNAs for access to it. The genus Flavivirus is a large group of positive, single-stranded RNA viruses that includes several important human pathogens, such as West Nile, Dengue and Zika virus. The genome of flaviviruses bears a type 1 cap structure at its 5′ end, needed for the main translation initiation mechanism. Several members of the genus also use a cap-independent translation mechanism. The present work provides evidence that the WNV 5′ end also promotes a cap-independent translation initiation mechanism in mammalian and insect cells, reinforcing the hypothesis that this might be a general strategy of flaviviruses. In agreement with previous reports, we show that this mechanism depends on the presence of the viral genomic 3′ UTR. The results also show that the 3′ UTR of the WNV genome enhances translation of the cap-dependent mechanism. Interestingly, WNV 3′ UTR can be replaced by the 3′ UTR of other flaviviruses and the translation enhancing effect is maintained, suggesting a molecular mechanism that does not involve direct RNA-RNA interactions to be at work. In addition, the deletion of specific structural elements of the WNV 3′ UTR leads to increased cap-dependent and cap-independent translation. These findings suggest the 3′ UTR to be involved in a fine-tuned translation regulation mechanism.

Hepatitis C virus (HCV) translation is mediated by a highly conserved internal ribosome entry site (IRES), mainly located at the 5"untranslatable region (

Hepatitis C virus (HCV) replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE), located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80%) of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents.

The RNA genome of the hepatitis C virus (HCV) encodes a single open reading frame (ORF) containing numerous functional elements. Among these, the cis-acting replication element (CRE) at the 3′ end of the viral ORF, has become of increasing interest given its dual role as a viral translation repressor and replication enhancer. Long-range RNA-RNA contacts mediated by the CRE build the structural scaffold required for its proper functioning. The recruitment of different cellular factors, many related to the functioning of the translation machinery, might aid in the CRE-exerted downregulation of viral translation. The present data show that the CRE promotes a defect in polysome production, and hinders the assembly of the 80S complex, likely through the direct, high affinity recruitment of the 40S ribosomal subunit. This interaction involves the highly conserved 5BSL3.1 and 5BSL3.3 domains of the CRE, and is strictly dependent on RNA-protein contacts, particularly with the ribosomal proteins RPSA and RPS29. These observations support a model in which the CRE-mediated inhibition of viral translation is a multifactorial process defined by the establishment of long-range RNA-RNA interactions between the 5′ and 3′ ends of the viral genome, the sequestration of the 40S subunit by the CRE, and the subsequent stalling of polysome elongation at the 3′ end of the ORF, all governed by the highly stable hairpin domains 5BSL3.1 and 5BSL3.3. The present data thus suggest a new managerial role in HCV translation for these 5BSL3.1 and 5BSL3.3 domains.

Abstract:Background: Hepatitis C virus (HCV) contains a (+) ssRNA genome with highly conserved structural, functional RNA domains, many of them with unknown roles for the consecution of the viral cycle. Such genomic domains are candidate therapeutic targets. This study reports the functional characterization of a set of aptamers targeting the cis-acting replication element (CRE) of the HCV genome, an essential partner for viral replication and also involved in the regulation of protein synthesis. Methods: Forty-four aptamers were tested for their ability to interfere with viral RNA synthesis in a subgenomic replicon system. Some of the most efficient inhibitors were further evaluated for their potential to affect the recruitment of the HCV RNA-dependent RNA polymerase (NS5B) and the viral translation in cell culture. Results: Four aptamers emerged as potent inhibitors of HCV replication by direct interaction with functional RNA domains of the CRE, yielding a decrease in the HCV RNA levels higher than 90%. Concomitantly, one of them also induced a significant increase in viral translation (>50%). The three remaining aptamers efficiently competed with the binding of the NS5B protein to the CRE. Conclusions: Present findings confirm the potential of the CRE as an anti-HCV target and support the use of aptamers as molecular tools for investigating the functionality of RNA domains in viral genomes.

The 3 ′ ′ ′ ′ ′ X domain of hepatitis C virus has been reported to control viral replication and translation by modulating the exposure of a nucleotide segment involved in a distal base-pairing interaction with an upstream 5BSL3.2 domain. To study the mechanism of this molecular switch, we have analyzed the structure of 3 ′ ′ ′ ′ ′ X mutants that favor one of the two previously proposed conformations comprising either two or three stem-loops. Only the two-stem conformation was found to be stable and to allow the establishment of the distal contact with 5BSL3.2, and also the formation of 3 ′ ′ ′ ′ ′ X domain homodimers by means of a universally conserved palindromic sequence. Nucleotide changes disturbing the two-stem conformation resulted in poorer replication and translation levels, explaining the high degree of conservation detected for this sequence. The switch function attributed to the 3 ′ ′ ′ ′ ′ X domain does not occur as a result of a transition between two-and three-stem conformations, but likely through the sequestration of the 5BSL3.2-binding sequence by formation of 3 ′ ′ ′ ′ ′ X homodimers.