Inés Nicieza

Variant Rabbit Hemorrhagic Disease Virus in Young Rabbits, Spain

Outbreaks of rabbit hemorrhagic disease have occurred recently in young rabbits on farms on the Iberian Peninsula where rabbits were previously vaccinated. Investigation identified a rabbit hemorrhagic disease virus variant genetically related to apathogenic rabbit caliciviruses. Improved antivirus strategies are needed to slow the spread of this pathogen.

Etiology of Sporadic Cases of Pediatric Acute Gastroenteritis in Asturias, Spain, and Genotyping and Characterization of Norovirus Strains Involved

ABSTRACT From November 2000 to October 2001, a reverse transcription-PCR using primers directed to the norovirus RNA polymerase coding region was included in a viral and bacterial routine screening to diagnose sporadic cases of acute gastroenteritis among children in Asturias, Spain. The role of noroviruses (8.6% of the positively diagnosed cases) as the cause of sporadic pediatric gastroenteritis was evaluated with respect to the detection rates of other gastroenteritis-associated viruses and bacteria. The results indicated that noroviruses were less common than rotaviruses (36.9%), Campylobacter spp. (28.8%), and Salmonella spp. (18.4%) but more frequent than astroviruses (4.3%), adenoviruses (3.8%), and Yersinia spp. (2.2%). Mixed infections involving noroviruses were rarely observed (0.5%). The presence of a norovirus-associated pediatric gastroenteritis peak in summer, as well as the complete absence of norovirus-associated cases in colder months, challenges the view that norovirus infections exclusively have wintertime seasonality. On the other hand, phylogenetic analysis of the amplified fragments showed that the norovirus strains responsible were closely related. A further study using the full-length capsid region showed that these strains could be included into genogroup II, Bristol/Lorsdale cluster, and were closely related to the 1995 and 1996 U.S. subset of strains associated with outbreaks recorded worldwide between 1995 and 1996.

Spread of new variant RHDV in domestic rabbits on the Iberian Peninsula

Rabbit hemorrhagic disease outbreaks in young rabbits have been recently observed in Spain. In this study we have tracked the spread of variant RHDV in samples collected from rabbit farms over a period of more than one year using RT-PCR and antigen-capture ELISA. The isolates were sequenced and compared to classic and variant RHDV strains and phylogenetic analyses were conducted. Mortalities have been observed in kits as young as 11 days. More than 50% of the dead rabbits had been previously vaccinated against RHDV using commercially available inactivated vaccines indicating a putative lack of protection against the variant RHDV. The large majority of the studied outbreaks (94.5%) in Spanish farms during 2012 were due to variant RHDV and only 3 out of the 55 farms were affected by classic RHDV. The data demonstrates that the variant RHDV has spread through a large number of Spanish provinces in a relatively short period of time, largely replacing the previously predominant G1 RHDV genotypes. Considering the lack of efficient vaccines against the variant RHDV strains strict disease control and greater vigilance measures should be put in place.

Bioactivity-guided Fractionation of Phyllanthus orbicularis and Identification of the Principal Anti HSV-2 Compounds

The antiherpes virus properties of Phyllanthus orbicularis Kunth, a Cuban‐endemic medicinal plant, have been reported previously but data on its phytochemical profile and identification of antiviral metabolites as well as their mechanisms of action are still lacking. In this work, a bioactivity‐guided phytochemical analysis was performed in order to isolate anti HSV‐2 compounds. P. orbicularis contained mainly phenolic acids derivatives and flavonoids. The antiviral effects were attributed to (−)‐epicatechin‐3‐O‐gallate (EC50 = 11.7 µg/mL), procyanidins B1 and B2 (EC50 = 32.8 µg/mL and 24.2 µg/mL, respectively) as well as oligomeric and polymeric procyanidins and their gallate derivatives. The antiviral mechanisms of the active P. orbicularis extracts and fractions were also investigated and the inhibition of several HSV‐2 early replication events and DNA synthesis were observed. This is the first study of extensive fractionation and phytochemical characterization of phenolic compounds from this species. Copyright

Apple Pomace, a By-Product from the Asturian Cider Industry, Inhibits Herpes Simplex Virus Types 1 and 2 In Vitro Replication: Study of Its Mechanisms of Action

The anti-herpes simplex virus type 1 and anti-herpes simplex virus type 2 effects of apple pomace, a by-product from the cider-processing industry, were investigated. The mechanisms of antiviral action were assessed using a battery of experiments targeting sequential steps in the viral replication cycle. The anti-herpetic mechanisms of apple pomaces included the inhibition of virus attachment to the cell surface and the arrest of virus entry and uncoating. Quercitrin and procyanidin B2 were found to play a crucial role in the antiviral activity.

Molecular characterisation of virulence graded field isolates of myxoma virus.

BackgroundMyxoma virus (MV) has been endemic in Europe since shortly after its deliberate release in France in 1952. While the emergence of more resistant hosts and more transmissible and attenuated virus is well documented, there have been relatively few studies focused on the sequence changes incurred by the virus as it has adapted to its new host. In order to identify regions of variability within the MV genome to be used for phylogenetic studies and to try to investigate causes of MV strain attenuation we have molecularly characterised nine strains of MV isolated in Spain between the years 1992 and 1995 from wide ranging geographic locations and which had been previously graded for virulence by experimental infection of rabbits.ResultsThe findings reported here show the analysis of 16 genomic regions accounting for approximately 10% of the viral genomes. Of the 20 genes analysed 5 (M034L, M069L, M071L, M130R and M135R) were identical in all strains and 1 (M122R) contained only a single point mutation in an individual strain. Four genes (M002L/R, M009L, M036L and M017L) showed insertions or deletions that led to disruption of the ORFs.ConclusionsThe findings presented here provide valuable tools for strain differentiation and phylogenetic studies of MV isolates and some clues as to the reasons for virus attenuation in the field.

Vaccine breaks: Outbreaks of myxomatosis on Spanish commercial rabbit farms

Despite the success of vaccination against myxoma virus, myxomatosis remains a problem on rabbit farms throughout Spain and Europe. In this study we set out to evaluate possible causes of myxoma virus (MYXV) vaccine failures addressing key issues with regard to pathogen, vaccine and vaccination strategies. This was done by genetically characterising MYXV field isolates from farm outbreaks, selecting a representative strain for which to assay its virulence and measuring the protective capability of a commercial vaccine against this strain. Finally, we compare methods (route) of vaccine administration under farm conditions and evaluate immune response in vaccinated rabbits. The data presented here show that the vaccine tested is capable of eliciting protection in rabbits that show high levels of seroconversion. However, the number of animals failing to seroconvert following subcutaneous vaccination may leave a large number of rabbits unprotected following vaccine administration. Successful vaccination requires the strict implication of workable, planned, on farm programs. Following this, analysis to confirm seroconversion rates may be advisable. Factors such as the wild rabbit reservoir, control of biting insects and good hygienic practices must be taken into consideration to prevent vaccine failures from occurring.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted. In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×108molecules/mL particles. The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease.

Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread

RHDVb has become the dominant RHDV on the Iberian Peninsula. A better understanding of its pathogenicity is required to aid control measures. Thus, the clinical course, humoral immune response, viraemia and kinetics of RHDV-N11 (a Spanish RHDVb isolate) infection in different tissues at both viral RNA and protein levels were studied in experimentally infected young and adult rabbits. The case fatality rate differed between the two age groups, with 21% of kits succumbing while no deaths were observed in adults. Fever and viremia were strongly associated with death, which occurred 48 h post infection (PI) too fast for an effective humoral immune response to be mounted. A significant effect on the number of viral RNA copies with regard to the variables age, tissue and time PI (p < 0.0001 in all cases) was detected. Histological lesions in infected rabbits were consistently more frequent and severe in liver and spleen and additionally intestine in kits, these tissues containing the highest levels of viral RNA and protein. Although no adults showed lesions or virus antigen in intestine, both kits and adults maintained steady viral RNA levels from days 1 to 7 PI in this organ. Analysis revealed the fecal route as the main dissemination route of RHDV-N11. Subclinically infected rabbits had detectable viral RNA in their faeces for up to seven days and thus may play an important role spreading the virus. This study allows a better understanding of the transmission of this virus and improvement of the control strategies for this disease.