Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo.

BACKGROUND: To date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions. RESULTS: The genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates. CONCLUSIONS: Genome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.

Field and experimental data indicate that the eastern cottontail (Sylvilagus floridanus) is susceptible to infection with European brown hare syndrome (EBHS) virus and not with rabbit haemorrhagic disease (RHD) virus.

The eastern cottontail (Sylvilagus floridanus) is an American lagomorph. In 1966, it was introduced to Italy, where it is currently widespread. Its ecological niche is similar to those of native rabbits and hares and increasing overlap in distribution brings these species into ever closer contact. Therefore, cottontails are at risk of infection with the two lagoviruses endemically present in Italy: Rabbit Haemorrhagic Disease virus (RHDV) and European Brown Hare Syndrome Virus (EBHSV). To verify the susceptibility of Sylvilagus to these viruses, we analyzed 471 sera and 108 individuals from cottontail populations in 9 provinces of north-central Italy from 1999 to 2012. In total, 15-20% of the cottontails tested seropositive for EBHSV; most titres were low, but some were as high as 1/1280. All the cottontails virologically tested for RHDV and EBHSV were negative with the exception of one individual found dead with hares during a natural EBHS outbreak in December 2009. The cottontail and the hares showed typical EBHS lesions, and the EBHSV strain identified was the same in both species (99.9% identity). To experimentally confirm the diagnosis, we performed two trials in which we infected cottontails with both EBHSV and RHDV. One out of four cottontails infected with EBHSV died of an EBHS-like disease, and the three surviving animals developed high EBHSV antibody titres. In contrast, neither mortality nor seroconversion was detected after infection with RHDV. Taken together, these results suggest that Sylvilagus is susceptible to EBHSV infection, which occasionally evolves to EBHS-like disease; the eastern cottontail could therefore be considered a "spill over" or "dead end" host for EBHSV unless further evidence is found to confirm that it plays an active role in the epidemiology of EBHSV.

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare.

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres.

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.

The new French 2010 Rabbit Hemorrhagic Disease Virus causes an RHD-like disease in the Sardinian Cape hare (Lepus capensis mediterraneus).

Lagovirus is an emerging genus of Caliciviridae, which includes the Rabbit Hemorrhagic Disease Virus (RHDV) of rabbits and the European brown hare syndrome virus (EBHSV) of hares that cause lethal hepatitis. In 2010, a new RHDV related virus (RHDV2) with a unique genetic and antigenic profile and lower virulence was identified in France in rabbits. Here we report the identification of RHDV2 as the cause in Sardinia of several outbreaks of acute hepatitis in rabbits and Cape hare (Lepus capensis mediterraneus). This is the first account of a lagovirus that causes fatal hepatitis in both rabbits and hares.

Emergence of a new lagovirus related to Rabbit Haemorrhagic Disease Virus.

Since summer 2010, numerous cases of Rabbit Haemorrhagic Disease (RHD) have been reported in north-western France both in rabbitries, affecting RHD-vaccinated rabbits, and in wild populations. We demonstrate that the aetiological agent was a lagovirus phylogenetically distinct from other lagoviruses and which presents a unique antigenic profile. Experimental results show that the disease differs from RHD in terms of disease duration, mortality rates, higher occurrence of subacute/chronic forms and that partial cross-protection occurs between RHDV and the new RHDV variant, designated RHDV2. These data support the hypothesis that RHDV2 is a new member of the Lagovirus genus. A molecular epidemiology study detected RHDV2 in France a few months before the first recorded cases and revealed that one year after its discovery it had spread throughout the country and had almost replaced RHDV strains. RHDV2 was detected in continental Italy in June 2011, then four months later in Sardinia.

Status of Infectious Diseases in Free-Ranging European Brown Hares (Lepus europaeus) Found Dead between 2017 and 2020 in Schleswig-Holstein, Germany.

The European brown hare (Lepus europaeus) is a quite adaptable species, but populations have been decreasing for several decades in different countries, including Germany. To investigate infectious diseases as possible influences on observed population decline in the German federal state Schleswig-Holstein, 118 deceased free-ranging European brown hares were collected between 2017 and 2020 and underwent detailed postmortem examination with extended sampling. Infectious diseases were a major cause of death (34.7%). The number of juveniles found exceeded the adult ones. The main pathomorphological findings were hepatitis (32.8%), pneumonia (22.2%), nephritis (19.1%), liver necrosis (12.9%), and enteritis (40.7%). An unusual main finding was steatitis (20.9%) of unknown origin. Animals were mainly emaciated and showed high infection rates with Eimeria spp. (91.3%) and Trichostrongylus spp. (36.2%). European Brown Hare Syndrome Virus reached an epidemic status with few fatal infections (4.2%) and high seroprevalence (64.9%), whereas the prevalence of Rabbit Haemorrhagic Disease Virus 2 was very low (0.8%) in hares in Schleswig-Holstein. Pathogens such as Yersinia pseudotuberculosis (5.9%), Pasteurella multocida (0.8%), and Staphylococcus aureus (3.4%) only caused sporadic deaths. This study illustrates the wide distribution of various infectious pathogens with high mortality and even zoonotic potential. Infectious diseases need to be considered as an important influence on population dynamics in Schleswig-Holstein.

Pathological changes and viral antigen distribution in tissues of Iberian hare (Lepus granatensis) naturally infected with the emerging recombinant myxoma virus (ha-MYXV).

BACKGROUND: A cross-species jump was confirmed in 2018, when a novel recombinant myxoma virus (MYXV) (ha-MYXV) caused high mortality in Iberian hare (Lepus granatensis) in the Iberian Peninsula. METHOD: The aim of this study was to evaluate the main lesions, tissular distribution and target cells of ha-MYXV in Iberian hare. Gross postmortem examinations and histological and immunohistochemical studies to detect ha-MYXV were carried out in 28 animals that were confirmed as ha-MYXV positive by PCR. RESULTS: The main macroscopic lesions were bilateral blepharoconjunctivitis, epistaxis, intense congestion and oedema in several organs and some internal haemorrhages. Visible myxomas were not found. Histopathological examination revealed hyperplastic epidermis with predominant hyperkeratosis and myxoid matrix in the dermis. ha-MYXV-positive keratinocytes showed hydropic degeneration and cytoplasmic inclusion bodies. Alveolar oedema, interstitial pneumonia, dramatic lymphoid depletion in the spleen and necrosis in the liver and testis were observed. ha-MYXV was mainly detected in epithelial and myxoma cells in the skin, and also in macrophages, lymphocytes, fibroblasts and endothelial cells in several organs, as well as in hepatocytes and Leydig cells. LIMITATIONS: A non-homogeneous number of samples were included in all the animals. Future experimental studies with controlled variables are necessary. CONCLUSION: These findings correspond to an unusual form of myxomatosis, characterised by an acute or hyperacute presentation.

Pathomorphological Findings and Infectious Diseases in Selected European Brown Hare (Lepus europaeus Pallas, 1778) Populations from Schleswig-Holstein, Germany.

In the northernmost German federal state Schleswig-Holstein, populations of European brown hares (Lepus europaeus Pallas, 1778) show diverse densities and varying courses over the years. To examine differences in pathomorphological findings and infectious diseases as possible reasons for varying population dynamics, we assessed 155 hunted hares from three locations in Schleswig-Holstein from 2016 to 2020. We investigated the association of location, year, age, and sex of animals to certain pathomorphological findings and infectious diseases. Frequent pathomorphological findings were intestinal parasites (63.9%), hepatitis (55.5%), nephritis (31.0%), steatitis (23.2%), enteritis (13.5%), and pneumonia (5.2%). Body condition differed significantly between locations, and the prevalence of pneumonia was significantly higher in females. Enteritis was not detected in 2019, when much more juveniles were sampled. Hepatitis and nephritis occurred significantly more often in 2016 and among adults. Additionally, more adults showed hepatitis with concurrent serotitre for European brown hare syndrome virus (EBHSV), while intestinal parasitosis as well as high excretion rates of coccidia were more common in juveniles. Sampled animals showed high infection rates with Eimeria spp. (96.1%), Trichostrongylus spp. (52.0%), Graphidium strigosum (41.2%), and a high seroprevalence (90.9%) for EBHSV, without severe symptoms. This study revealed a low prevalence of infectious pathogens, but a high prevalence of chronic inflammations of unknown origin in the tested brown hare populations. Overall, our results indicate a rather minor importance of infectious diseases for observed population dynamics of analysed hare populations in Schleswig-Holstein.

West Nile virus: characterization and diagnostic applications of monoclonal antibodies.

BACKGROUND: Diagnosis of West Nile virus (WNV) infections is often difficult due to the extensive antigenic cross-reactivity among flaviviruses, especially in geographic regions where two or more of these viruses are present causing sequential infections. The purpose of this study was to characterize a panel of monoclonal antibodies (MAbs) produced against WNV to verify their applicability in WNV diagnosis and in mapping epitope targets of neutralizing MAbs. METHODS: Six MAbs were produced and characterized by isotyping, virus-neutralization, western blotting and MAb-epitope competition. The MAb reactivity against various WNVs belonging to lineage 1 and 2 and other related flaviviruses was also evaluated. The molecular basis of epitopes recognized by neutralizing MAbs was defined through the selection and sequencing of MAb escape mutants. Competitive binding assays between MAbs and experimental equine and chicken sera were designed to identify specific MAb reaction to epitopes with high immunogenicity. RESULTS: All MAbs showed stronger reactivity with all WNVs tested and good competition for antigen binding in ELISA tests with WNV-positive equine and chicken sera. Four MAbs (3B2, 3D6, 4D3, 1C3) resulted specific for WNV, while two MAbs (2A8, 4G9) showed cross-reaction with Usutu virus. Three MAbs (3B2, 3D6, 4D3) showed neutralizing activity. Sequence analysis of 3B2 and 3D6 escape mutants showed an amino acid change at E307 (Lys → Glu) in the E protein gene, whereas 4D3 variants identified mutations encoding amino acid changed at E276 (Ser → Ile) or E278 (Thr → Ile). 3B2 and 3D6 mapped to a region on the lateral surface of domain III of E protein, which is known to be a specific and strong neutralizing epitope for WNV, while MAb 4D3 recognized a novel specific neutralizing epitope on domain II of E protein that has not previously been described with WNV MAbs. CONCLUSIONS: MAbs generated in this study can be applied to various analytical methods for virological and serological WNV diagnosis. A novel WNV-specific and neutralizing MAb (4D3) directed against the unknown epitope on domain II of E protein can be useful to better understand the role of E protein epitopes involved in the mechanism of WNV neutralization.

Changes in European wild rabbit population dynamics and the epidemiology of rabbit haemorrhagic disease in response to artificially increased viral transmission.

European wild rabbit (Oryctolagus cuniculus) populations are severely affected by rabbit haemorrhagic disease (RHD), currently aggravated by the spread of the new lagovirus serotype RHDV2 that replaced the classical RHDV strains (RHDV/RHDVa). This virus causes high mortality in both adult and young rabbits and to date, there is no management tool to effectively reduce its impact on wild rabbit populations. This hinders the success of common strategies, such as habitat management or restocking, in areas where rabbits are native. However, the present study, conducted on enclosed wild rabbit populations, showed that spreading RHDV2 on baits during breeding periods induced infection of young rabbits, reducing mortality rates, presumably due to maternal antibody protection. This reduced the young rabbit mortality hazard by a third, and more juvenile rabbits immune to RHDV2 were recruited into the adult breeding population. Young rabbits from populations in which the force of infection of RHDV2 was increased, however, exhibited considerably higher susceptibility to infection by RHDV than those from non-treated control populations. Since co-circulation of classical RHDVs was ruled out, differences in the type and degree of immunization, the level of cross-protection and/or other unknown factors, such as the circulation of undetected non-pathogenic lagoviruses, arose as possible explanations. This meant that although the present study demonstrated the possibility of successfully modulating the impact of RHD in wild populations, the epidemiological complexity of the situation where several lagoviruses circulate requires additional research to determine final applicability of the proposed method.

Retrospective serological and molecular survey of myxoma or antigenically related virus in the Iberian hare, Lepus granatensis.

The 2018 outbreak of myxomatosis in the Iberian hare (Lepus granatensis) has been hypothesized to originate from a species jump of the rabbit-associated myxoma virus (MYXV), after natural recombination with an unknown poxvirus. Iberian hares were long considered resistant to myxomatosis as no prior outbreaks were reported. To provide insights into the emergence of this recombinant virus (ha-MYXV), we investigated serum samples from 451 Iberian hares collected over two time periods almost two decades apart, 1994-1999 and 2017-2019 for the presence of antibodies and MYXV-DNA. First, we screened all serum samples using a rabbit commercial indirect ELISA (iELISA) and then tested a subset of these samples in parallel using indirect immunofluorescence test (IFT), competitive ELISA (cELISA) and qPCR targeting M000.5L/R gene conserved in MYXV and ha-MYXV. The cut-off of iELISA relative index 10 = 6.1 was selected from a semiparametric finite mixture analysis aiming to minimize the probability of false positive results. Overall, MYXV related-antibodies were detected in 57 hares (12.6%) including 38 apparently healthy hares (n = 10, sampled in 1994-1999, none MYXV-DNA positive, and n = 28 sampled in 2017-2019 of which four were also ha-MYXV-DNA positive) and 19 found-dead and ha-MYXV-DNA-positive sampled in 2018-2019. Interestingly, four seronegative hares sampled in 1997 were MYXV-DNA positive by qPCR, the result being confirmed by sequencing of three of them. For the Iberian hares hunted or live trapped (both apparently health), seroprevalence was significantly higher in 2017-2019 (13.0%, CI95% 9.2-18.2%) than in 1994-1999 (5.4%, CI95% 3.0-9.6%) (p = .009). Within the second period, seroprevalence was significantly higher in 2019 compared to 2017 (24.7 vs 1.7% considering all the sample, p = .007), and lower during the winter than the autumn (p < .001). While our molecular and serological results show that Iberian hares have been in contact with MYXV or an antigenically similar virus at least since 1996, they also show an increase in seroprevalence in 2018-2019. The remote contact with MYXV may have occurred with strains that circulated in rabbits, or with unnoticed strains already circulating in Iberian hare populations. This work strongly suggests the infection of Iberian hares with MYXV or an antigenically related virus, at least 20 years before the severe virus outbreaks were registered in 2018.

Early circulation of rabbit haemorrhagic disease virus type 2 in domestic and wild lagomorphs in southern California, USA (2020-2021).

Rabbit haemorrhagic disease virus type 2 (RHDV2) causes a severe systemic disease with hepatic necrosis. Differently from classic RHDV, which affects only European rabbits (Oryctolagus cuniculus), RHDV2 can affect many leporid species, including hares (Lepus spp.) and cottontail rabbits (Sylvilagus spp.). RHDV2 emerged in Europe in 2010 and spread worldwide. During the last 5 years, there have been multiple outbreaks in North America since the first known event in 2016 in Quebec, Canada, including several detections in British Columbia, Canada, between 2018 and 2019, Washington State and Ohio, USA, in 2018 and 2019, and New York, USA, in 2020. However, the most widespread outbreak commenced in March 2020 in the southwestern USA and Mexico. In California, RHDV2 spread widely across several southern counties between 2020 and 2021, and the aim of this study was to report and characterize these early events of viral incursion and circulation within the state. Domestic and wild lagomorphs (n = 81) collected between August 2020 and February 2021 in California with a suspicion of RHDV2 infection were tested by reverse transcription quantitative real-time PCR on the liver, and histology and immunohistochemistry for pan-lagovirus were performed on liver sections. In addition, whole genome sequencing from 12 cases was performed. During this period, 33/81 lagomorphs including 24/59 domestic rabbits (O. cuniculus), 3/16 desert cottontail rabbits (Sylvilagus audubonii), and 6/6 black-tailed jackrabbits (Lepus californicus) tested positive. All RHDV2-positive animals had hepatic necrosis typical of pathogenic lagovirus infection, and the antigen was detected in sections from individuals of the three species. The 12 California sequences were closely related (98.9%-99.95%) to each other, and also very similar (99.0%-99.4%) to sequences obtained in other southwestern states during the 2020-2021 outbreak; however, they were less similar to strains obtained in New York in 2020 (96.7%-96.9%) and Quebec in 2016 (92.4%-92.6%), suggesting that those events could be related to different viral incursions. The California sequences were more similar (98.6%-98.7%) to a strain collected in British Columbia in 2018, which suggests that that event could have been related to the 2020 outbreak in the southwestern USA.

Comparative susceptibility of eastern cottontails and New Zealand white rabbits to classical rabbit haemorrhagic disease virus (RHDV) and RHDV2.

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild-caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP-GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post-inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus.

Analysis of gene expression in white blood cells of cattle orally challenged with bovine amyloidotic spongiform encephalopathy.

Bovine amyloidotic spongiform encephalopathy (BASE) is one of the recently discovered atypical forms of BSE, which is transmissible to primates, and may be the bovine equivalent of sporadic Creutzfeldt-Jacob disease (CJD) in humans. Although it is transmissible, it is unknown whether BASE is acquired through infection or arises spontaneously. In the present study, the gene expression of white blood cells (WBCs) from 5 cattle at 1 yr after oral BASE challenge was compared with negative controls using a custom microarray containing 43,768 unique gene probes. In total, 56 genes were found to be differentially expressed between BASE and control animals with a log fold change of 2 or greater. Of these, 39 were upregulated in BASE animals, while 17 were downregulated. The majority of these genes are related to immune function. In particular, BASE animals appeared to have significantly modified expression of genes linked to T- and B-cell development and activation, and to inflammatory responses. The potential impacts of these gene expression changes are described.

Widespread occurrence of the non-pathogenic hare calicivirus (HaCV Lagovirus GII.2) in captive-reared and free-living wild hares in Europe.

The Lagovirus genus comprises both pathogenic viruses as European brown hare syndrome virus (EBHSV- GII.1) and rabbit hemorrhagic disease viruses (RHDV-GI.1 and RHDV2-GI.2), that principally infect European brown hares (Lepus europeaus) and European rabbits (Oryctolagus cuniculus), respectively, causing severe necrotic hepatitis, spleen enlargement and disseminated haemorrhage. This genus includes also non-pathogenic agents, such as rabbit calicivirus (RCV-E1 - GI.3) and the non-pathogenic hare Lagovirus, provisionally named hare calicivirus (HaCV - GII.2). The latter had been identified for the first time in 2012 in the gut contents and faeces of healthy young hares raised in a breeding farm. In this study, we further investigated the presence of HaCV by testing the intestinal tract of 621 wild hares collected between 2010 and 2018 in Northern and Central Italy, and in 2011 in Austria, Germany and Spain. These wild hares were found dead for causes other than EBHS or were healthy hares shot during the hunting season. Forty-three out of 322 hare samples from Italy and 14 out of 299 samples from Austria and Germany were positive for HaCV-GII.2 by RT-PCR using universal primers for lagoviruses and primers specific for HaCV. Sequence analysis of the full capsid protein gene conducted on 12 strains representative of different years and locations indicated that these viruses belong to the same, single cluster as the prototype strain initially identified at the hares' farm (HaCV_Bs12_1). The relatively high level of genetic variation (88% nt identity) within this cluster suggests HaCVs may have been circulating widely in Europe for some time.

Intraspecies transmission of BASE induces clinical dullness and amyotrophic changes.

The disease phenotype of bovine spongiform encephalopathy (BSE) and the molecular/ biological properties of its prion strain, including the host range and the characteristics of BSE-related disorders, have been extensively studied since its discovery in 1986. In recent years, systematic testing of the brains of cattle coming to slaughter resulted in the identification of at least two atypical forms of BSE. These emerging disorders are characterized by novel conformers of the bovine pathological prion protein (PrP(TSE)), named high-type (BSE-H) and low-type (BSE-L). We recently reported two Italian atypical cases with a PrP(TSE) type identical to BSE-L, pathologically characterized by PrP amyloid plaques and known as bovine amyloidotic spongiform encephalopathy (BASE). Several lines of evidence suggest that BASE is highly virulent and easily transmissible to a wide host range. Experimental transmission to transgenic mice overexpressing bovine PrP (Tgbov XV) suggested that BASE is caused by a prion strain distinct from the BSE isolate. In the present study, we experimentally infected Friesian and Alpine brown cattle with Italian BSE and BASE isolates via the intracerebral route. BASE-infected cattle developed amyotrophic changes accompanied by mental dullness. The molecular and neuropathological profiles, including PrP deposition pattern, closely matched those observed in the original cases. This study provides clear evidence of BASE as a distinct prion isolate and discloses a novel disease phenotype in cattle.

Retrospective serological analysis reveals presence of the emerging lagovirus RHDV2 in Australia in wild rabbits at least five months prior to its first detection.

The lagovirus rabbit haemorrhagic disease virus (RHDV) has been circulating in Australia since the mid-1990s when it was released to control overabundant rabbit populations. In recent years, the viral diversity of different RHDVs in Australia has increased, and currently four different types of RHDV are known to be circulating. To allow for ongoing epidemiological studies and impact assessments of these viruses on Australian wild rabbit populations, it is essential that serological tools are updated. To this end, reference sera were produced against all four virulent RHDVs (RHDV, RHDV2 and two different strains of RHDVa) known to be present in Australia and tested in a series of available immunological assays originally developed for the prototype RHDV, to assess patterns of cross-reactivity and the usefulness of these assays to detect lagovirus antibodies, either in a generic or specific manner. Enzyme-linked immunosorbent assays (ELISAs) developed to detect antibody isotypes IgM, IgA and IgG were sufficiently cross-reactive to detect antibodies raised against all four virulent lagoviruses. For the more specific detection of antibodies to the antigenically more different RHDV2, a competition ELISA was adapted using RHDV2-specific monoclonal antibodies in combination with Australian viral antigen. Archival serum banks from a long-term rabbit monitoring site where rabbits were sampled quarterly over a period of 6 years were re-screened using this assay and revealed serological evidence for the arrival of RHDV2 in this population at least 5 months prior to its initial detection in Australia in a dead rabbit in May 2015. The serological methods and reference reagents described here will provide valuable tools to study presence, prevalence and impact of RHDV2 on Australian rabbit populations; however, the discrimination of different antigenic variants of RHDVs as well as mixed infections at the serological level remains challenging.

Characterization of the IgA response to PRRS virus in pig oral fluids.

Porcine Reproductive and Respiratory Syndrome (PRRS) is a complex model of host/virus relationship. Disease control measures often includes "acclimatization", i.e. the exposure of PRRS-naïve gilts and sows to PRRSV-infected pigs and premises before the breeding period. In this respect, we had repeatedly observed an association between PRRSV-specific IgA responses in oral fluids (OF) of gilts and block of PRRSV spread. Therefore, we set out to investigate in vitro the inhibition of PRRSV replication by OF samples with different titers of PRRSV-specific IgA and IgG antibody, using Real-time RT PCR. PRRSV yield reduction in monocyte-derived macrophages was associated with the IgA content in OF samples, whereas the IgG-rich samples were sometimes associated with antibody-dependent enhancement (ADE) of replication. Accordingly, we could discriminate between ADE-positive and ADE-negative PRRSV strains. Next, we separated Ig isotypes in OF samples of PRRSV-infected pigs by means of protein A and size exclusion chromatography. The above results were confirmed by using separated Ig isotypes. Both dimeric and monomeric IgA were associated with the strongest reduction of PRRSV replication. The treatment of pig macrophages with separated OF antibodies before PRRSV infection was also associated with PRRSV yield reduction, along with clear changes of both CD163 and CD169 surface expression. Our results point at a role of mucosal IgA in the control of PRRSV replication by extra- and/or intracellular interaction with PRRSV, as well as by induction of signals leading to a reduced susceptibility of macrophages to PRRSV infection.

Characterization of the Maternally Derived Antibody Immunity against Rhdv-2 after Administration in Breeding Does of an Inactivated Vaccine.

Inactivated strain-specific vaccines have been successfully used to control rabbit haemorrhagic disease (RHD) caused by RHDV-2 in the rabbit industry. It is unknown whether and how vaccination of breeding does contributed to protect the population of young susceptible rabbit kits. The present study investigates whether the immunity against RHDV-2 produced by vaccination of breeding does is transmitted to their progeny and its dynamic once inherited by kits. For this purpose, New Zealand female rabbits of 8-9 weeks of age were allocated into 2 groups of 40 subjects each and bred during 6 reproductive cycles. The first experimental group was vaccinated with a commercially available inactivated vaccine against RHDV-2 whereas the second group was inoculated with PBS. Moreover, the present study was also meant to identify the mechanisms of transmission of that maternal immunity. For this reason, rabbit kits of vaccinated and non-vaccinated breeding does were cross-fostered before milk uptake. The RHDV-2 antibody response was monitored in the blood serum of breeding does and of their kits by competition ELISA (cELISA) and solid-phase ELISA (spELISA). Since it has been clearly demonstrated that cELISA positive rabbits are protected from RHD, we avoided the resorting of the challenge of the kits with RHDV-2. Results showed that RHDV-2 antibodies were inherited by kits up to one year from vaccination of breeding does. Once inherited, the maternally derived antibody response against RHDV-2 lasted at least until 28 days of life. Finally, the study also elucidated that the major contribution to the maternal derived immunity against RHDV-2 in kits was provided during gestation and probably transmitted through transplacental mechanisms although lactation provided a little contribution to it. The present study contributed to elucidate the characteristics of the maternal antibody immunity produced by vaccination and its mechanisms of transmission.