Virulence evaluation of Israeli Marek's disease virus isolates from commercial poultry using their meq gene sequence.

Fifty-seven Gallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019) from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysis exemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strains kept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

Marek's disease in genetically susceptible Cochin chickens in Italy: a case report.

The present study investigates an outbreak of classical Marek's disease (MD) in backyard Cochin chickens reared for hobby in Italy. Examined chickens showed spastic paralysis of the legs and at necropsy, enlargement and discoloration of the peripheral nerves and plexuses that matched microscopic A­ and B­ type MD lesions. Molecular analysis of the meq gene of the detected Gallid alphaherpesvirus 2 (GaHV­2) strain, showed typical markers of low virulence and the strain shared the entire meq gene sequence with strains circulating in Italian backyard chickens. Furthermore, the haplotype B19 of the major histocompatibility complex (MHC) was defined in the affected chickens, showing that the birds possessed a genetic profile of high susceptibility to MD, allowing the appearance of a classical nervous clinical form after infection with an apparently low pathogenicity GaHV­2 strain. Trade of live ornamental purebred chickens occurs frequently between hobby farmers and biosecurity practices, such as quarantine periods, should be applied to avoid the introduction of infected animals. Veterinarians should raise awareness of this issue and promote the use of vaccines against MD.

The Complexity of Swine Caliciviruses. A Mini Review on Genomic Diversity, Infection Diagnostics, World Prevalence and Pathogenicity.

Caliciviruses are single stranded RNA viruses, non-enveloped structurally, that are implicated in the non-bacterial gastroenteritis in various mammal species. Particularly in swine, viral gastroenteritis represents a major problem worldwide, responsible for significant economic losses for the pig industry. Among the wide range of viruses that are the proven or suspected etiological agents of gastroenteritis, the pathogenicity of the members of Caliciviridae family is among the less well understood. In this context, the present review presents and discusses the current knowledge of two genera belonging to this family, namely the Norovirus and the Sapovirus, in relation to swine. Aspects such as pathogenicity, clinical evidence, symptoms, epidemiology and worldwide prevalence, genomic diversity, identification tools as well as interchanging hosts are not only reviewed but also critically evaluated. Generally, although often asymptomatic in pigs, the prevalence of those microbes in pig farms exhibits a worldwide substantial increasing trend. It should be mentioned, however, that the factors influencing the symptomatology of these viruses are still far from well established. Interestingly, both these viruses are also characterized by high genetic diversity. These high levels of molecular diversity in Caliciviridae family are more likely a result of recombination rather than evolutionary or selective adaptation via mutational steps. Thus, molecular markers for their detection are mostly based on conserved regions such as the RdRp region. Finally, it should be emphasized that Norovirus and the Sapovirus may also infect other domestic, farm and wild animals, including humans, and therefore their surveillance and clarification role in diseases such as diarrhea is a matter of public health importance as well.

First report of canine Astrovirus and Sapovirus in Greece, hosting both asymptomatic and gastroenteritis symptomatic dogs.

BACKGROUND: Astrovirus, Norovirus and Sapovirus are widely distributed viruses in humans and animals worldwide. They have frequently been associated with disease, mainly of gastroenteric nature. In dogs, these viruses have been detected both in symptomatic and asymptomatic animals, mainly of young age. METHODS: In the present epidemiologic study, we investigated the presence of canine Astrovirus (CAstV), canine Norovirus (canine NoV) and canine Sapovirus (Canine SaV) in saliva and stools of 201 domestic dogs originating from throughout Greece, based on two different molecular methods, i.e. conventional and SYBR-Green Real-time RT-PCR. The samples derived from young and adult asymptomatic and symptomatic animals. CAstV was detected in 15/201 (7.5%) and 29/201 (15%) of the examined dogs using conventional RT-PCR and SYBR-Green Real time RT-PCR, respectively. RESULTS: The prevalence of the virus was higher at healthy dogs, with a slight discrepancy of the two methods on the aspect of age (67% young dogs with the method of conventional RT-PCR, versus 52% adult positive dogs with the method of SYBR-Green Real-time RT-PCR). Canine SaV was detected in 52/201 (23%) of the dogs (mainly young and asymptomatic), with the method of SYBR-Green Real-time RT-PCR only, while canine NoV was not detected in any sample with either of the two methods applied. Sequencing of the CAstV positive samples resulted in the acquisition of one CAstV sequence. Phylogenetic analysis confirmed the results, clustering the CAstV sequence with homologous canine hosting sequences from other countries. CONCLUSIONS: CAstV and Canine SaV were proved to circulate in Greek dogs. SYBR-Green Real time RT-PCR showed greater sensitivity in the detection of these viruses. Additionally, we were able to specify the CAstV strain that circulates in Greece, through phylogenetic analysis. To our knowledge, this is the first epidemiological study of CAstV and canine SaV in dogs in Greece, as well as the first time detected in dogs from Greece.

Epidemiology of Astrovirus, Norovirus and Sapovirus in Greek pig farms indicates high prevalence of Mamastrovirus suggesting the potential need for systematic surveillance.

BACKROUND: Astrovirus, Norovirus and Sapovirus exhibit a wide distribution in swine pig herds worldwide. However, the association of porcine Astrovirus (PAstV), porcine Norovirus (PoNoV) and porcine Sapovirus (PoSaV) with disease in pigs remains uncertain. In this study, we investigated the prevalence of PAstV, PoNoV and PoSaV in Greek pig farms using both conventional RT-PCR and SYBR-Green Real-time RT-PCR in an effort to compare the sensitivity of the two methods. We examined 1400 stool samples of asymptomatic pigs originating from 28 swine farms throughout Greece in pools of five. RESULTS: PAstV was detected in all 28 swine farms examined, with an overall prevalence of 267/280 positive pools (95.4%). Porcine Caliciviruses prevalence was found at 36 and 57 out of the 280 examined samples, by the conventional and SYBR-Green Real time RT-PCR, respectively. Sequencing and phylogenetic analysis of the positive samples revealed that the detected PAstV sequences are clustered within PAstV1, 3 and 4 lineages, with PAstV3 being the predominant haplotype (91.2%). Interestingly, sequencing of the Calicivirus positive samples demonstrated the presence of non-target viruses, i.e. Sapovirus, Kobuvirus and Sapelovirus sequences and one sequence highly similar to bat Astrovirus, while no Norovirus sequence was detected. CONCLUSIONS: The high prevalence of PAstV in Greek pig farms poses a necessity for further investigation of the pathogenicity of this virus and its inclusion in surveillance programs in case that it proves to be important. To our knowledge, this is the first epidemiological study of these viruses in pig farms in Greece.

A two-branched upgrade to demonstrate ITV transmission by blood-sucking insects.

The enveloped flavivirus Israel turkey meningoencephalitis virus (ITV) causes a neuroparalytic disease in adult turkeys leading to morbidity and mortality. This study reevaluates the role of blood-sucking insects in the transmission of ITV. We demonstrate the crucial importance of two factors in detecting viruses carried by blood-sucking insects: first, enhanced molecular detection of ITV in insects by nested qRT-PCR and second, collection and maintenance of live insects until their molecular examination. These upgrades allowed overcoming the small virus quantities contained in the insects and detecting ITV for the first time in field-collected Culex pipiens.

Israeli Rousettus aegyptiacus Pox Virus (IsrRAPXV) Infection in Juvenile Egyptian Fruit Bat (Rousettus aegyptiacus): Clinical Findings and Molecular Detection.

During 2019, five carcasses of juvenile Egyptian fruit bats (Rousettus aegyptiacus) were submitted to the Kimron Veterinary Institute. These bats exhibited typical poxvirus like lesion plaques of different sizes on the skin, abdomen and the ventral side of the wings. Clinical and histopathological findings suggested a poxvirus infection. Infectious virus was isolated from skin swabs, skin tissue and tongue of the dead bats and was further confirmed to be a Poxvirus by molecular diagnosis using PCR with pan-chordopoxviruses primers. All the dead bats were found positive for two Poxvirus genes encoding a metalloproteinase and DNA dependent DNA polymerase. In this study, a novel real time quantitative PCR (qPCR) assay was established to further confirmed the presence of specific poxvirus viral DNA in all pathologically tested tissues. Moreover, according to sequence analysis, the virus was found to be highly similar to the recently discovered Israeli Rousettus aegyptiacus Pox Virus (IsrRAPXV).

Out of Sight, but Not Out of Mind: Aspects of the Avian Oncogenic Herpesvirus, Marek's Disease Virus.

Marek's disease virus is an economically important avian herpesvirus that causes tumors and immunosuppression in chickens and turkeys. The virus, disease, and vaccines have been known for more than 50 years, but as knowledge gaps still exists, intensive research is still ongoing. The understanding of MDV complexity can provide scientific insight in topics that cannot be experimented in humans, providing a unique model that is dually useful for the benefit of the poultry industry and for studying general herpesvirology. The present review presents the following topics: the MDV biology, the vaccine's and virulent virus' peculiar presence in feathers, protection by vaccination. In addition, two relatively behind the scenes topics are reviewed; first, the meq MDV oncogene and its recent implication in molecular epidemiology and in the MDV virulence determination, and second, the functionality of conformational epitopes of the MDV immunodominant protein, glycoprotein B. Our studies were particular, as they were the only ones describing three-dimensional MDV gB oligomers. MDV gB (glycoprotein B) continuous and discontinuous epitopes were shown to possess distinctive neutralization activities. In contrast, the significance of oligomerization of the viral membrane proteins for the creation of discontinuous epitopes in other herpesviruses was explored extensively.

A novel poxvirus isolated from an Egyptian fruit bat in Israel.

An Egyptian fruit bat (Rousettus aegyptiacus) from the Zoological Gardens, at Tel Aviv, Israel, showed pox-like clinical signs including vesicular and nodular skin lesions on the wings. Cell culture isolation, histopathology, electron microscopy and molecular analysis, revealed the presence of a novel bat poxvirus. Future research is needed to determine whether this virus can affect human health.

Marek's disease viruses circulating in commercial poultry in Italy in the years 2015-2018 are closely related by their meq gene phylogeny.

Marek's disease (MD) is a lymphoproliferative disease important to the poultry industry worldwide; it is caused by Gallid alphaherpesvirus 2 (GaHV-2). The virulence of GaHV-2 isolates has shifted over the years from mild to virulent, very virulent and very virulent +. Nowadays the disease is controlled by vaccination, but field strains of increased virulence are emerging worldwide. Economic losses due to MD are mostly associated with its acute form, characterized by visceral lymphomas. The present study aimed to molecularly classify a group of 13 GaHV-2 strains detected in vaccinated Italian commercial chicken flocks during acute MD outbreaks, and to scrutinize the ability of predicting GaHV-2 virulence, according to the meq gene sequence. The full-length meq genes were amplified, and the obtained amino acid (aa) sequences were analysed, focusing mainly on the number of stretches of four proline molecules (PPPP) within the transactivation domain. Phylogenetic analysis was carried out with the Maximum Likelihood method using the obtained aa sequences, and the sequences of Italian strains detected in backyard flocks and of selected strains retrieved from GenBank. All the analysed strains showed 100% sequence identity in the meq gene, which encodes a Meq protein of 339 aa. The Meq protein includes four PPPP motifs in the transactivation domain and an interruption of a PPPP motif due to a proline-to-serine substitution at position 218. These features are typically encountered in highly virulent isolates. Phylogenetic analysis revealed that the analysed strains belonged to a cluster that includes high-virulence GaHV-2 strains detected in Italian backyard flocks and a hypervirulent Polish strain. Our results support the hypothesis that the virulence of field isolates can be suggested by meq aa sequence analysis.

Molecular characterization of a Marek's disease virus strain detected in tumour-bearing turkeys.

Marek's disease (MD) is a lymphoproliferative disease caused by Gallid alphaherpesvirus 2 (GaHV-2), which primarily affects chickens. However, the virus is also able to induce tumours in turkeys, albeit less frequently than in chickens. This study reports the molecular characterization of a GaHV-2 strain detected in a flock of Italian meat-type turkeys exhibiting visceral lymphomas. Sequencing and phylogenetic analysis of the meq gene revealed that the turkey GaHV-2 has molecular features of high virulence and genetic similarity with GaHV-2 strains recently detected in Italian commercial and backyard chickens. GaHV-2 is ubiquitous among chickens despite vaccination, and chicken-to-turkey transmission is hypothesized due to the presence of broilers in neighbouring pens.RESEARCH HIGHLIGHTS A GaHV-2 strain from Italian turkeys was molecularly characterized.The turkey strain presented molecular characteristics of high virulence in its meq gene.The turkey strain was closely related to previously detected chicken strains.

Biotic concerns in generating molecular diagnosis matrixes for 4 avian viruses with emphasis on Marek's disease virus.

The great advance in the field of diagnosis of avian viruses is reflecting the highly sophisticated molecular assays of the human and general virology in providing highly sensitive and fast methods of diagnosis. The present review will discuss the biotic factors and the complexities that became evident with the evolution of the novel molecular diagnostic assays with emphasis on 4 avian viruses, chicken anemia, infectious laryngotracheitis, turkey meningoencephalitis, but mainly on Marek's disease virus. To create a biologically meaningful diagnosis, attention should be dedicated to various biotic factors and not only of the diagnostic assay. Included among the important factors are, (a) the sample examined and the sampling strategy, (b) the outcomes of the pathogen amplification ex vivo, (c) the sampling time and its reflection on the disease diagnosis, (d) the impact of simultaneous multiple virus-infections regarding the ability to demonstrate all pathogens and inter- and intra-interactions between the pathogens. A concerted consideration of the relevant factors and the use of advanced molecular diagnostic assay would yield biologically significant diagnosis in real-time that would beneficiate the poultry industry.

Study of the underlying mechanisms and consequences of pathogenicity differences between two in vitro selected G1-H9N2 clones originating from a single isolate.

The G1-H9N2 avian influenza virus (AIV) has caused significant economic losses in the commercial poultry industry due to reduced egg production and increased mortality. The field observations have shown that H9N2 viruses circulate and naturally mix with other pathogens and these simultaneous infections can exacerbate disease. To avoid an incorrect virus characterization, due to co-infection, isolates were purified by in vitro plaque assays. Two plaque purified G1-H9N2 clones, selected on different cell types, named MDCK-and CEF-clone in regards to the cell culture used, were studied in vivo, revealing two different virulence phenotypes. Subsequently, the underlying mechanisms were studied. Specifically, the phenotypical outcome of SPF bird infection by the two clones resulted in completely different clinical outcomes. These differences in clinical outcome were used to study the factors behind this output in more detail. Further studies demonstrated that the more severe disease outcome associated with the MDCK-clone involves a strong induction of pro-inflammatory cytokines and a lack of type I interferon production, whereas the mild disease outcome associated with the CEF-clone is related to a greater antiviral cytokine response. The immunosuppressive effect of the MDCK-clone on splenocytes was further demonstrated via ChIFN-γ lack production after ex vivo mitogenic stimulation. Genome sequencing of the two clones identified only four amino acid differences including three in the HA sequence (HA-E198A, HA-R234L, HA-E502D-H9 numbering) and one in the NA sequence (NA-V33M). In the present study, valuable insights on the mechanisms responsible for AI pathogenicity and molecular mechanisms of H9N2 infections in chicken were obtained while highlighting the impact of the cells viruses are grown on their virulence.

A G1-lineage H9N2 virus with oviduct tropism causes chronic pathological changes in the infundibulum and a long-lasting drop in egg production.

Since 1997, G1-lineage H9N2 avian influenza viruses have been circulating in Asia and later on in the Middle East, and they have been associated to mild respiratory disease, drops in egg production and moderate mortality in chickens, in particular in the presence of concurrent infections. In this study, we investigated the importance of the G1-lineage H9N2 A/chicken/Israel/1163/2011 virus as a primary pathogen in layers, analyzing its tropism and binding affinity for the oviduct tissues, and investigating the long-term impact on egg production. Besides causing a mild respiratory infection, the virus replicated in the oviduct of 60% of the hens causing different degrees of salpingitis throughout the organ, in particular at the level of the infundibulum, where the detection of the virus was associated with severe heterophilic infiltrate, and necrosis of the epithelium. Binding affinity assays confirmed that the infundibulum was the most receptive region of the oviduct. The drop in egg production was at its peek at 2 weeks post-infection (pi) (60% decrease) and continued up to 80 days pi (35% decrease). On day 80 pi, non-laying birds showed egg yolk peritonitis, and histopathological analyses described profound alteration of the infundibulum architecture, duct ectasia and thinning of the epithelium, while the rest of the oviduct and ovary appeared normal. Our results show that this H9N2 virus is a primary pathogen in layer hens, and that its replication in the infundibulum is responsible for acute and chronic lesions that limits the effective functionality of the oviduct, compromising the commercial life of birds.

Middle East respiratory syndrome coronavirus specific antibodies in naturally exposed Israeli llamas, alpacas and camels.

Thus far, no human MERS-CoV infections have been reported from Israel. Evidence for the circulation of MERS-CoV in dromedaries has been reported from almost all the countries of the Middle East, except Israel. Therefore, we aimed to analyze MERS-CoV infection in Israeli camelids, sampled between 2012 and 2017. A total of 411 camels, 102 alpacas and 19 llamas' sera were tested for the presence of antibodies to MERS-CoV. Our findings indicate a lower MERS-CoV seropositivity among Israeli dromedaries than in the surrounding countries, and for the first time naturally infected llamas were identified. In addition, nasal swabs of 661 camels, alpacas and lamas, obtained from January 2015 to December 2017, were tested for the presence of MERS-CoV RNA. All nasal swabs were negative, indicating no evidence for MERS-CoV active circulation in these camelids during that time period.

Monitoring the uptake of live avian vaccines by their detection in feathers.

Protection against diseases caused by the avian viruses, Marek's disease, Infectious laryngotracheitis, chicken anemia and turkey meningoencephalitis is achieved by live vaccines. The application quality is important to assure proper uptake in commercial flocks. We describe a novel evaluation method for the vaccination process by sequential monitoring the vaccine viruses in feathers. Feather collection is easy, non-invasive and non-lethal for the birds, therefore advantageous for monitoring purposes. To demonstrate the vaccine virus presence, an innovative assay of nested real-time amplification was approached because vaccine viruses presence in vivo is less abundant comparing to virulent wild-type isolates. The Marek's disease virus vaccine virus, Rispens/CVI988, in feathers of commercial flock was detected from 4 to 7 days and for at least 3 months post-vaccination, until the survey stopped. As the drinking water route was newly adopted for Infectious laryngotracheitis vaccination, one or two vaccine doses/bird were administered. The virus uptake was detected in feathers between 2 and 20 days-post-vaccination. With a doubled vaccine dose the positivity bird rate was higher. For the first time the chicken anemia vaccine virus presence in chicken feathers was demonstrated between 14 and 35 days-post-vaccination. No previous studies were available, thus in parallel to feathers the vaccine virus was demonstrated in the livers and spleens. The turkey meningoencephalitis vaccine virus uptake in turkey feather-pulps is even more innovative because this is the first turkey virus amplified from feather-pulps. The vaccine virus presence resemble the kinetics of the other 3 viruses, 3-21 days-post-vaccination. Detecting the specific antibodies following vaccination possessed a lower sensitivity than vaccine virus demonstration in feathers. In summary, the presented assay can be adopted for the quality evaluation of the vaccination process in poultry.

Differential amplification of Marek's disease CVI988 vaccine and of wild-type isolates from organs of commercial chickens using single or duplexed probes in real-time PCR.

The differentiation of Marek's disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay's applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.

Synergy or interference of a H9N2 avian influenza virus with a velogenic Newcastle disease virus in chickens is dose dependent.

Field observations indicate that the impact of velogenic Newcastle disease virus (vNDV) is more severe in countries with concomitant circulation of low pathogenicity avian influenza virus, as is the case in the Middle East, in particular in Israel, where H9N2 and NDV are endemic. In our study, we evaluated how the exposure of chickens to an H9N2 challenge either favours or interferes with a subsequent vNDV infection and its transmission to sentinels. For this purpose, single vNDV and sequential H9/NDV challenges were performed with increasing doses of vNDV (101-106 EID50). The H9N2 challenge made birds more susceptible to the vNDV, lowering the minimum dose required to cause an infection, exacerbating the clinical outcome, while delaying the onset of the disease and time of death. Interestingly, the presence and degree of these seemingly contrasting effects were dose-dependent and not mutually exclusive.

Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method for diagnosis of Turkey Meningoencephalitis Virus.

The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, drooping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA has now been upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the envelope and the non-structural NS5 gene, increasing the sensitivity by 10-100-fold compared to the previously existing assays. Based on the recent complete sequences of five TMEV isolates we have now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA assay was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009 and 2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least in experimentally infected turkeys and in three cases of disease encountered in commercial flocks.