Genetic characterization of newly emerging avian reovirus variants in chickens with viral arthritis/tenosynovitis in Israel.

In recent years, new avian reovirus (ARV) variants caused a variety of symptoms in chickens worldwide, the most important of which was Viral arthritis/tenosynovitis which caused substantial economic losses and has become a concern to the worldwide chicken industry. In this study, we characterized emerging ARV variants in Israel and analyzed their genetic relationship with reference strains. One hundred thirty-four ARV variants were isolated from tendons and synovial fluids of commercial broiler chickens with signs of arthritis/tenosynovitis. Phylogenetic analysis of the partial segment of the sigma C (σC) gene confirmed that these field isolates from Israel could be clustered into all six known clusters. The majority of ARV isolates in Israel belonged to the genotypic cluster 5 (GC5). The strains in this study had a low sequence identity when compared to the commercial vaccine (strain S1133). The findings of this study demonstrated the genetic diversity of ARV strains in Israel from 2015 to 2022. It is reasonable to conclude from the preliminary results of this investigation that Israel has not been subject to selection pressure or the emergence of new ARV variants since the introduction of the live vaccine (ISR-7585). Due to the ongoing emergence of ARV variants, a robust epidemiological monitoring program supported by molecular biology techniques is required to track ARV strains in Israeli poultry flocks.

Development of a wide-range real-time RT-PCR assay for detection of Avian reovirus (ARV).

Avian reovirus (ARV) is a common pathogen in chickens and other birds causing a variety of clinical symptoms such as arthritis and tenosynovitis but also enteric and respiratory symptoms. A rapid method that detects as many ARV genotypes as possible, will contribute to the early identification and control of the virus infection that causes high economic damage to the poultry industry worldwide. In this study, a real-time reverse transcription polymerase chain reaction (RT-qPCR) assay for the detection of ARV was developed. The RT-qPCR detection threshold for ARV genomic RNA standard cases was 10 copies/µL. Reproducibility of the RT-qPCR was confirmed by intra- and inter-assays. When the nucleic acids of different ARV genotypes and other common avian pathogens (IBDV, AIV, NDV, and IBV) were subjected to that RT-qPCR test, only ARV samples tested positive while all other pathogens tested negative. Due to the simplicity, convenience, high sensitivity, and specificity of the assay, the probe-based RT-qPCR is proposed to be used as an alternative diagnostic assay for the detection of ARVs in veterinary diagnostic laboratories.

First detection of avian influenza subtype H4N6 in Israel in a wild mallard (Anas platyrhynchos).

Avian influenza viruses (AIV) are a worldwide threat to animal and human health. As wild waterfowl circulate and spread these viruses around the world, investigations of AIV prevalence in wild populations are critical for understanding pathogen transmission, as well as predicting disease outbreaks in domestic animals and humans. Surveillance efforts in this study have isolated H4N6 for the first time in Israel from a faecal sample of a wild mallard (Anas platyrhynchos). Phylogenetic analyses of the HA and NA genes revealed that this strain is closely related to isolates from Europe and Asia. This Eurasian origin, together with Israel serving as an important migratory bottleneck of the mid Palearctic-African flyway, suggests a potential introduction of this strain by migratory birds. Additional phylogenetic analysis of the isolate's internal genes (PB1, PB2, PA, NP, M and NS) revealed high levels of phylogenetic relatedness with other AIV subtypes, indicating previous reassortment events. High reassortment rates are characteristic for H4N6 viruses, which, together with this subtype's ability to infect pigs and adaptability to the human receptor binding domain, raises the concern that it would potentially become zoonotic in the future. These results emphasize the importance of continuous AIV monitoring in migratory birds.

West Nile Virus in Common Wild Avian Species in Israel.

In order to evaluate the contribution of different wild bird species to West Nile virus (WNV) circulation in Israel, during the months preceding the 2018 outbreak that occurred in Israel, we randomly sampled 136 frozen carcasses of a variety of avian species. Visceral and central nervous system (CNS) tissue pools were tested using WNV NS2A RT qPCR assay; of those, 15 (11.03%, 95% CI: 6.31-17.54%) tissue pools were positive. A total of 13 out of 15 WNV RT qPCR positive samples were successfully sequenced. Phylogenetic analysis indicated that all WNV isolates were identified as lineage 1 and all categorized as cluster 2 eastern European. Our results indicated that WNV isolates that circulated within the surveyed wild birds in spring 2018 were closely related to several of the isolates of the previously reported 2018 outbreak in birds in Israel and that the majority of infected birds were of local species.

Protection against avian coronavirus conferred by oral vaccination with live bacteria secreting LTB-fused viral proteins.

The devastating impact of infectious bronchitis (IB) triggered by the IB virus (IBV), on poultry farms is generally curbed by livestock vaccination with live attenuated or inactivated vaccines. Yet, this approach is challenged by continuously emerging variants and by time limitations of vaccine preparation techniques. This work describes the design and evaluation of an anti-IBV vaccine comprised of E. coli expressing and secreting viral spike 1 subunit (S1) and nucleocapsid N-terminus and C-terminus polypeptides fused to heat-labile enterotoxin B (LTB) (LS1, LNN, LNC, respectively). Following chicken oral vaccination, anti-IBV IgY levels and cellular-mediated immunity as well as protection against virulent IBV challenge, were evaluated 14 days following the booster dose. Oral vaccination induced IgY levels that exceeded those measured following vaccination with each component separately. Following exposure to inactivated IBV, splenocytes isolated from chicks orally vaccinated with LNN or LNC -expressing bacteria, showed a higher percentage of CD8+ cells as compared to splenocytes isolated from chicks vaccinated with wild type or LTB-secreting E. coli and to chicks subcutaneously vaccinated. Significant reduction in viral load and percent of shedders in the vaccinated chicks was evident starting 3 days following challenge with 107.5 EID50/ml virulent IBV. Taken together, orally delivered LTB-fused IBV polypeptide-expressing bacteria induced virus-specific IgY antibody production and was associated with significantly shorter viral shedding on challenge with a live IBV. The proposed vaccine design and delivery route promise an effective and rapidly adaptable means of protecting poultry farms from devastating IB outbreaks.

Molecular characterization of the re-emerging West Nile virus in avian species and equids in Israel, 2018, and pathological description of the disease.

BACKGROUND: In this report we describe the molecular and pathological characteristics of West Nile virus (WNV) infection that occurred during the summer and fall of 2018 in avian species and equines. WNV is reported in Israel since the 1950s, with occasional outbreaks leading to significant morbidity and mortality in birds, high infection in horses and humans, and sporadic fatalities in humans. METHODS: Animal and avian carcasses in a suitable condition were examined by post-mortem analysis. Tissue samples were examined for WNV by RT-qPCR and the viral load was quantified. Samples with sufficient material quality were further analyzed by Endpoint PCR and sequencing, which was used for phylogenetic analysis. Tissue samples from positive animals were used for culturing the virus in Vero and C6/36 cells. RESULTS: WNV RNA was detected in one yellow-legged gull (Larus michahellis), two long-eared owls (Asio otus), two domesticated geese (Anser anser), one pheasant (Phasianus colchicus), four hooded crows (Corvus cornix), three horses and one donkey. Pathological and histopathological findings were characteristic of viral infection. Molecular analysis and viral load quantification showed varying degrees of infection, ranging between 70-1.4 × 106 target copies per sample. Phylogenetic analysis of a 906-bp genomic segment showed that all samples belonged to Lineage 1 clade 1a, with the following partition: five samples from 2018 and one sample detected in 2016 were of Cluster 2 Eastern European, two of Cluster 2 Mediterranean and four of Cluster 4. Four of the positive samples was successfully propagated in C6/36 and Vero cell lines for further work. CONCLUSIONS: WNV is constantly circulating in wild and domesticated birds and animals in Israel, necessitating constant surveillance in birds and equines. At least three WNV strains were circulating in the suspected birds and animals examined. Quantitative analysis showed that the viral load varies significantly between different organs and tissues of the infected animals.

Epidemiologic and phylogenetic analysis of the 2018 West Nile virus (WNV) outbreak in Israel demonstrates human infection of WNV lineage I.

As at 12 November 2018, an outbreak of West Nile virus (WNV) was responsible for 139 WNV infection cases in Israel. Here, we characterise the epidemiology of the outbreak and demonstrate that only WNV lineage I was circulating in mosquitoes and responsible for WNV infection in humans. This suggests that the concurrence of the outbreak in Israel with WNV outbreaks in several European countries is not due to a common, more virulent WNV genotype.

Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4.

BACKGROUND: Proventricular dilatation disease (PDD) is a fatal disorder of psittacine birds worldwide. The disease is characterized by lymphoplasmacytic infiltration of the central and peripheral nervous systems, leading to gastrointestinal motility and/or central nervous system dysfunction. Recently, we detected a significant association between avian bornavirus (ABV) infection and clinical signs of PDD in psittacines. However, it remains unclear whether ABV infection actually causes PDD. To address this question, we examined the impact of ABV inoculation on the cockatiel (Nymphicus hollandicus). RESULTS: Five cockatiels were inoculated via multiple routes (intramuscular, intraocular, intranasal, and oral) with a brain homogenate derived from either a PDD(+) avian bornavirus 4 (ABV4) (+) case (n = 3 inoculees) or from a PDD(-) ABV(-) control (n = 2 inoculees). The control birds remained free of clinical or pathological signs of PDD, and tested ABV(-) by RT-PCR and immunohistochemistry (IHC). In contrast, all three cockatiels inoculated with ABV4(+) brain homogenate developed gross and microscopic PDD lesions, and two exhibited overt clinical signs. In numerous tissues, ABV RT-PCR and sequence analysis demonstrated the presence of ABV4 RNA nearly identical to that in the inoculum. ABV was detected in the central nervous system of the three ABV-inoculees by IHC. Pyrosequencing to investigate the viral flora in the ABV4(+) inoculum uncovered 7 unique reads sharing 73-100% nucleotide sequence identity with previously identified ABV sequences and 24 reads sharing 40-89% amino acid sequence identity with viruses in the Retroviridae and Astroviridae families. Of these candidate viral species, only ABV RNA was recovered from tissues of the inoculated birds. CONCLUSION: In this study, the clinical and pathological manifestations of PDD were induced by inoculation of cockatiels with brain homogenates containing avian bornavirus 4. By using high throughput pyrosequencing an in-depth view of the viral content of the inoculum was achieved, revealing that of 3 candidate virus families detected, only the presence of ABV RNA correlated with the development of PDD. This study provides evidence of a causal association between ABV4 infection and PDD in cockatiels.

Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.

BACKGROUND: Proventricular dilatation disease (PDD) is a fatal disorder threatening domesticated and wild psittacine birds worldwide. It is characterized by lymphoplasmacytic infiltration of the ganglia of the central and peripheral nervous system, leading to central nervous system disorders as well as disordered enteric motility and associated wasting. For almost 40 years, a viral etiology for PDD has been suspected, but to date no candidate etiologic agent has been reproducibly linked to the disease. RESULTS: Analysis of 2 PDD case-control series collected independently on different continents using a pan-viral microarray revealed a bornavirus hybridization signature in 62.5% of the PDD cases (5/8) and none of the controls (0/8). Ultra high throughput sequencing was utilized to recover the complete viral genome sequence from one of the virus-positive PDD cases. This revealed a bornavirus-like genome organization for this agent with a high degree of sequence divergence from all prior bornavirus isolates. We propose the name avian bornavirus (ABV) for this agent. Further specific ABV PCR analysis of an additional set of independently collected PDD cases and controls yielded a significant difference in ABV detection rate among PDD cases (71%, n = 7) compared to controls (0%, n = 14) (P = 0.01; Fisher's Exact Test). Partial sequence analysis of a total of 16 ABV isolates we have now recovered from these and an additional set of cases reveals at least 5 distinct ABV genetic subgroups. CONCLUSION: These studies clearly demonstrate the existence of an avian reservoir of remarkably diverse bornaviruses and provide a compelling candidate in the search for an etiologic agent of PDD.