A novel array of real-time RT-PCR assays for the rapid pathotyping of type I avian paramyxovirus (APMV-1).

Newcastle disease (ND) caused by virulent avian paramyxovirus type I (APMV-1) is a WOAH and EU listed disease affecting poultry worldwide. ND exhibits different clinical manifestations that may either be neurological, respiratory and/or gastrointestinal, accompanied by high mortality. In contrast, mild or subclinical forms are generally caused by lentogenic APMV-1 and are not subject to notification. The rapid discrimination of virulent and avirulent viruses is paramount to limit the spread of virulent APMV-1. The appropriateness of molecular methods for APMV-1 pathotyping is often hampered by the high genetic variability of these viruses that affects sensitivity and inclusivity. This work presents a new array of real-time RT-PCR (RT-qPCR) assays that enable the identification of virulent and avirulent viruses in dual mode, i.e., through pathotype-specific probes and subsequent Sanger sequencing of the amplification product. Validation was performed according to the WOAH recommendations. Performance indicators on sensitivity, specificity, repeatability and reproducibility yielded favourable results. Reproducibility highlighted the need for assays optimization whenever major changes are made to the procedure. Overall, the new RT-qPCRs showed its ability to detect and pathotype all tested APMV-1 genotypes and its suitability for routine use in clinical samples.

Redesign and Validation of a Real-Time RT-PCR to Improve Surveillance for Avian Influenza Viruses of the H9 Subtype.

Avian influenza viruses of the H9 subtype cause significant losses to poultry production in endemic regions of Asia, Africa and the Middle East and pose a risk to human health. The availability of reliable and updated diagnostic tools for H9 surveillance is thus paramount to ensure the prompt identification of this subtype. The genetic variability of H9 represents a challenge for molecular-based diagnostic methods and was the cause for suboptimal detection and false negatives during routine diagnostic monitoring. Starting from a dataset of sequences related to viruses of different origins and clades (Y439, Y280, G1), a bioinformatics workflow was optimized to extract relevant sequence data preparatory for oligonucleotides design. Analytical and diagnostic performances were assessed according to the OIE standards. To facilitate assay deployment, amplification conditions were optimized with different nucleic extraction systems and amplification kits. Performance of the new real-time RT-PCR was also evaluated in comparison to existing H9-detection methods, highlighting a significant improvement of sensitivity and inclusivity, in particular for G1 viruses. Data obtained suggest that the new assay has the potential to be employed under different settings and geographic areas for a sensitive detection of H9 viruses.

Herpetic Pneumonia in Indian Ringneck Parrots (Psittacula krameri): First Report of Novel Psittacid Alphaherpesvirus-5 Infection in Europe.

The first two European outbreaks of herpetic pneumonia caused by Psittacid alphaherpesvirus-5 were diagnosed based on gross pathology findings, histological examination, transmission electron microscopy visualization and genome sequencing. The outbreaks, characterized by high morbidity and high mortality rates, involved two parrot species, namely the Indian ringneck parrot (Psittacula krameri) and the Alexandrine parakeet (Psittacula eupatria). Clinical signs observed were ruffled feathers, dyspnea, tail bobbing, open wings while breathing, depression and anorexia. Necropsy was performed on Indian ringneck parrots only, and the most evident and serious gross lesion found in all the birds was a diffuse marked consolidation of the lungs associated with parenchyma congestion and oedema. Histological examination confirmed the existence of bronchopneumonia characterized by the presence of syncytial cells with intranuclear inclusion bodies. In one bird, fibrinous airsacculitis was observed as well. Lung tissue inspection through electron microscopy revealed the presence of virus particles resembling herpesviruses. Viral DNA was extracted, amplified using primers for Alloherpesviridae DNA polymerase gene detection, and then sequenced. BLAST analysis showed a 100% identity with the only previously reported sequence of PsHV-5 (MK955929.1).

Genetic characterization of highly pathogenic avian Influenza H5Nx clade 2.3.4.4b reveals independent introductions in nigeria.

Among recurrent sanitary emergencies able to spread rapidly worldwide, avian influenza is one of the main constraints for animal health and food security. In West Africa, Nigeria has been experiencing repeated outbreaks of different strains of avian influenza virus (AIV) since 2006 and is also recognized as a hot spot in the region for the introduction of emerging strains by migratory wild birds. Here, we generated complete genomes of 20 highly pathogenic avian influenza (HPAI) H5N8 viruses collected during active surveillance in Nigerian live bird markets (LBM) and from outbreaks reported in the country between 2016 and 2019. Phylogenetic analysis reveals that the Nigerian viruses cluster into four separate genetic groups within HPAI H5 clade 2.3.4.4b. The first group includes 2016-2017 Nigerian viruses with high genetic similarity to H5N8 viruses detected in Central African countries, while the second includes Nigerian viruses collected both in LBM and poultry farms (2018-2019), as well as in Cameroon, Egypt and Siberia. A natural reassortant strain identified in 2019 represents the third group: H5N8 viruses with the same gene constellation were identified in 2018 in South Africa. Finally, the fourth introduction represents the first detection in the African continent of the H5N6 subtype, which is related to European viruses. Bayesian phylogeographic analyses confirmed that the four introductions originated from different sources and provide evidence of the virus spread within Nigeria, as well as diffusion beyond its borders. The multiple epidemiological links between Nigeria, Central and Southern African countries highlight the need for harmonized and coordinated surveillance system to control AIV impact. Improved surveillance at the Wetlands, LBMs and early warning of outbreaks are crucial for prevention and control of AIV, which can be potentially zoonotic and be a threat to human health.

Development of a Novel Assay Based on Plant-Produced Infectious Bursal Disease Virus VP3 for the Differentiation of Infected From Vaccinated Animals.

Infectious bursal disease virus is the causative agent of Gumboro disease, a severe infection that affects young chickens and is associated with lymphoid depletion in the bursa of Fabricius. Traditional containment strategies are based either on inactivated or live-attenuated vaccines. These approaches have several limitations such as residual virulence or low efficacy in the presence of maternally derived antibodies (MDA) but, most importantly, the impossibility to detect the occurrence of natural infections in vaccinated flocks. Therefore, the development of novel vaccination strategies allowing the differentiation of infected from vaccinated animals (DIVA) is a priority. Recently, commercial vectored and experimental subunit vaccines based on VP2 have been proved effective in protecting from clinical disease and posed the basis for the development of novel DIVA strategies. In this study, an engineered version of the VP3 protein of IBDV (His-VP3) was produced in plants, successfully purified from Nicotiana benthamiana leaves, and used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-VP3 antibodies. The His-VP3 ELISA was validated with a panel of 180 reference sera and demonstrated to have 100% sensitivity (95% CI: 94.7-100.0) and 94.17% specificity (95% CI: 88.4-97.6). To evaluate the application of His-VP3 ELISA as a DIVA test, the novel assay was used to monitor, in combination with a commercial kit, detecting anti-VP2 antibodies, the immune response of chickens previously immunized with an inactivated IBDV vaccine, a recombinant Turkey herpes virus carrying the VP2 of IBDV (HVT-ND-IBD) or with plant-produced VP2 particles. The combined tests correctly identified the immune status of the vaccinated specific pathogen free white-leghorn chickens. Moreover, the His-VP3 ELISA correctly detected MDA against VP3 in commercial broiler chicks and showed that antibody titers fade with time, consistent with the natural decrease of maternally derived immunity. Finally, the novel assay, in combination with a VP2-specific ELISA, demonstrated its potential application as a DIVA test in chickens inoculated with VP2-based vaccines, being able to detect the seroconversion after challenge with a very virulent IBDV strain.

Heterogeneity of Early Host Response to Infection with Four Low-Pathogenic H7 Viruses with a Different Evolutionary History in the Field.

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV's major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered.

Effect of assay choice, viral concentration and operator interpretation on infectious bronchitis virus detection and characterization.

Despite the efforts to achieve a consistent classification scheme based on the complete S1 gene, the genetic characterization of infectious bronchitis virus (IBV) is often performed on partial S1 regions due to economic and time constraints in the diagnostic routine. Sanger sequencing remains the most common and cost-effective option even if the analysis of samples where multiple field and vaccine strain populations coexist can lead to partial or misleading results. The present study aimed to evaluate the different diagnostic outcomes of three commonly used RT-PCR methods targeting two regions of the S1 gene. A possible bias in IBV detection and characterization was investigated in relation to the adopted method, the strain concentration as well as their ratio in mixed samples. Thirty samples were prepared by artificially mixing two vaccine strains, combined at different ratios and selected among four different IBV lineages, i.e. GI-1 (Mass), GI-13 (793/B), GI-19 (QX), GI-23 (Israeli Variant 2). Sequence analysis was conducted both manually and with bioinformatic methods. The result agreement among methods, replicates and analysis approaches was statistically evaluated. Consistent results emerged among the three assays, with a few discrepancies likely caused by primer affinity and target amount. This study confirms the complexity of IBV strain identification and highlights the importance of evaluating and updating the available diagnostic assays for a reliable detection of all circulating IBV strains.

Detection of avian influenza virus: a comparative study of the in silico and in vitro performances of current RT-qPCR assays.

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.

Genomic Sequence of a New Alphavirus Detected in Comber (Serranus cabrilla).

The comber alphavirus was isolated from a fish cell line from the brain of an apparently healthy Serranus cabrilla specimen collected during wild fish surveillance in southern Italy. The comber alphavirus is a new member of the genus Alphavirus, family Togaviridae.

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.

Detection and characterization of a rhabdovirus causing mortality in black bullhead catfish, Ameiurus melas.

This study fully describes a severe disease outbreak occurred in 2016 in black bullhead catfish farmed in Italy. Affected fish showed nervous clinical signs as well as emaciations and haemorrhagic petechiae on the skin at the fin bases, abdomen and gills. Viral isolation in cell culture allowed the subsequent identification of a rhabdovirus, tentatively named ictalurid rhabdovirus (IcRV), through electron microscopy, immunofluorescence and whole genome sequencing (WGS). The newly isolated virus, together with 14 additional viral strains stored in our repository and detected during similar mortality episodes in the period 1993-2016, was phylogenetically analysed on the basis of the nucleoprotein and the glycoprotein nucleotide and amino acid sequences. The genetic distances among Italian IcRV strains were also estimated. Our results show that all the IcRV strains belong to the genus Sprivivirus and are closely related to the tench rhabdovirus (TenRV). Italian catfish production is constantly decreasing, mainly due to viral infections, which include the newly characterized IcRV. Data presented in this work will assist to investigate the molecular epidemiology and the diffusive dynamics of this virus and to develop adequate surveillance activities.