Experimental Usutu Virus Infection in Domestic Canaries Serinus canaria.

Usutu virus (USUV) is a neurotropic flavivirus closely related to West Nile virus (WNV). Its enzootic cycle mainly involves mosquitoes and birds. Human infection can occur with occasional, but sometimes severe, neurological complications. Since its emergence and spread in Europe over the last two decades, USUV has been linked to significant avian outbreaks, especially among Passeriformes, including European blackbirds (Turdus merula). Strikingly, no in vivo avian model exists so far to study this arbovirus. The domestic canary (Serinus canaria) is a passerine, which is considered as a highly susceptible model of infection by WNV. Here, we experimentally challenged domestic canaries with two different doses of USUV. All inoculated birds presented detectable amounts of viral RNA in the blood and RNA shedding via feathers and droppings during the early stages of the infection, as determined by RT-qPCR. Mortality occurred in both infected groups (1/5 and 2/5, respectively) and was not necessarily correlated to a pure neurological disease. Subsequent analyses of samples from dead birds showed histopathological changes and virus tropism mimicking those reported in naturally infected birds. A robust seroconversion followed the infection in almost all the surviving canaries. Altogether, these results demonstrate that domestic canaries constitute an interesting experimental model for the study of USUV pathogenesis and transmission.

Domestic canaries (Serinus canaria forma domestica) are susceptible to low pathogenic avian influenza virus infections.

Avian influenza viruses have been isolated from many bird species; however, little is known about the susceptibility of pet birds to low pathogenic avian influenza (LPAI) viruses. To address this research gap, domestic canaries (Serinus canaria forma domestica) were experimentally infected with H5 and H7 LPAI viruses to determine susceptibility and to evaluate samples for diagnostic purposes. Clinical evidence of infection (e.g. ruffled plumage and apathy) and mortality were noted for the canaries inoculated with chicken-adapted LPAI viruses. Real-time reverse transcription-polymerase chain reaction (RRT-PCR) demonstrated higher viral RNA levels in buccal compared to faecal samples. No clinical signs or mortality were observed in canaries inoculated with LPAI virus originating from wild birds; however, the canaries in this group did have evidence of viral RNA in buccal and faecal samples. Overall, this study showed that domestic canaries are susceptible to LPAI virus infections and that they can shed large amounts of viral RNA, primarily through the respiratory route. Thus, buccal swabs might be better samples than faeces for efficient detection of some LPAI virus infections in these birds. Although canaries have not been identified as a significant reservoir for LPAI viruses, they may be infected by LPAI viruses. Thus, the importance of the control of domestic canaries for detection of LPAI viruses should not be underestimated, especially in the contexts of international commercial exchange and outbreaks. RESEARCH HIGHLIGHTS Canaries are susceptible to infection with H5/H7 LPAI viruses. Canaries inoculated with LPAI viruses excrete large amounts of viral RNA. Buccal swabs may be appropriate specimens for AI virus detection in canaries. The control of canaries for LPAI virus detection should not be overlooked.

Detection and Characterization of Circovirus in Canary Flocks.

Circovirus infections have been documented in adult and nestling canaries (Fringillidae) but the distribution of the virus in the world is not yet known. In captive canary flocks, Circovirus infections have been reported based on the clinical observations. In this study, the presence of both canary circovirus (CaCV) and chicken anemia virus (CAV) in canary flocks was investigated. Virus strains were detected by PCR and direct sequencing of amplified products. Nucleotide sequences were aligned and compared with existing data in GenBank. PCR identified CaCV-positive birds, giving an overall positivity rate of 25%, but all samples were negative for CAV. According to the sequencing data, three distinct strains were identified. Our results indicated a relationship between genetic variation in the replicase gene ( rep) and the geographic regions as well as the feasibility of using the rep gene for virus detection and molecular epidemiology investigations. We are reporting detection and characterization of canary circovirus based on the rep gene. Sequencing results and sequence identity analysis revealed that the rep gene could be used for detecting and discriminating the members of family Circoviridae. This manuscript is the first report of canary circovirus in Iran and of three new strains in the world.

West Nile Virus Infection in American Singer Canaries: An Experimental Model in a Highly Susceptible Avian Species.

This study investigated the susceptibility of American singer canaries ( Serinus canaria) to West Nile virus (WNV) infection. Adult canaries were inoculated with 105, 102, and 101 plaque forming units (PFU) of WNV. All birds became infected and mortality occurred by 5 days postinoculation. The load of viral RNA as determined by RT-qPCR was dose dependent, and was higher at all doses than the level of viral RNA detected in American crows ( Corvus brachyrhynchos) challenged with 105 PFU of WNV. In a subset of birds, viremia was detected by virus isolation; canaries inoculated with 101 PFU of WNV developed viremia exceeding 1010 PFU/mL serum, a log higher than American crows inoculated with 105 PFU of virus. In canaries euthanized at 3 days postinoculation, WNV was isolated at >107 PFU of virus/100 mg of lung, liver, heart, spleen, and kidney tissues. Pallor of the liver and splenomegaly were the most common macroscopic observations and histologic lesions were most severe in liver, spleen, and kidney, particularly in canaries challenged with 102 and 101 PFU. Immunoreactivity to WNV was pronounced in the liver and spleen. IgG antibodies to WNV were detected in serum by enzyme immunoassay in 11 of 21 (52%) challenged canaries and, in 4 of 5 (20%) of these sera, neutralization antibodies were detected at a titer ≥ 1:20. American singer canaries provide a useful model as this bird species is highly susceptible to WNV infection.

Chapparvoviruses occur in at least three vertebrate classes and have a broad biogeographic distribution.

Chapparvoviruses are a highly divergent group of parvoviruses (family Parvoviridae) that have recently been identified via metagenomic sampling of animal faeces. Here, we report the sequences of six novel chapparvoviruses identified through both metagenomic sampling of bat tissues and in silico screening of published vertebrate genome assemblies. The novel chapparvoviruses share several distinctive genomic features and group together as a robustly supported monophyletic clade in phylogenetic trees. Our data indicate that chapparvoviruses have a broad host range in vertebrates and a global distribution.

Viral vector vaccines expressing nucleoprotein and phosphoprotein genes of avian bornaviruses ameliorate homologous challenge infections in cockatiels and common canaries.

Avian bornaviruses are causative agents of proventricular dilatation disease (PDD), an often fatal disease of parrots and related species (order Psittaciformes) which is widely distributed in captive psittacine populations and may affect endangered species. Here, we established a vaccination strategy employing two different well described viral vectors, namely recombinant Newcastle disease virus (NDV) and modified vaccinia virus Ankara (MVA) that were engineered to express the phosphoprotein and nucleoprotein genes of two avian bornaviruses, parrot bornavirus 4 (PaBV-4) and canary bornavirus 2 (CnBV-2). When combined in a heterologous prime/boost vaccination regime, NDV and MVA vaccine viruses established self-limiting infections and induced a bornavirus-specific humoral immune response in cockatiels (Nymphicus hollandicus) and common canaries (Serinus canaria forma domestica). After challenge infection with a homologous bornavirus, shedding of bornavirus RNA and viral loads in tissue samples were significantly reduced in immunized birds, indicating that vaccination markedly delayed the course of infection. However, cockatiels still developed signs of PDD if the vaccine failed to prevent viral persistence. Our work demonstrates that avian bornavirus infections can be repressed by vaccine-induced immunity. It represents a first crucial step towards a protective vaccination strategy to combat PDD in psittacine birds.

Phylogenetic Analysis Supports Horizontal Transmission as a Driving Force of the Spread of Avian Bornaviruses.

BACKGROUND: Avian bornaviruses are a genetically diverse group of viruses initially discovered in 2008. They are known to infect several avian orders. Bornaviruses of parrots and related species (Psittaciformes) are causative agents of proventricular dilatation disease, a chronic and often fatal neurologic disease widely distributed in captive psittacine populations. Although knowledge has considerably increased in the past years, many aspects of the biology of avian bornaviruses are still undiscovered. In particular, the precise way of transmission remains unknown. AIMS AND METHODS: In order to collect further information on the epidemiology of bornavirus infections in birds we collected samples from captive and free-ranging aquatic birds (n = 738) and Passeriformes (n = 145) in Germany and tested them for the presence of bornaviruses by PCR assays covering a broad range of known bornaviruses. We detected aquatic bird bornavirus 1 (ABBV-1) in three out of 73 sampled free-ranging mute swans (Cygnus olor) and one out of 282 free-ranging Eurasian oystercatchers (Haematopus ostralegus). Canary bornavirus 1 (CnBV-1), CnBV-2 and CnBV-3 were detected in four, six and one out of 48 captive common canaries (Serinus canaria forma domestica), respectively. In addition, samples originating from 49 bornavirus-positive captive Psittaciformes were used for determination of parrot bornavirus 2 (PaBV-2) and PaBV-4 sequences. Bornavirus sequences compiled during this study were used for phylogenetic analysis together with all related sequences available in GenBank. RESULTS OF THE STUDY: Within ABBV-1, PaBV-2 and PaBV-4, identical or genetically closely related bornavirus sequences were found in parallel in various different avian species, suggesting that inter-species transmission is frequent relative to the overall transmission of these viruses. Our results argue for an important role of horizontal transmission, but do not exclude the additional possibility of vertical transmission. Furthermore we defined clearly separated sequence clusters within several avian bornaviruses, providing a basis for an improved interpretation of transmission events within and between wild bird populations and captive bird collections.

No contact transmission of avian bornavirus in experimentally infected cockatiels (Nymphicus hollandicus) and domestic canaries (Serinus canaria forma domestica).

Avian bornaviruses (ABV) are the causative agents of proventricular dilatation disease (PDD), a widely distributed disease of parrots. Distinct ABV lineages were also found in various non-psittacine avian species, such as canaries, but the pathogenic role of ABV in these species is less clear. Despite the wide distribution of ABV in captive parrots and canaries, its mode of transmission is poorly understood: both horizontal transmission via the urofaecal-oral route and vertical transmission are discussed to play a role. In this study we investigated pathology and horizontal transmission of ABV in domestic canaries (Serinus canaria forma domestica) and cockatiels (Nymphicus hollandicus), two natural host species commonly used for experimental ABV infections. ABV inoculation resulted in persistent infection of all inoculated animals from both species. ABV-infected cockatiels exhibited PDD-like symptoms, such as neurologic signs or shedding of undigested seeds. In contrast, infected domestic canaries did not develop clinical disease. Interestingly, we did not detect viral RNA in cloacal swabs and organ samples or ABV-specific antibodies in serum samples of contact-exposed sentinel birds from either species at any time during a four months observation period. Our results strongly indicate that horizontal transmission of ABV by direct contact is inefficient in immunocompetent fully fledged domestic canaries and cockatiels.

Whole-genome characterization of a novel polyomavirus detected in fatally diseased canary birds.

Polyomaviruses of birds are aetiological agents of acute inflammatory diseases in non-immunocompromised hosts, which is in contrast to mammalian polyomaviruses. VP4, an additional structural protein encoded by the viral genomes of the known avian polyomaviruses, has been suggested to contribute to pathogenicity through loss of cells following induction of apoptosis. Four distinct bird polyomaviruses have been identified so far, which infect crows, finches, geese and parrots. Using broad-spectrum PCR, a novel polyomavirus, tentatively designated canary polyomavirus (CaPyV), was detected in diseased canary birds (Serinus canaria) that died at an age of about 40 days. Intranuclear inclusion bodies were found in the liver, spleen and kidneys. The entire viral genome was amplified from a tissue sample using rolling-circle amplification. Phylogenetic analysis of the genome sequence indicated a close relationship between CaPyV and other avian polyomaviruses. Remarkably, an ORF encoding VP4 could not be identified in the CaPyV genome. Therefore, the mechanism of pathogenicity of CaPyV may be different from that of the other avian polyomaviruses.

Novel avian bornavirus in a nonpsittacine species (Canary; Serinus canaria) with enteric ganglioneuritis and encephalitis.

A canary bird (Serinus canaria) died with nonsuppurative ganglioneuritis of the proventriculus and gizzard and encephalitis, lesions comparable to proventricular dilatation disease (PDD) of psittacine birds. Recently, several genotypes of a novel avian bornavirus have been linked to PDD. In the canary, bornaviral antigen was detected by immunohistochemistry in both neural and extraneural tissues. The widespread viral dissemination was confirmed by reverse transcription-PCR. Sequence analysis revealed a unique genotype of avian bornavirus. This observation suggests that bornaviruses are natural pathogens of several avian species and that the family Bornaviridae comprises more viral genotypes (or viral species) than previously assumed.

Circovirus inclusion bodies in intestinal muscle cells of a canary.

Multiple cytoplasmic inclusion bodies were observed in the intestinal smooth muscle cells of an adult canary from an aviary with a history of high mortality (50%) both in adult and young birds. Grossly, a mild enteritis was the only lesion appreciable. Smears of the proventricular contents contained a few megabacteria (Macrorhabdus ornithogaster). The intestinal inclusions were found in very high numbers in all parts of the tract examined. They appeared round to oval, amphophilic and hyaline in sections stained with haematoxylin and eosin, and magenta with Feulgen stain. Inclusions of the same type were occasionally detectable in the wall of a few splenic and pancreatic arteries. No inclusions or lesions were seen in the other organs examined. Transmission electron microscopy of the intestinal wall revealed circovirus-like particles either in paracrystalline arrays or loose arrangements, mostly within the cytoplasm of the intestinal muscule cells. Polymerase chain reaction amplification and sequence analysis confirmed infection with canary circovirus.

A retrospective description of a highly pathogenic avian influenza A virus (H7N1/Carduelis/Germany/72) in a free-living siskin (Carduelis spinus Linnaeus, 1758) and its accidental transmission to yellow canaries (Serinus canaria Linnaeus, 1758).

A haemagglutinating virus was isolated in summer 1972 from a single free-living siskin (Carduelis spinus Linnaeus, 1758) in embryonated chicken eggs. Additional cases of morbidity or mortality were not observed in the area were the sick siskin was found. The virus was characterized as an avian influenza A virus of the subtype H7N1 and designated H7N1/Carduelis/Germany/72. The virus induced following experimental inoculation of chicken embryos a high rate mortality (mean death time approximately 24 hours), formed plaques in chicken embryo fibroblast cultures without addition of trypsin and has an intracerebral pathogenicity index (ICPI) of 1.80. Therefore, this virus is considered as a highly pathogenic avian influenza A virus. Canaries (Serinus canarius Linnaeus, 1758), that were housed in the same room with the siskin were accidentially exposed by contact to the sick siskin which resulted in virus transmission followed by conjunctivitis, apathy, anorexia and a high rate mortality.

Cross-protection test of an avian poxvirus isolated from houbara bustards.

An avian poxvirus was isolated previously from the houbara bustard (Chlamydotis undulata). We carried out a cross-protection test on 66 captive-bred canaries. Thirty-five canaries were vaccinated with a commercial canary poxvirus (CP) vaccine. Three weeks later all 66 birds were assigned randomly to six different groups: group Ia (n = 14) was vaccinated and challenged with houbara bustard poxvirus (HP) strain; group Ib (n = 13) was vaccinated and challenged with a CP strain; group Ic (n = 7) was vaccinated and not inoculated; group IIa (n = 14) was nonvaccinated and challenged with HP strain; group IIb (n = 11) was nonvaccinated and challenged with a CP strain; and group IIc (n = 7) was not vaccinated and not challenged. Vaccinated groups (Ia, Ib, Ic) had no losses and remained healthy. All of the birds (100%) in group IIb died within 10 days, and 10 birds (71.4%) of group IIa died within 20 days. The nonvaccinated control group (IIc) remained healthy. Poxvirus was isolated from the liver, digestive tract, lungs, and inoculation lesions of nonvaccinated dead CP- and HP-challenged birds. Secondary bacterial infections were higher among nonvaccinated HP-challenged birds (85.7%) than in nonvaccinated CP-challenged birds (25%). The results of this experiment reveal a degree of immunogenic relatedness between CP and HP strain and support the recommendation that houbara bustards be vaccinated with a CP vaccine.

Development and application of an immunohistochemical staining technique to detect avian polyomaviral antigen in tissue sections.

An immunohistochemical staining technique was developed to detect polyomaviral antigens of budgerigar fledgling disease in formalin-fixed tissue sections. This technique used an indirect avidin-biotin, alkaline phosphatase labeling system with a mixture of monoclonal antibodies developed against the virus major capsid protein. The staining technique was applied retrospectively to 24 avian accessions which were originally diagnosed as budgerigar fledgling disease or avian polyomavirus infection based on microscopic findings including typical intranuclear inclusions. Immunohistochemical staining resulted in positive reactions in some tissues from 17 of 24 cases. The tissues most frequently containing typical intranuclear inclusions or positive immunohistochemical staining were the spleen, liver, and kidney. Neither of the 2 nonpsittacine cases was positive immunohistochemically. This technique may be used wither as a rapid test on routinely processed diagnostic samples to confirm the presence of avian polyomavirus or for pathogenesis research studies.

Biological and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in non-avian species.

A canarypox-based (ALVAC) recombinant expressing the rabies G glycoprotein has been utilized to assess in vitro and in vivo biological properties of the canarypox virus vector system. In vitro studies have shown that no replication of the virus can be detected on six human-derived cell lines, nor can the virus be readily adapted to replicate on non-avian cells. Expression of the rabies G can be detected on all cell lines analyzed in the absence of productive viral replication. Analysis of viral-specific DNA accumulation indicated that the block in the replication cycle in the human cell lines analyzed occurred prior to DNA replication. The exact nature of the block, however, remains unknown. The concept of using a non-replicating immunization vehicle has been demonstrated through extensive in vivo studies in a range of species including non-human primates and humans. The results of such in vivo studies have exemplified the safety and immunogenicity of the ALVAC vaccine vector.