Detection of Avian bornavirus 5 RNA in Eclectus roratus with feather picking disorder

Avian bornavirus (ABV) was discovered recently in parrots with proventricular dilatation disease (PDD), a fatal neurological disease. Although ABV has been shown to be a causative agent of PDD, its virological characteristics are largely unknown. Here we report the detection of ABV genotype 5 RNA in an Eclectus roratus with feather picking disorder (FPD). Interestingly, although the bird was persistently infected with ABV5 for at least 8 months, it had no clinical signs of PDD. Although it remains unclear whether ABV5 is associated with FPD, these findings raise the importance of epidemiological studies of birds with diseases other than PDD.     

Respiratory viral infections detected by multiplex PCR among pediatric patients with lower respiratory tract infections seen at an urban hospital in Delhi from 2005 to 2007

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.     

Complete genome sequence of avian bornavirus genotype 1 from a Macaw with proventricular dilatation disease

Avian bornaviruses (ABV) were first detected and described in 2008. They are the etiologic agents of proventricular dilatation disease (PDD), a frequently fatal neurologic disease of captive parrots. Seven ABV genogroups have been identified worldwide from a variety of sources, and that number may increase as surveillance for novel bornaviruses continues. Here, we report the first complete sequence of a genogroup 1 avian bornavirus (ABV1).     

Fine Structure and Morphogenesis of Borna Disease Virus

Borna disease virus (BDV), a negative nonsegmented single-stranded RNA virus, has not been fully characterized morphologically. Here we present what is to our knowledge the first data on the fine ultrastructure and morphogenesis of BDV. The supernatant of MDCK cells persistently infected with BDV treated with n-butyrate contained many virus-like particles and more BDV-specific RNA than that of untreated samples. The particles were spherical, enveloped, and approximately 130 nm in diameter; had spikes 7 nm in length; and reacted with BDV p40 antibody. A thin nucleocapsid, 4 nm in width, was present peripherally in contrast to the thick nucleocapsid of hemagglutinating virus of Japan. The BDV particles reproduced by budding on the cell surface.     

Identification and Characterization of a Distinct Strain of Beak and Feather Disease Virus in Southeast China

Beak and feather disease virus(BFDV) is an infectious agent responsible for feather degeneration and beak deformation in birds. In March 2017, an epidemic of psittacine beak and feather disease(PBFD) struck a farm in Fuzhou in the Fujian Province of southeast China, resulting in the death of 51 parrots. In this study, the disease was diagnosed and the pathogen was identified by PCR and whole genome sequencing. A distinct BFDV strain was identified and named as the FZ strain.This BFDV strain caused severe disease symptoms and pathological changes characteristic of typical PBFD in parrots, for example, loss of feathers and deformities of the beak and claws, and severe pathological changes in multiple organs of the infected birds. Phylogenetic analysis showed that the FZ strain was more closely related to the strain circulating in New Caledonia than the strains previously reported in China. Nucleotide homology between the FZ strain and other 43 strains of BFDV ranged from 80.0% to 92.0%. Blind passage experiment showed that this strain had limited replication capability in SPF Chicken Embryos and DF-1 Cells. Furthermore, the capsid(Cap) gene of this FZ strain was cloned into the p GEX-4 T-1 expression vector to prepare the polyclonal anti-Cap antibody. Western blotting analysis using the anti-Cap antibody further confirmed that the diseased parrots were infected with BFDV. In this study, a PBFD and its pathogen was identified for the first time in Fujian Province of China, suggesting that future surveillance of BFDV should be performed.     

Evolutionarily Conserved Interaction between the Phosphoproteins and X Proteins of Bornaviruses from Different Vertebrate Species

Bornavirus, a non-segmented, negative-strand RNA viruses, is currently classified into several genetically distinct genotypes, such as Borna disease virus (BDV) and avian bornaviruses (ABVs). Recent studies revealed that bornavirus genotypes show unique sequence variability in the putative 5′ untranslated region (5′ UTR) of X/P mRNA, a bicistronic mRNA for the X protein and phosphoprotein (P). In this study, to understand the evolutionary relationship among the bornavirus genotypes, we investigated the functional interaction between the X and P proteins of four bornavirus genotypes, BDV, ABV genotype 4 and 5 and reptile bornavirus (RBV), the putative 5′ UTRs of which exhibit variation in the length. Immunofluorescence and immunoprecipitation analyses using mammalian and avian cell lines revealed that the X proteins of bornaviruses conserve the ability to facilitate the export of P from the nucleus to the cytoplasm via interaction with P. Furthermore, we showed that inter-genotypic interactions may occur between X and P among the genotypes, except for X of RBV. In addition, a BDV minireplicon assay demonstrated that the X and P proteins of ABVs, but not RBV, can affect the polymerase activity of BDV. This study demonstrates that bornaviruses may have conserved the fundamental function of a regulatory protein during their evolution, whereas RBV has evolved distinctly from the other bornavirus genotypes.     

The Bicolored White-Toothed Shrew Crocidura leucodon (HERMANN 1780) Is an Indigenous Host of Mammalian Borna Disease Virus

Borna disease (BD) is a sporadic neurologic disease of horses and sheep caused by mammalian Borna disease virus (BDV). Its unique epidemiological features include: limited occurrence in certain endemic regions of central Europe, yearly varying disease peaks, and a seasonal pattern with higher disease frequencies in spring and a disease nadir in autumn. It is most probably not directly transmitted between horses and sheep. All these features led to the assumption that an indigenous virus reservoir of BDV other than horses and sheep may exist. The search for such a reservoir had been unsuccessful until a few years ago five BDV-infected shrews were found in a BD-endemic area in Switzerland. So far, these data lacked further confirmation. We therefore initiated a study in shrews in endemic areas of Germany. Within five years 107 shrews of five different species were collected. BDV infections were identified in 14 individuals of the species bicolored white-toothed shrew (Crocidura leucodon, HERMANN 1780), all originating from BD-endemic territories. Immunohistological analysis showed widespread distribution of BDV antigen both in the nervous system and in epithelial and mesenchymal tissues without pathological alterations. Large amounts of virus, demonstrated by presence of viral antigen in epithelial cells of the oral cavity and in keratinocytes of the skin, may be a source of infection for natural and spill-over hosts. Genetic analyses reflected a close relationship of the BDV sequences obtained from the shrews with the regional BDV cluster. At one location a high percentage of BDV-positive shrews was identified in four consecutive years, which points towards a self-sustaining infection cycle in bicolored white-toothed shrews. Analyses of behavioral and population features of this shrew species revealed that the bicolored white-toothed shrew may indeed play an important role as an indigenous host of BDV.     

A new eimerian species (Apicomplexa: Eimeriidae) from the blue-fronted Amazon parrot Amazona aestiva L. (Aves: Psittacidae) in Brazil

The Neotropical psittacine species Amazona aestiva, commonly known as the blue-fronted Amazon, is one of the most common and best-known psittacine birds kept as a pet worldwide. However, very little is known about the diseases or parasites of these birds. In this study, we describe a new species, Eimeria aestivae, associated with these parrots. The new species is characterized by: ovoid smooth oocysts (n = 60), 36.8 (33.2-41.5) × 23.7 (21.7-25.7) μm, length/width ratio = 1.55; polar granule present; ellipsoidal sporocysts (n = 25), 19.8 (17.5-21.6) × 9.3 (8.3-9.9) μm; Stieda, sub-Stieda body, and sporocyst residuum present. Sporozoites (n = 20), 2 per sporocyst, elongate and curved, 17.6 (15.8-19.2) × 3.8 (3.2-4.8) μm; each with 2 refractile bodies. The oocysts of the other 2 eimerian species described for Amazona are larger than those of the presented species, but they all seem to be closely related because of some similarities among them.     

Guidelines and ethical considerations for housing and management of psittacine birds used in research

The Psittaciformes are a large order of landbirds comprising over 350 species in about 83 genera. In 2009, 141 published studies implicated parrots as research subjects; in 31 of these studies, 483 individuals from 45 different species could be considered laboratory animals. Amazons and budgerigars were by far the most represented psittacine species. The laboratory research topics were categorized as either veterinary medicine and diagnostics (bacteriology, hematology, morphology, and reproduction; 45%) or behavioral and sensory studies (behavior, acoustics, and vision; 17%). Confinement of psittacine species for research purposes is a matter of concern as scientifically based species-specific housing guidelines are scarce. The aim of this article is to provide scientific information relevant to the laboratory confinement of Psittaciformes to promote the refinement of acquisition, housing, and maintenance practices of these birds as laboratory animals. We briefly discuss systematics, geographical distribution, legislation, and conservation status as background information on laboratory parrot confinement. The following section presents welfare concerns related to captive containment (including domestication status) and psittacine cognition. We then discuss considerations in the acquisition of laboratory parrots and review important management issues such as nutrition, zoonoses, housing, and environmental enrichment. The final section reviews indications of distress and compromised welfare.     

Cell Entry of Borna Disease Virus Follows a Clathrin-Mediated Endocytosis Pathway That Requires Rab5 and Microtubules

Borna disease virus (BDV), the prototypic member of the Bornaviridae family within the order Mononegavirales, exhibits high neurotropism and provides an important and unique experimental model system for studying virus-cell interactions within the central nervous system. BDV surface glycoprotein (G) plays a critical role in virus cell entry via receptor-mediated endocytosis, and therefore, G is a critical determinant of virus tissue and cell tropism. However, the specific cell pathways involved in BDV cell entry have not been determined. Here, we provide evidence that BDV uses a clathrin-mediated, caveola-independent cell entry pathway. We also show that BDV G-mediated fusion takes place at an optimal pH of 6.0 to 6.2, corresponding to an early-endosome compartment. Consistent with this finding, BDV cell entry was Rab5 dependent but Rab7 independent and exhibited rapid fusion kinetics. Our results also uncovered a key role for microtubules in BDV cell entry, whereas the integrity and dynamics of actin cytoskeleton were not required for efficient cell entry of BDV.Borna disease virus (BDV) causes central nervous system disease in a variety of vertebrate species that is frequently manifested by behavioral abnormalities (27, 59). BDV is the causative agent of Borna disease, an often fatal immune-mediated neurological disease naturally occurring mainly in horses and sheep (21, 26, 47). However, current evidence indicates that the natural host range, prevalence, and geographic distribution of BDV are wider than originally thought (25, 31). Experimentally, BDV has a wide host range, and both host and viral factors contribute to a variable period of incubation and heterogeneity in the symptoms and pathology associated with BDV infection (20, 23, 34, 50). Notably, cases of proventricular dilatation disease affecting different species of psittacine birds have recently been linked to infection with avian bornaviruses (24, 29), a finding that expands the natural host range of bornavirus infections associated with clinical manifestations.BDV is an enveloped virus with a nonsegmented negative-strand RNA genome (11, 33, 53, 55) whose gene organization [3′-N-P-p10 (X)-M-G-L-5′] is characteristic of mononegaviruses. However, on the basis of its unique genetic and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae, within the order Mononegavirales.The BDV surface glycoprotein G plays a key role in receptor recognition and cell entry (20, 46). The G gene directs the synthesis of a precursor, GPC, with a predicted M_r of ca. 56 kDa, but due to its extensive glycosylation, GPC migrates with an M_r of 84 to 94 kDa. GPC is posttranslationally cleaved by the cellular protease furin into GP-1 and GP-2, corresponding to the N-and C-terminal regions, respectively, of G (2, 8, 19, 49). GP-1 has been shown to be sufficient for virus cell entry via receptor-mediated endocytosis (46), whereas GP-2 likely mediates the pH-dependent fusion event between BDV and cell membranes required for a BDV productive infection (19). In vivo, neurons are the initial target of BDV, suggesting a restricted expression pattern of a yet-unidentified virus receptor. Late in infection, BDV is detected in many tissues and organs as a consequence of its centrifugal spread via the axoplasm of peripheral nerve tissues. Receptor-independent mechanisms also contribute to cell-to-cell propagation of BDV (8).The paucity of cell-free virus associated with BDV infection has hindered studies aimed at the elucidation of the mechanisms involved in BDV cell entry. To overcome this problem, we generated a replication-competent recombinant vesicular stomatitis virus expressing BDV G (rVSVΔG*/BDVG) (45). Cells infected with rVSVΔG*/BDVG produced high titers (10⁷ PFU/ml) of cell-free virus progeny. Notably, rVSVΔG*/BDVG recreated the cell tropism and entry pathway of bona fide BDV, thus providing a unique tool for the investigation of BDV G-mediated cell entry.Viruses that enter cells via receptor-mediated endocytosis mainly use trafficking pathways mediated by either clathrin or caveola, although alternative entry pathways have been also reported (36). Nevertheless, clathrin-mediated endocytosis (CME) is the route most commonly used by enveloped viruses for cell internalization (35). The initial virus-cell surface receptor interaction results in the activation of different signaling pathways leading to the accumulation of clathrin coated-pits and subsequent formation of endocytotic vesicles (43). Another major endocytotic pathway used by several viruses, including Ebola virus (16) and SV40 (44), uses caveolae for viral internalization into the cell. This endocytotic pathway is strictly dependent on recruitment of lipid rafts to the cell surface, an event mediated by cholesterol. In this regard, we have recently documented the requirements of cholesterol and structural integrity of cell surface lipid rafts for efficient cell entry of BDV (9).In this work, we provide evidence for the first time that BDV cell entry follows a CME-dependent, caveola-independent pathway. Moreover, we show that BDV entry is Rab5 dependent but Rab7 independent and that BDV G-mediated fusion has a rapid kinetics and an optimal pH between 6.0 and 6.2. These findings indicate that BDV G-mediated fusion occurs within the early-endosome compartment. We also provide evidence that microtubules, but not actin dynamics, play a role in BDV cell entry likely by mediating trafficking of BDV-containing endosomes to the subcellular location where viral and endosomal membranes fuse.     

Duplex shuttle PCR for differential diagnosis of budgerigar fledgling disease and psittacine beak and feather disease

Two common viral diseases in psittacine birds including budgerigar fledgling disease (BFD), generally called avian polyomavirus (APV) infection, and psittacine beak and feather disease (PBFD) have similar clinical manifestations characterized by feather disorders. A duplex shuttle PCR was developed for detection of APV and PBFD virus (PBFDV). Two pairs of oligonucleotide primers were designed to amplify a 298-bp fragment of the t/T antigen region of APV genome and a 495-bp fragment of the capsid protein region encoded by open reading frame (ORF) C1 of PBFDV genome, respectively. In the present study, APV and PBFDV were detected simultaneously in one tube by duplex shuttle PCR using these two pairs of primers. The detection limits were 2 viral copies of APV and 3 viral copies of PBFDV. In the clinical application, we detected 16 APV-positive, 15 PBFDV-positive, and 3 mixed infected samples in 39 samples examined. Sequences of the amplified products were read. The t/T antigen region was conserved in the APV-positive samples as expected. ORF C1 of PBFDV genome showed diversity. Phylogenic analysis indicated that PBFDV ORF C1 consisted of 6 clusters which were related to subfamilies of psittacine birds. Our duplex shuttle PCR could be a useful method for differential diagnosis and molecular epidemiology of BFD and PBFD.     

Cell-to-cell spread of Borna disease virus proceeds in the absence of the virus primary receptor and furin-mediated processing of the virus surface glycoprotein

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of Mononegavirales. BDV cell entry follows a receptor-mediated endocytosis pathway, which is initiated by the recognition of an as-yet-unidentified receptor at the cell surface by the virus glycoprotein G. BDV G is synthesized as a precursor (GPC) that is cleaved by the cellular protease furin to produce the mature glycoproteins GP1 and GP2, which have been implicated in receptor recognition and pH-dependent fusion events, respectively. BDV is highly neurotropic and its spread in cultured cells proceeds in the absence of detectable extracellular virus or syncytium formation. BDV spread has been proposed to be strictly dependent on the expression and correct processing of BDV G. Here we present evidence that cell-to-cell spread of BDV required neither the expression of cellular receptors involved in virus primary infection, nor the furin-mediated processing of BDV G. We also show that in furin-deficient cells, the release of BDV particles induced by the treatment of BDV-infected cells with hypertonic buffer was not significantly affected, while virion infectivity was dramatically impaired, correlating with the decreased incorporation of BDV G species into viral particles. These findings support the view that the propagation of BDV within the central nervous systems of infected hosts involves both a primary infection that follows a receptor-mediated endocytosis pathway and a subsequent cell-to-cell spread that is independent of the expression of the primary receptor and does not require the processing of BDV G into GP1 and GP2.     

Borna Disease Virus Phosphoprotein Impairs the Developmental Program Controlling Neurogenesis and Reduces Human GABAergic Neurogenesis

It is well established that persistent viral infection may impair cellular function of specialized cells without overt damage. This concept, when applied to neurotropic viruses, may help to understand certain neurologic and neuropsychiatric diseases. Borna disease virus (BDV) is an excellent example of a persistent virus that targets the brain, impairs neural functions without cell lysis, and ultimately results in neurobehavioral disturbances. Recently, we have shown that BDV infects human neural progenitor cells (hNPCs) and impairs neurogenesis, revealing a new mechanism by which BDV may interfere with brain function. Here, we sought to identify the viral proteins and molecular pathways that are involved. Using lentiviral vectors for expression of the bdv-p and bdv-x viral genes, we demonstrate that the phosphoprotein P, but not the X protein, diminishes human neurogenesis and, more particularly, GABAergic neurogenesis. We further reveal a decrease in pro-neuronal factors known to be involved in neuronal differentiation (ApoE, Noggin, TH and Scg10/Stathmin2), demonstrating that cellular dysfunction is associated with impairment of specific components of the molecular program that controls neurogenesis. Our findings thus provide the first evidence that a viral protein impairs GABAergic human neurogenesis, a process that is dysregulated in several neuropsychiatric disorders. They improve our understanding of the mechanisms by which a persistent virus may interfere with brain development and function in the adult.     

Two Different Epidemiological Scenarios of Border Disease in the Populations of Pyrenean chamois (Rupicapra p. pyrenaica) after the First Disease Outbreaks

Since 2001 several outbreaks of a new disease associated with Border disease virus (BDV) infection have caused important declines in Pyrenean chamois (Rupicapra pyrenaica) populations in the Pyrenees. The goal of this study was to analyze the post-outbreak BDV epidemiology in the first two areas affected by disease with the aim to establish if the infection has become endemic. We also investigated if BDV infected wild and domestic ruminants sharing habitat with chamois. Unexpectedly, we found different epidemiological scenarios in each population. Since the disease outbreaks, some chamois populations recuperated quickly, while others did not recover as expected. In chamois from the first areas, prevalence was high (73.47%) and constant throughout the whole study period and did not differ between chamois born before and after the BDV outbreak; in all, BDV was detected by RT-PCR in six chamois. In the other areas, prevalence was lower (52.79%) and decreased during the study period; as well, prevalence was significantly lower in chamois born after the disease outbreak. No BDV were detected in this population. A comparative virus neutralisation test performed with four BDV strains and one Bovine viral diarrhoea virus (BVDV) strain showed that all the chamois had BDV-specific antibodies. Pestivirus antibodies were detected in all the rest of analyzed species, with low prevalence values in wild ruminants and moderate values in domestic ruminants. No viruses were detected in these species. These results confirm the hypothesis that outbreaks of BDV infection only affect the Pyrenean chamois, although other wild ruminants can occasionally be infected. In conclusion, two different scenarios have appeared since the first border disease outbreaks in Pyrenean chamois: on the one hand frequent BDV circulation with possible negative impact on population dynamics in some areas and on the other, lack of virus circulation and quick recovery of the chamois population.     

The evolution of the spindlin gene in birds: sequence analysis of an intron of the spindlin W and Z gene reveals four major divisions of the Psittaciformes

The Psittaciformes (parrots, parakeets) are among the most widely held captive birds. Yet, their evolution and their phylogenetic relationships have been relatively little studied. This paper describes the phylogenetic relationships between a number of Psittaciformes as derived from the sequences of the third intron of the Z-chromosomal and W-chromosomal spindlin genes. The Z-chromosomal sequences of the kakapo (Strigops habroptilus), the kea (Nestor notabilis), and the kaka (Nestor meridionalis) from New Zealand form a cluster which is the sister group to all other Psittaciformes. The results show further that the Z-chromosomal sequences of the other species can be divided into two groups based on the occurrence of a sequence element ACCCT. The group with the insert (A) is mainly from species with an Australasian geographical distribution and includes such species as the Lories (Lorius, etc.), the budgerigar (Melospittacus undulatus), and the rosellas (Platycercus). It also includes the African lovebirds (Agapornidae), which are the only representative of group A outside Australasia. Group B, without the insert, includes the neotropical parrots and parakeets such as the amazons (Amazona, etc.), the macaws (Ara, etc.), and the conures (Aratinga, etc.), the Australian Cacatuini and the African species such as the African grey parrot (Psittacus erithacus) as well as Coracopsis vasa from Madagascar and Psittrichas fulgidus from New Guinea. The W-chromosomal sequence data show that another division of the Psittacidae is found in the replacement of a pyrimidine-rich segment occurring in many non-psittacines as well as the kakapo (S. habroptilus), the kea (N. notabilis), the kaka (N. meridionalis), and the Cacatuini by a microsatellite consisting of a variable number of TATTA monomers in the other Psittaciformes. The results support a Gondwanan origin of the Psittaciformes and the suggestion that paleogeographic events were a major force in psittacine divergence.     

A Novel Psittacine Adenovirus Identified During an Outbreak of Avian Chlamydiosis and Human Psittacosis: Zoonosis Associated with Virus-Bacterium Coinfection in Birds

Chlamydophila psittaci is found worldwide, but is particularly common among psittacine birds in tropical and subtropical regions. While investigating a human psittacosis outbreak that was associated with avian chlamydiosis in Hong Kong, we identified a novel adenovirus in epidemiologically linked Mealy Parrots, which was not present in healthy birds unrelated to the outbreak or in other animals. The novel adenovirus (tentatively named Psittacine adenovirus HKU1) was most closely related to Duck adenovirus A in the Atadenovirus genus. Sequencing showed that the Psittacine adenovirus HKU1 genome consists of 31,735 nucleotides. Comparative genome analysis showed that the Psittacine adenovirus HKU1 genome contains 23 open reading frames (ORFs) with sequence similarity to known adenoviral genes, and six additional ORFs at the 3′ end of the genome. Similar to Duck adenovirus A, the novel adenovirus lacks LH1, LH2 and LH3, which distinguishes it from other viruses in the Atadenovirus genus. Notably, fiber-2 protein, which is present in Aviadenovirus but not Atadenovirus, is also present in Psittacine adenovirus HKU1. Psittacine adenovirus HKU1 had pairwise amino acid sequence identities of 50.3–54.0% for the DNA polymerase, 64.6–70.7% for the penton protein, and 66.1–74.0% for the hexon protein with other Atadenovirus. The C. psittaci bacterial load was positively correlated with adenovirus viral load in the lung. Immunostaining for fiber protein expression was positive in lung and liver tissue cells of affected parrots, confirming active viral replication. No other viruses were found. This is the first documentation of an adenovirus-C. psittaci co-infection in an avian species that was associated with a human outbreak of psittacosis. Viral-bacterial co-infection often increases disease severity in both humans and animals. The role of viral-bacterial co-infection in animal-to-human transmission of infectious agents has not received sufficient attention and should be emphasized in the investigation of disease outbreaks in human and animals.     

Psittacid herpesvirus 1 and infectious laryngotracheitis virus: Comparative genome sequence analysis of two avian alphaherpesviruses

Psittacid herpesvirus 1 (PsHV-1) is the causative agent of Pacheco's disease, an acute, highly contagious, and potentially lethal respiratory herpesvirus infection in psittacine birds, while infectious laryngotracheitis virus (ILTV) is a highly contagious and economically significant avian herpesvirus which is responsible for an acute respiratory disease limited to galliform birds. The complete genome sequence of PsHV-1 has been determined and compared to the ILTV sequence, assembled from published data. The PsHV-1 and ILTV genomes exhibit similar structural characteristics and are 163,025 bp and 148,665 bp in length, respectively. The PsHV-1 genome contains 73 predicted open reading frames (ORFs), while the ILTV genome contains 77 predicted ORFs. Both genomes contain an inversion in the unique long region similar to that observed in pseudorabies virus. PsHV-1 is closely related to ILTV, and it is proposed that it be assigned to the Iltovirus genus. These two avian herpesviruses represent a phylogenetically unique clade of alphaherpesviruses that are distinct from the Marek's disease-like viruses (Mardivirus). The determination of the complete genomic nucleotide sequences of PsHV-1 and ILTV provides a tool for further comparative and functional analysis of this unique class of avian alphaherpesviruses.     

Referential learning of French and Czech labels in African grey parrots (Psittacus erithacus): Different methods yield contrasting results

Some African grey parrots (Psittacus erithacus), the most famous being Pepperberg's parrot Alex, are able to imitate human speech and produce labels referentially. In this study, the aim was to teach ten African grey parrots from two laboratories to label items. Training three parrots from the first laboratory for several months with the Model/Rival method, developed by Pepperberg, in which two humans interact in front of the subject to demonstrate the use of a label, led to disappointing results. Similarly, seven parrots from the second laboratory, having been trained with several variants of Model/Rival attained little success. After the informal observation of the efficiency of other methods (i.e. learning to imitate labels either spontaneously or with specific learning methods and use of these labels referentially), four different teaching methods were tested with two birds: the Model/Rival; Repetition/Association which consisted of repeating a label and presenting the item only when the parrot produced the label; Intuitive in which the experimenter handled an item and repeated its name in front of the subject; Diffusion in which labels with either variable or flat intonation were played back daily to parrots. One bird learned three labels, one of which was used referentially, with the Repetition/Association method. He learned one label non-referentially with the Model/Rival but no labels were acquired using the other methods. The second bird did not learn any labels. This study demonstrates that different methods can be efficient to teach labels referentially and it suggests that rearing conditions and interindividual variability are important features when assessing learning ability of African grey parrots.