Detection and partial genetic characterisation of novel avi- and siadenoviruses in racing and fancy pigeons (Columba livia domestica).

Up to now, only a single adenovirus (AdV) isolate seemingly specific for pigeons, hence named pigeon AdV-1 (PiAdV-1), has been characterised at DNA sequence level. In the present work, the prevalence and diversity of AdVs occurring in domestic pigeon were examined by a survey performed on randomly collected samples using a very efficient, consensus nested PCR targeting the viral DNA polymerase gene. The newly detected viruses were characterised by sequencing and phylogeny analysis. Amplification of additional genome fragments was attempted by the use of several other PCR methods aiming at the hexon gene. During a 4-year survey, samples from dead or live, healthy pigeons originating from 27 lofts were examined in Hungary. Almost 50% of the samples (48 out of 97) proved to be positive for AdV. Sequence analysis revealed the presence of four hitherto unknown pigeon AdV types. PiAdV-1 was also identified in one sample. Two novel viruses named PiAdV-2 and -3 were found to belong to the genus Aviadenovirus, and two other novel types (PiAdV-4 and -5) to the genus Siadenovirus. This is the first report on the occurrence of siadenoviruses in birds belonging to the order Columbiformes. Approximately two-thirds of the PiAdV-2 genome was sequenced and analysed.

Proteomic analysis of purified turkey adenovirus 3 virions.

Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesis. Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified TAdV-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. Our analysis resulted in the identification of 13 viral proteins associated with TAdV-3 virions including a novel uncharacterized TaV3gp04 protein. Further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. Notably, seven of these host proteins have not yet been reported to be present in any other purified virus. In addition, five of these proteins are known antiviral host restriction factors. The availability of reagents allowed us to identify two cellular proteins (collagen alpha-1 (VI) chain and haemoglobin) in the purified TAdV-3 preparations. These results represent the first comprehensive proteomic profile of TAdV-3 and may provide information for illustrating TAdV-3 replication and pathogenesis.

Turkey adenovirus 3: ORF1 gene sequence comparison between vaccine-like and field strains.

Haemorrhagic enteritis is an economically significant disease reported in the majority of the countries where turkeys are raised intensively; it is caused by Turkey adenovirus 3 (TAdV-3). The aim of this study was to analyse and compare the ORF1 gene 3' region from turkey haemorrhagic enteritis virus (THEV) vaccine-like and field strains in order to develop a molecular diagnostic method to differentiate the strains from each other. Eighty samples were analysed by sequencing and phylogenetic analyses using a new set of polymerase chain reaction (PCR) primers targeting a genomic region spanning the partial ORF1, hyd and partial IVa2 gene sequences. A commercial live vaccine was also included in the analysis. The results showed that 56 of the 80 sequences obtained in this study showed ≥99.8% nucleotide identity with the homologous vaccine strain sequence. Three non-synonymous mutations - ntA1274G (aaI425V), ntA1420C (aaQ473H) and ntG1485A (aaR495Q) - were detected in the THEV field strains but not in the vaccine strain. Phylogenetic analysis confirmed the clustering of the field and vaccine-like strains in different phylogenetic branches. In conclusion, the method employed in this study could be a useful tool towards making a correct diagnosis. The data could contribute to the knowledge of field distribution of THEV strains and increase the limited existing information available on native isolates around the world.

A screening of wild bird samples enhances our knowledge about the biodiversity of avian adenoviruses.

Wild birds are threatened by anthropic effects on a global scale, and their adenoviruses might contribute to their endangerment. Thus, it is important to reveal the real biodiversity of avian adenoviruses, as, unfortunately, this research topic is far from being prioritized. The turkey hemorrhagic enteritis is an economically important disease causing high mortalities, and its causative siadenoviral agent is only distantly related to other avian siadenoviruses in phylogenetic analyses. Both to enhance our knowledge about the biodiversity of wild bird adenoviruses and to possibly trace back the origin of the turkey hemorrhagic enteritis virus, numerous Hungarian wild bird samples were screened for adenoviruses using PCR, and the detected strains were typed molecularly. The screening revealed numerous new adenovirus types, several of which represent novel adenovirus species as well, in the genera Atadenovirus, Aviadenovirus and Siadenovirus.

The genome and phylogenetic analyses of tit siadenoviruses reveal both a novel avian host and viral species.

In both a Eurasian blue tit (Cyanistes caeruleus) and a great tit (Parus major), found dead in North Rhine-Westphalia, Germany, intranuclear inclusion bodies were observed in the kidneys during the histologic examination. Siadenoviruses were detected in both samples, and the nucleotide sequence of the partial DNA polymerase, obtained from the blue tit, was almost identical with that of great tit adenovirus type 1, reported from Hungary previously. The sequence, derived from the German great tit sample was more similar to great tit adenovirus 2, yet divergent enough to forecast the possible establishment of a novel viral type and species. Therefore, the complete genome was subjected to next generation sequencing. The annotation revealed a typical siadenoviral genome layout, and phylogenetic analyses proved the distinctness of the novel virus type: great tit adenovirus 3. We propose the establishment of a new species (Siadenovirus carbocapituli) within the genus Siadenovirus to contain great tit adenovirus types 2 and 3. As both of the newly-detected viruses originated from histologically confirmed cases, and several siadenoviruses have been associated with avian nephritis earlier, we assume that the renal pathology might have been also of adenoviral origin. Additionally, we performed structural studies on two virus-coded proteins. The viral sialidase and the fiber knob were modeled using the AlphaFold2 program. According to the results of the sialic acid docking studies, the fiber trimer of the new great tit adenovirus 3 binds this acid with good affinity. As sialic acid is expressed in the kidney, it can be hypothesized that it is used during the receptor binding and entry of the virus. Strong binding of sialic acid was also predictable for the viral sialidase albeit its enzymatic activity remains disputable since, within its catalytic site, an asparagine residue was revealed instead of the conserved aspartic acid.

Circulation and Molecular Characterization of Hemorrhagic Enteritis Virus in Commercial Turkey and Meat Chicken Flocks in Australia.

Currently, there is no available vaccine against hemorrhagic enteritis virus (HEV) in Australia. Although it is assumed that subclinical HEV infections occur and may be associated with an increase in colibacillosis in Australian commercial turkey flocks, the prevalence of infection with this virus in the country is largely unknown. The aims of this study were to determine the extent of HEV infection in commercial flocks in Australia and to investigate the diversity of Australian HEV strains. Serum and spleen samples were collected from breeder and grower turkeys and serum was collected from breeder and grower chickens by the two major poultry integrator companies in Australia. Of the turkey samples, 727/849 (86%) sera were positive for anti-HEV antibodies by ELISA. HEV DNA was detected in 215/278 (77%) spleen samples positive by PCR. Of the meat chicken sera, 115/144 (80%) samples were seropositive. Sequencing the whole genome of three HEV field isolates showed that the Australian strains are highly similar and cluster separately from strains from other geographic regions although several point mutations were shared with HEV strains considered to be virulent. In conclusion, HEV infection is ubiquitous in Australian commercial poultry flocks. The impact of the many genomic point mutations detected in Australian HEV strains on virus pathogenicity is unclear.

Circulación y caracterización molecular del virus de la enteritis hemorrágica en parvadas comerciales de pavo y pollos de engorde en Australia. Actualmente, no existe una vacuna disponible contra el virus de la enteritis hemorrágica (HEV) en Australia. Aunque se supone que se producen infecciones subclínicas por el virus de la enteritis hemorrágica y pueden estar asociadas con un aumento de la colibacilosis en las parvadas comerciales de pavos australianos, se desconoce en gran medida la prevalencia de la infección por este virus en el país. Los objetivos de este estudio fueron determinar la diseminación de la infección por el virus de la enteritis hemorrágica en parvadas comerciales en Australia e investigar la diversidad de cepas del virus de la enteritis hemorrágica australianas. Se recolectaron muestras de suero y bazo de pavos reproductores y de engorda y las dos principales empresas integradoras avícolas de Australia recolectaron suero de pollos reproductores y de engorde. De las muestras de pavo, 727/849 (86%) sueros fueron positivos para anticuerpos contra la enteritis hemorrágica por ELISA. Se detectó ADN del virus de la enteritis hemorrágica en 215/278 (77%) muestras de bazo positivas por PCR. De los sueros de carne de pollo, 115/144 (80%) muestras fueron seropositivas. La secuenciación del genoma completo de tres aislados de campo del virus de la enteritis hemorrágica mostró que las cepas australianas son muy similares y se agrupan por separado de las cepas de otras regiones geográficas, aunque se compartieron varias mutaciones puntuales con las cepas del virus de la enteritis hemorrágica consideradas virulentas. En conclusión, la infección por el virus de la enteritis hemorrágica es ubicua en las parvadas avícola comerciales australianas. No está claro el impacto de las diferentes mutaciones puntuales genómicas detectadas en las cepas australianas del virus de la enteritis hemorrágica sobre la patogenicidad del virus.

Genomic Characterisation of a Highly Divergent Siadenovirus (Psittacine Siadenovirus F) from the Critically Endangered Orange-Bellied Parrot (Neophema chrysogaster).

Siadenoviruses have been detected in wild and captive birds worldwide. Only nine siadenoviruses have been fully sequenced; however, partial sequences for 30 others, many of these from wild Australian birds, are also described. Some siadenoviruses, e.g., the turkey siadenovirus A, can cause disease; however, most cause subclinical infections. An example of a siadenovirus causing predominately subclinical infections is psittacine siadenovirus 2, proposed name psittacine siadenovirus F (PsSiAdV-F), which is enzootic in the captive breeding population of the critically endangered orange-bellied parrot (OBP, Neophema chrysogaster). Here, we have fully characterised PsSiAdV-F from an OBP. The PsSiAdV-F genome is 25,392 bp in length and contained 25 putative genes. The genome architecture of PsSiAdV-F exhibited characteristics similar to members within the genus Siadenovirus; however, the novel PsSiAdV-F genome was highly divergent, showing highest and lowest sequence similarity to skua siadenovirus A (57.1%) and psittacine siadenovirus D (31.1%), respectively. Subsequent phylogenetic analyses of the novel PsSiAdV-F genome positioned the virus into a phylogenetically distinct sub-clade with all other siadenoviruses and did not show any obvious close evolutionary relationship. Importantly, the resulted tress continually demonstrated that novel PsSiAdV-F evolved prior to all known members except the frog siadenovirus A in the evolution and possibly the ancestor of the avian siadenoviruses. To date, PsSiAdV-F has not been detected in wild parrots, so further studies screening PsSiAdV-F in wild Australian parrots and generating whole genome sequences of siadenoviruses of Australian native passerine species is recommended to fill the siadenovirus evolutionary gaps.

Molecular Characterization of Hemorrhagic Enteritis Virus (HEV) Obtained from Clinical Samples in Western Canada 2017-2018.

Hemorrhagic enteritis virus (HEV) is an immunosuppressive adenovirus that causes an acute clinical disease characterized by hemorrhagic gastroenteritis in 4-week-old turkeys and older. Recurrent incidence of secondary infections (e.g., systemic bacterial infections, cellulitis, and elevated mortality), may be associated with the presence of field-type HEV in Canadian turkey farms. We speculate that field-type HEV and vaccine/vaccine-like strains can be differentiated through analysis of the viral genomes, hexon genes, and the specific virulence factors (e.g., ORF1, E3, and fib knob domain). Nine out of sixteen spleens obtained from cases suspected of immunosuppression by HEV were analyzed. The limited data obtained showed that: (1) field-type HEV circulates in many non-vaccinated western Canadian flocks; (2) field-type HEV circulates in vaccinated flocks with increased recurrent bacterial infections; and (3) the existence of novel point mutations in hexon, ORF1, E3, and specially fib knob domains. This is the first publication showing the circulation of wild-type HEV in HEV-vaccinated flocks in Western Canada, and the usefulness of a novel procedure that allows whole genome sequencing of HEV directly from spleens, without passaging in cell culture or passaging in vivo. Further studies focusing more samples are required to confirm our observations and investigate possible vaccination failure.

Confirmation of a novel siadenovirus species detected in raptors: partial sequence and phylogenetic analysis.

Partial genome characterisation of a novel adenovirus, found recently in organ samples of multiple species of dead birds of prey, was carried out by sequence analysis of PCR-amplified DNA fragments. The virus, named as raptor adenovirus 1 (RAdV-1), has originally been detected by a nested PCR method with consensus primers targeting the adenoviral DNA polymerase gene. Phylogenetic analysis with the deduced amino acid sequence of the small PCR product has implied a new siadenovirus type present in the samples. Since virus isolation attempts remained unsuccessful, further characterisation of this putative novel siadenovirus was carried out with the use of PCR on the infected organ samples. The DNA sequence of the central genome part of RAdV-1, encompassing nine full (pTP, 52K, pIIIa, III, pVII, pX, pVI, hexon, protease) and two partial (DNA polymerase and DBP) genes and exceeding 12 kb pairs in size, was determined. Phylogenetic tree reconstructions, based on several genes, unambiguously confirmed the preliminary classification of RAdV-1 as a new species within the genus Siadenovirus. Further study of RAdV-1 is of interest since it represents a rare adenovirus genus of yet undetermined host origin.

Systemic adenovirus infection in Sulawesi tortoises (Indotestudo forsteni) caused by a novel siadenovirus.

A novel siadenovirus was identified in the Sulawesi tortoise (Indotestudo forsteni). A group of 105 Sulawesi tortoises was obtained by the Turtle Survival Alliance. Many of the tortoises were in poor health. Clinical signs included anorexia, lethargy, mucosal ulcerations and palatine erosions of the oral cavity, nasal and ocular discharge, and diarrhea. Initial diagnostic tests included fecal testing for parasites, complete blood count and plasma biochemical analysis, mycoplasma serology, and polymerase chain reaction (PCR) testing for intranuclear coccidia and chelonian herpesvirus. Treatment included administration of antibiotics, antiparasitic medications, parenteral fluids, and nutritional support. Tissue samples from animals that died were submitted for histopathologic evaluation. Histopathologic examination revealed systemic inflammation and necrosis associated with intranuclear inclusions consistent with a systemic viral infection in 35 tortoises out of 50 examined. Fecal testing results and histopathologic findings revealed intestinal and hepatic amoebiasis and nematodiasis in 31 animals. Two of 5 tortoises tested by PCR were positive for Chlamydophila sp. Aeromonas hydrophila and Escherichia coli were cultured from multiple organs of 2 animals. The mycoplasma serology and PCR results for intranuclear coccidia and chelonian herpesvirus were negative. Polymerase chain reaction testing of tissues, plasma, and choanal/cloacal samples from 41 out of 42 tortoises tested were positive for an adenovirus, which was characterized by sequence analysis and molecular phylogenetic inference as a novel adenovirus of the genus Siadenovirus. The present report details the clinical and anatomic pathologic findings associated with systemic infection of Sulawesi tortoises by this novel Siadenovirus, which extends the known reptilian adenoviruses to the chelonians and extends the known genera of reptilian Adenoviridae beyond Atadenovirus to include the genus Siadenovirus.

In vitro growth kinetics and gene expression analysis of the turkey adenovirus 3, a siadenovirus.

Turkey adenovirus 3 (TAdV-3) belongs to the genus Siadenovirus, family Adenoviridae. Previously, nucleotide sequencing and annotation of the Virginia avirulent strain (VAS) of TAdV-3 genome, isolated in our laboratory, indicated the presence of a total of 23 genes and open reading frames (ORFs). The goals of this study were 1) to delineate the growth kinetics of the virus using a qPCR-based infectivity assay, and 2) to determine the virus gene expression profile during the early and late phases of infection in target B lymphocytes. The one-step growth curve experiment demonstrated 3 phases of virus replication cycle: a lag phase lasted for 12-18 h post-infection (h.p.i.), in which the virus titer declined; a log phase from 18 to 120 h.p.i., in which the number of infectious virus particles increased over 20,000 folds, and a brief decline phase thereafter. Southern blot analysis indicated that the synthesis of new viral DNA started by 8 h.p.i. Gene-specific RT-PCR analysis revealed the expression of mRNAs from the 23 TAdV-3 genes/ORFs. According to the temporal transcriptional profiling of TAdV-3 genome, genes could be divided into 3 groups based on the time of transcription initiation: group 1 showed detectable levels of transcription at 2 h.p.i and included 7 genes, i.e., hyd, III, pX, pVI, II, 100 K, and 33 K; group 2 included 12 genes whose mRNAs were detected for the first time at 4 h.p.i., i.e., ORF1, IVa2, pol, pTP, pIIIa, EP, DBP, E3, U exon, IV, ORF7, and ORF8; group 3 of transcripts were detectable starting 8 h.p.i. and included only 4 genes, i.e., 52 K, 22 K, pVII, and pVIII. Our data suggest that the transcriptional kinetics of genus Siadenovirus differ from that observed in other adenoviral genera; however, a few TAdV-3 genes showed similar expression patterns to their adenoviral homologs.

Histopathologic Changes, Ultrastructure, and Molecular Characterization of an Adenovirus in a Sun Conure (Aratinga solstitialis).

In this case report, we describe the pathologic changes and the ultrastructural and molecular characteristics of an adenovirus in a sun conure (Aratinga solstitialis) that presented with a history of sudden death. On histologic examination, there was multifocal hepatic and splenic necrosis. Within some hepatocytes and unidentified cells in the spleen, renal interstitial fibroblasts, and ovarian stroma were intranuclear amphophilic inclusion bodies. Electron microscopy of affected tissue showed intranuclear icosahedral viral particles with an inner capsid (29.2-33.8 nm in diameter) and an outer capsid (70.2-71.7 nm in diameter). Next-generation sequencing and BLAST analysis of complementary DNA synthesized from RNA extracted from formalin-fixed tissues showed an adenovirus, designated sun conure adenovirus (SCAdv). A DNA in situ hybridization (ISH) probe, constructed from the SCAdv and similar sequences from GenBank, was also positive in the intranuclear inclusion bodies, whereas standard ISH for psittacine adenovirus 1 was negative. These results show that ancillary diagnostic testing, such as next-generation sequencing, even using formalin-fixed, paraffin-embedded tissues, along with ISH, can be useful in identifying additional, unknown viruses that show similar pathology to commonly known viruses but do not show up as positive on routine diagnostic tests.

Reporte de caso- Cambios histopatológicos, ultraestructura y caracterización molecular de un adenovirus en una cotorra solar (Aratinga solstitialis). En este reporte de caso, se describen los cambios patológicos y las características ultraestructurales y moleculares de un adenovirus en una cotorra solar (Aratinga solstitialis) que se presentó con un historial de muerte súbita. En el examen histológico, hubo necrosis hepática y esplénica multifocal. Dentro de algunos hepatocitos y células no identificadas en el bazo, los fibroblastos intersticiales renales y en el estroma ovárico se encontraron cuerpos de inclusión anfofílicos intranucleares. La microscopía electrónica del tejido afectado mostró partículas víricas intranucleares icosaédricas con una cápside interna (de 29.2 a 33.8 nm de diámetro) y una cápside externa (de 70.2 a 71.7 nm de diámetro). Mediante el análisis de secuenciación de segunda generación y por la Herramienta de Búsqueda de Alineaciones Local Básica (con siglas en inglés BLAST) del ADN complementario sintetizado a partir de ARN extraído de tejidos fijados con formalina mostraron un adenovirus, denominado adenovirus de cotorra solar (SCAdv). Se construyó una sonda de ADN para hibridación in situ (ISH), a partir de la secuencia del virus SCAdv y de secuencias similares de GenBank, que generó reacción positiva en los cuerpos de inclusión intranucleares, mientras que la hibridación in situ estándar para el adenovirus I de psitácidos fue negativa. Estos resultados muestran que las pruebas de diagnóstico complementarias, como la secuenciación de segunda generación, utilizando tejidos fijados con formalina e incluidos en parafina junto con la hibridación in situ pueden ser útiles para identificar virus adicionales desconocidos que muestran una patología similar a los virus comúnmente conocidos, pero que no se detectan con las pruebas diagnósticas de rutina.

Novel siadenovirus infection in a cockatiel with chronic liver disease.

A 15-year-old female cockatiel (Nymphicus hollandicus) undergoing long term management for hepatopathy died and underwent necropsy. Microscopic findings were consistent with chronic liver disease characterized by distorted hepatic architecture, fibrosis and biliary proliferation. The additional finding of large intranuclear inclusion bodies within hepatocytes and renal tubular epithelium prompted diagnostic next generation sequencing. The assembled sequences isolated from pooled kidney and liver were related to siadenoviruses. The genus Siadenovirus, within the family Adenoviridae, includes several species of viruses that pathogenically infect avian species including hemorrhagic enteritis virus of turkeys and marble spleen virus of pheasants. Siadenoviruses have previously been reported in seven psittacine species: a plum-headed parakeet (Psittacula cyanocephala), an umbrella cockatoo (Cacatua alba) budgerigars (Melopsittacus undulates), an eastern rosella (Platycercus eximius), a scarlet chested parrot (Neophema splendida), a cockatiel (Nymphicus hollandicus), and a red-crowned parakeet (Cyanoramphus novaezelandiae). This report describes a novel siadenovirus in a cockatiel that is highly identical to budgerigar adenovirus 1 and distinct from PsAdV-2 in cockatiels. We report the clinical pathologic, gross, and histopathologic findings in a cockatiel with chronic hepatitis and a novel siadenovirus, PsAdV-5. The sequencing data is presented with a phylogenetic analysis.

Investigation of field outbreaks of turkey haemorrhagic enteritis in Hungary.

Epidemiological, pathological, serological and virological investigations are reported on turkey haemorrhagic enteritis virus (THEV) infection in Hungarian turkey flocks. The pathogenesis of infection in experimentally infected turkeys and chickens, as well as the usefulness of polymerase chain reaction (PCR)/sequencing method for epidemiological investigation and for the differentiation of vaccine and field strains of THEV was also studied. Since the first recognition of the disease in Hungary in the late 1970s, until recently the disease has been diagnosed sporadically in its mild form. In the last few years (2000-2005), however, the number of outbreaks and the severity of the disease increased (9-23 affected flocks/year). Most of the outbreaks occurred at the age of 6 to 8 weeks and was complicated with Escherichia coli infection. The antibody levels to THEV in turkey flocks gradually declined till 5-7 weeks of age, and then they increased sharply due to natural infection with THEV. The immune response to vaccination (at 5 weeks of age) showed no significant antibody level increase one week postvaccination, but four weeks later the antibody level reached high values and then remained at this high level. The agar gel immunodiffusion (AGID) test to detect turkey adenovirus A (TAdV-A) antigen and PCR methods for THEV-specific DNA gave similarly positive results if spleens with pathognomonic lesions were tested; however, PCR proved to be more sensitive in cases with less characteristic pathological lesions. Nucleotide sequence alignment of PCR products amplified from Hungarian field strains and the Domermuth vaccine strain and that of the published THEV hexon sequences in GenBank database revealed slight differences between the sequences.

Haemorrhagic enteritis of turkeys - current knowledge.

Haemorrhagic enteritis virus (HEV), an adenovirus associated with acute haemorrhagic gastro-intestinal disease of 6-11-week old turkeys predominantly hampers both humoral and cellular immunity. Affected birds are more prone to secondary complications (e.g. colibacillosis and clostridiosis) and failure to mount an effective vaccine-induced immune response. HEV belongs to the new genus Siadenovirus. Feco-oral transmission is the main route of entry of the virus and it mainly colonizes bursa, intestine and spleen. Both naturally occurring virulent and avirulent strains of HEVs are serologically indistinguishable. Recent findings revealed that ORF1, E3 and fib genes are the key factors affecting virulence. The adoption of suitable diagnostic tools, proper vaccination and biosecurity measures have restrained the occurrence of disease epidemics. For diagnostic purposes, the best source of HEV is either intestinal contents or samples from spleen. For rapid detection highly sensitive and specific tests such as quantitative real-time PCR based on Taq man probe has been designed. Avirulent strains of HEV or MSDV can be effectively used as live vaccines. Novel vaccines include recombinant hexon protein-based subunit vaccines or recombinant virus-vectored vaccines using fowl poxvirus (FPV) expressing the native hexon of HEV. Notably, subunit vaccines and recombinant virus vectored vaccines altogether offer high protection against challenge or field viruses. Herein, we converse a comprehensive analysis of the HEV genetics, disease pathobiology, advancements in diagnosis and vaccination along with appropriate prevention and control strategies.

Molecular Characterization of Hemorrhagic Enteritis Viruses (HEV) Detected in HEV-Vaccinated Commercial Turkey Flocks in Germany.

Despite the application of live hemorrhagic enteritis virus (HEV) vaccines, HEV field outbreaks are suspected to still occur in turkey flocks in Germany. Increasing secondary bacterial infections in HEV-vaccinated flocks suggest that vaccines may be losing efficacy or, possibly, that vaccine strains are causing disease. Thus, the goal of the current study was to investigate the diversity of HEV isolates from fattening turkey flocks between 2008 and 2012 by characterizing the open reading frame (ORF)1 gene at its 5' and 3' ends. Analyses of ORF1 sequences of field isolates and comparison with sequences present in databases revealed that in many cases (13 out of 16 samples), vaccine (avirulent) strains were present. In addition, data indicated the circulation of suspected virulent field isolates and these isolates (3 out of 16) cluster with an early isolate from Germany in the 1980s, but show some mutations in the predicted amino acid (aa) sequences of ORF1 compared to the early isolate. These virulent isolates clearly differ from the spleen-derived avirulent Domermuth vaccine strain used in Germany. In this study, a unique isolate was identified and showed unusual nucleotide mutations that resulted in aa exchanges at the 5' end of ORF1 between aa positions 34 and 174. This genetic drift suggests evolution of HEV including virulent and vaccine-derived strains in the field. This may lead to evasion of vaccinal immunity by drifted viruses and/or an increase in the virulence of field strains.

Porcine adenovirus as a delivery system for swine vaccines and immunotherapeutics.

Porcine adenovirus (PAdV) has many qualities which make it an ideal choice for use as a delivery vector in swine. It is a low grade pathogen, present almost world-wide in a number of serotypes varying in their virulence and tissue tropism, which may allow for serotype specific vaccine targeting. PAdV is species specific having only been isolated from swine, reducing the possibility of its spread to other animals or man following administration. When engineered to contain a foreign gene, recombinant PAdV (rPAdV) can be grown to high titres in tissue culture cells making it cheap to produce. Knowledge of the complete nucleotide sequence of the PAdV genome has enabled rationally directed insertions of foreign genes which remain stably inserted in the genome and can be expressed at high levels following delivery to the target host. Importantly, recombinant PAdV can be administered by injection or by the oral route in feed or drinking water. We have delivered a range of antigens and immunomodulatory molecules to commercially available pigs using rPAdV and found it to be a very effective delivery system. Significantly, recombinant PAdV serotype 3 is highly effective as a delivery vehicle even when administered in the face of high levels of artificially induced serotype specific neutralising antibody to the vector.

Structure and Sialyllactose Binding of the Carboxy-Terminal Head Domain of the Fibre from a Siadenovirus, Turkey Adenovirus 3.

The virulent form of turkey adenovirus 3 (TAdV-3), also known as turkey hemorrhagic enteritis virus (THEV), is an economically important poultry pathogen, while the avirulent form is used as a vaccine. TAdV-3 belongs to the genus Siadenovirus. The carboxy-terminal region of its fibre does not have significant sequence similarity to any other adenovirus fibre heads of known structure. Two amino acid sequence differences between virulent and avirulent TAdV-3 map on the fibre head: where virulent TAdV-3 contains Ile354 and Thr376, avirulent TAdV-3 contains Met354 and Met376. We determined the crystal structures of the trimeric virulent and avirulent TAdV-3 fibre head domains at 2.2 Å resolution. Each monomer contains a beta-sandwich, which, surprisingly, resembles reovirus fibre head more than other adenovirus fibres, although the ABCJ-GHID topology is conserved in all. A beta-hairpin insertion in the C-strand of each trimer subunit embraces its neighbouring monomer. The avirulent and virulent TAdV-3 fibre heads are identical apart from the exact orientation of the beta-hairpin insertion. In vitro, sialyllactose was identified as a ligand by glycan microarray analysis, nuclear magnetic resonance spectroscopy, and crystallography. Its dissociation constant was measured to be in the mM range by isothermal titration calorimetry. The ligand binds to the side of the fibre head, involving amino acids Glu392, Thr419, Val420, Lys421, Asn422, and Gly423 binding to the sialic acid group. It binds slightly more strongly to the avirulent form. We propose that, in vivo, the TAdV-3 fibre may bind a sialic acid-containing cell surface component.

A novel adenovirus in Chinstrap penguins (Pygoscelis antarctica) in Antarctica.

Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

A novel siadenovirus detected in the kidneys and liver of Gouldian finches (Erythura gouldiae).

A novel siadenovirus was found in six captive Gouldian finches (Erythrura gouldiae) in the United States and Hungary. Histopathological examination revealed inclusions in the kidney of the captive Gouldian finch in the United States, and virions morphologically consistent with adenoviruses were seen by electron microscopy. Partial sequence of the DNA-dependent DNA polymerase gene was gained by consensus PCR and sequencing in all six finches, and all proved to be identical. In one Hungarian finch, additional sequence was obtained from the DNA polymerase gene, the pre-terminal protein (pTP) gene, the 52k gene, and the hexon gene. Bayesian, maximum likelihood, and distance-based analyses showed the novel virus clusters with the siadenoviruses, and is herein referred to as Gouldian finch adenovirus 1. The genes looked at in this study had low G+C percentages, which is common in the genus Siadenovirus, and suggestive of recent host switch. The significance of this virus' presence is unknown at this time as clinical signs of positive birds varied.