DNA Polymerase Gene Sequences Research Articles

Prevalence and Sequence Analysis of Equine Rhinitis Viruses among Horses in Poland.

Equine rhinitis A (ERAV) and B (ERBV) viruses are respiratory pathogens with worldwide distribution. The current study aimed to determine the frequency of infection of ERAV and ERBV among horses and foals at Polish national studs, and to determine genetic variability within the viruses obtained. Virus-specific quantitative RT-PCR assays targeting a 5' untranslated region were used to screen nasal swabs collected from 621 horses at 16 national horse studs from throughout Poland, including 553 healthy horses and 68 horses with respiratory disease. A partial DNA polymerase gene was amplified and sequenced from the qRT-PCR-positive samples. The obtained sequences were analysed using phylogeny and genetic network analysis. None of the nasal swabs were positive for ERAV, whereas ERBV was found in 11/621 (1.78%) samples collected from 10 healthy horses and one foal affected by respiratory disease. Partial DNA polymerase gene sequence variability was correlated with individual horses and studs from which samples were collected when only Polish sequences were analysed, but there was no correlation between country of origin and ERBV sequence when Polish and international sequences were included in the network. The report presents the first detection of ERBV in Poland.

Analisis filogenetik Hyposidra talaca nucleopolyhedrovirus (HytaNPV) yang diisolasi dari perkebunan teh Gunung Mas, Bogor, Jawa Barat dan virulensinya terhadap Hyposidra talaca Walker

Hyposidra talaca (Walker) is an important pest of tea plant. H. talaca can cause losses of between 40–100% in the dry season if proper control is not carried out. H. talaca has natural enemies such as predators, parasitoids, and pathogens. One of the entomopathogens is NPV. This study aims to obtain molecular characteristics through DNA polymerase sequences and determine the virulence level of NPV isolates from H. talaca. The same species from different locations can have genetic variability. Therefore, molecular characterization by polymerase chain reaction (PCR) on DNA polymerase sequences is one way to study the genetics of HytaNPV. NPV was isolated from infected H. talaca larvae collected from the field. The DNA isolates were used as templates for PCR for DNA polymerase gene amplification with an amplicon target of ±1,000 bp. A sequencing process followed the PCR provides nucleotide sequence. HytaNPV DNA polymerase sequencing results were aligned with GenBank's BLAST data to provide information on the relationship of HytaNPV to NPVs isolated from other regions. Based on molecular character analysis using DNA polymerase gene sequence, HytaNPV Bogor has a homology level of 93.9% with HytaNPV isolated from India. HytaNPV Bogor has a genetic relationship with the NPV that infects Buzura suppressaria from China and Australia. HytaNPV Bogor is similar to the NPV that infects H. talaca from India. The bioassay of HytaNPV isolate against H. talaca showed the highest LT50 value of 1.92 days was found in concentration of 1.58 x 107 POBs/ml in second instar larvae.

Adenoviral infection in 5 red-tailed hawks and a broad-winged hawk.

Adenoviral infections among raptors are best described in falcons and are characterized most commonly by necrotizing hepatitis and splenitis; only one case has been reported in a hawk. Five red-tailed hawks (Buteo jamaicensis) and a broad-winged hawk (Buteo platypterus) had an adenoviral infection based on history, histopathology, negative-stain electron microscopy, and PCR. All birds had acute onset of illness resulting in death; 3 had evidence of a concurrent bacterial infection. Microscopically, all 6 birds had solitary, pale eosinophilic-to-amphophilic, intranuclear inclusion bodies within presumed hematopoietic cells in bone marrow and macrophages in spleen. Five of the 6 birds had similar inclusions within hepatocytes and Kupffer cells. All but one bird had severe bone marrow necrosis. There was moderate splenic necrosis (3 of 6) and mild-to-marked hepatic necrosis (4 of 6). Negative-stain electron microscopy demonstrated adenoviral particles in bone marrow (5 of 6), liver (1 of 5), and/or spleen (1 of 5). PCR was positive for adenovirus in bone marrow (3 of 5), liver (1 of 3), spleen (4 of 6), and/or intestinal contents (2 of 3). Viral DNA polymerase gene sequences clustered within the Siadenovirus genus. There was 99% nucleotide identity to one another and 90% nucleotide identity with the closest related adenovirus (Harris hawk, EU715130). Our case series expands on the limited knowledge of adenoviral infections in hawks. The splenic and hepatic necrosis, and particularly the hitherto unreported bone marrow necrosis, suggest that adenoviral infection is clinically relevant and potentially fatal in hawks.

Isolation, Identification and Characterization of a Novel Megalocytivirus from Cultured Tilapia (Oreochromis spp.) from Southern California, USA.

Simple SummaryAquaculture is the world’s fastest-growing food production sector, with tilapia (Oreochromis spp.) among the most important cultured species. First reported in red seabream, Pagrus major, in 1990, an increasing number of megalocytiviruses are responsible for losses that threaten the production and economic sustainability of important cultured fish species, including tilapia. In the current study, we describe an epizootic in tilapia fingerlings from Southern California, USA and report the discovery of a novel megalocytivirus associated with the mortalities. In spring 2019, diseased four-month-old tilapia (Oreochromis spp.) from an aquaculture farm in Southern California, USA were received for diagnostic evaluation with signs of lethargy, anorexia, abnormal swimming, and low-level mortalities. At necropsy, non-specific external lesions were noted including fin erosion, cutaneous melanosis, gill pallor, and coelomic distension. Internal changes included ascites, hepatomegaly, renomegaly, splenomegaly, and multifocal yellow-white nodules in the spleen and kidney. Cultures of spleen and kidney produced bacterial colonies identified as Francisella orientalis. Homogenized samples of gill, brain, liver, spleen, and kidney inoculated onto Mozambique tilapia brain cells (OmB) developed cytopathic effects, characterized by rounding of cells and detaching from the monolayer 6–10 days post-inoculation at 25 °C. Transmission electron microscopy revealed 115.4 ± 5.8 nm icosahedral virions with dense central cores in the cytoplasm of OmB cells. A consensus PCR, targeting the DNA polymerase gene of large double-stranded DNA viruses, performed on cell culture supernatant yielded a sequence consistent with an iridovirus. Phylogenetic analyses based on the concatenated full length major capsid protein and DNA polymerase gene sequences supported the tilapia virus as a novel species within the genus Megalocytivirus, most closely related to scale drop disease virus and European chub iridovirus. An intracoelomic injection challenge in Nile tilapia (O. niloticus) fingerlings resulted in 39% mortality after 16 days. Histopathology revealed necrosis of head kidney and splenic hematopoietic tissues.

Novel herpesviruses in neotropical bats and their relationship with other members of the Herpesviridae family

In the past decade, a large number of studies have detected herpesvirus sequences from many bat species around the world. Nevertheless, the discovery of bat herpesviruses is geographically uneven. Of the various bat species tested to date, only a few were from the New World. Seeking to investigate the distribution and diversity of herpesviruses circulating in neotropical bats, we carried out molecular screening of 195 blood DNA samples from 11 species of three bat families (Phyllostomidae, Mormoopidae, and Molossidae). Using polymerase chain reaction amplification, with degenerate consensus primers targeting highly conserved amino acid motifs of the herpesvirus DNA polymerase and Glycoprotein B genes, we characterized novel viral sequences from all tested species. BLAST searches, pairwise nucleotide and amino acid sequence comparisons, as well as phylogenetic analyses confirmed that they all belonged to the Herpesviridae family, of the Beta- and Gammaherpesvirinae subfamilies. Fourteen partial DNA polymerase gene sequences, of which three beta- and 11 gamma-herpesviruses, were detected. A total of 12 partial Glycoprotein B gene sequences, all gamma-herpesviruses, were characterized. Every sequence was specific to a bat species and in some species (Desmodus rotundus, Carollia perspicillata, and Pteronotus rubiginosus) multiple viruses were found. Phylogenetic analyses of beta- and gammaherpesvirus sequences led to the identification of bat-specific clades. Those composed of sequences obtained from different bat species belonging to distinct subfamilies follow the taxonomy of bats. This study confirms the astonishing diversity of bat herpesviruses and broadens our knowledge of their host range. Nevertheless, it also emphasizes the fact that, to better appreciate the evolutionary history of these viruses, much remains to be done at various taxonomic levels.

Psittacid herpesvirus 3 infection in rose-ringed parakeets in southern Brazil

We diagnosed disease caused by psittacid herpesvirus 3 (PsHV-3), a novel psittacid pathogen, in rose-ringed parakeets (Psittacula krameri) housed in an exotic psittacine breeding colony in southern Brazil. The disease affected several adult birds. Clinical signs included apathy, tachypnea, and wheezing. Four birds were autopsied, and sections of lungs and liver were examined histologically and by electron microscopy (EM), revealing pulmonary congestion, bronchopneumonia, or multifocal necrosis of tertiary bronchi, with syncytial cells and eosinophilic intranuclear inclusion bodies. Viral particles morphologically compatible with herpesviruses were observed by EM in lung sections. PCR with pan-herpesvirus primers performed on total DNA extracted from paraffinized tissue resulted in a 278-bp product. Sequencing of the amplicon revealed 93% nucleotide identity with a PsHV-3 sequence available in GenBank. Phylogenetic analysis grouped the obtained sequence with the only PsHV-3 DNA polymerase gene sequence available (GenBank accession JX028240) and separated the sequence from psittacid herpesviruses 1 and 2. The clinical, pathologic, and molecular findings support the association of PsHV-3 with pneumonia found in these rose-ringed parakeets in southern Brazil.

Seasonal emergence of benign epidermal tumor in farm-reared adult grass carp (Ctenopharyngodon idella) caused by lymphocystis disease virus at Uttarakhand, India

From 2017‐2019, during the winter months (November–January), epidermal papilloma like benign tumor was observed in farm-reared adult grass carp, Ctenopharyngodon idella at the northern part of India. An investigation was undertaken to establish the causative agent of this epizootiology. Based on the clinical signs and ineffectiveness of antibiotics to control this disease, viral infection was suspected. A primer was designed to PCR amplify the partial DNA polymerase gene of lymphocystis disease virus (LCDV) from the tumor of moribund grass carp, which yielded 460 bp amplicon. After sequencing this 460 bp amplicon, the viral agent from the tumor of grass carp was identified as lymphocystis disease virus and given strain designation LRI-18 (Genbank accession number-MK347473). Phylogenetic analysis based on the nucleotide sequence of DNA polymerase gene of LCDV revealed the close association of LCDV LRI-18 to LCDV strain from Israel with 99.0% and 96.5% nucleotide and amino acid sequence identity, respectively. LCDV LRI-18 form single (type A; 0.5–2.0 μm) or multiple (type B; 0.5–2.0 μm) intracytoplasmic inclusion bodies on lymphocystis and erythrocyte cells of infected grass carp. Transmission Electron Microscopy (TEM) of blood revealed the presence of enveloped icosahedral virions of approximately 280 nm in diameter with electron dense core and an outer capsid. Histopathological study of various organs of LCDV infected grass carp revealed extensive hypertrophy and fusion of secondary gill lamellae, focal hemorrhage in the eye, pyknotic nuclei in the liver, inflammation in the myocardium, fused villi of intestine and degeneration of the glomeruli in the kidney. Experimental infection by a dip to grass carp fingerlings (average body weight 4.4 ± 0.9 g) using 0.45 μm filtered LCDV crude suspension did not reproduce the disease, and also no mortality were observed. This is the first report of the occurrence of lymphocystis disease in grass carp in India.

The Environment and Cyanophage Diversity: Insights From Environmental Sequencing of DNA Polymerase.

Globally distributed and abundant cyanophages in the family Myoviridae have dsDNA genomes with variable gene content, including host-derived auxiliary metabolic genes (AMGs) that potentially can facilitate viral replication. However, it is not well understood how this variation in gene content interacts with environmental variables to shape cyanomyovirus communities. This project correlated the genetic repertoire of cyanomyoviruses with their phyologeny, and investigated cyanomyovirus ecotype distribution as a function of environmental conditions across locations and seasons. Reference cyanomyovirus genomes were compared for their overlap in gene content to infer phyologenetic distances, and these distances were compared to distances calculated based on DNA polymerase (gp43) gene sequences. In turn, gp43 partial gene sequences amplified from natural cyanophage communities were used to describe cyanomyovirus community composition and to assess the relationship between environmental variables. The results showed the following: (1) DNA polymerase gene phylogeny generally correlated with the similarity in gene content among reference cyanomyoviruses, and thus can be used to describe environmental cyanomyovirus communities; (2) spatial and seasonal patterns in cyanomyovirus communities were related to environmental variables; (3) salinity and temperature, combined with nutrient concentration were predictors of cyanomyovirus richness, diversity and community composition. This study shows that environmental variables shape viral communities by drawing on a diverse seed bank of viral genotypes. From these results it is evident that that viral ecotypes with their corresponding genetic repertoires underlie selection pressures. However, the mechanisms involved in selecting for specific viral genotypes remain to be fully understood.

Molecular detection and characterization of fowl adenovirus associated with inclusion body hepatitis from broiler chickens in Egypt.

A case-control study was performed to assess prescence of inclusion body hepatitis (IBH) caused by fowl adenoviruses (FAdVs) at Kafr EL-Shiekh Governorate, Egypt, during spring, 2017. The case group consisted of 100 liver and spleen samples collected from 10 broiler chickens flocks (10 samples from each flock) suspected to be infected with IBH depending on clinical manefestations and necropsy examination. Controls were randamly selected from chickens without clinical sings or evidence of the disease on postmortem examination. Molecular screening of the disease disease in collected samples based on the DNA polymerase gene of FAdVs was carried out. Furthermore, the DNA polymerase gene sequence was determined and analyzed with published reference sequences on GeneBank. Respectively, enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm existence of co-infection with chicken infectious anemia virus (CIAV) and/or infectious bursal disease virus (IBDV in flocks involved in the study. Using PCR, FAdV genome was detected in seven flocks in the case group and one in the control group. FAdV identified in this study revealed close genetic relationship with FAdVs-D previously identified in UK and Canada, suggesting potential virus transmission from these countries. All tested serum samples from diseased chickens were positive for CIAV infection via ELISA while none of the collected bursa of Fabricius samples tested IBDV positive by RT-PCR. Therefore, results obtained from the current study highlighted the importance of implementation of control measures against FAdV and CIAV in Egyptian poultry flocks. This study opens the door for future work toward specific identification of FAdV serotypes circulating in Egyptian poultry farms and molecular characterization of the virus based on hexon gene or full genome sequencing for better understanding of genetic diversity among FAdVs in Egypt at higher reolution.

An alloherpesvirus infection of European perch Perca fluviatilis in Finland.

The order Herpesvirales includes viruses that infect aquatic and terrestrial vertebrates and several aquatic invertebrates (i.e. mollusks), and share the commonality of possessing a double-stranded DNA core surrounded by an icosahedral capsid. Herpesviruses of the family Alloherpesviridae that infect fish and amphibians, including channel catfish virus and koi herpesvirus, negatively impact aquaculture. Here, we describe a novel herpesvirus infection of wild European perch from lakes in Finland. Infected fish exhibited white nodules on the skin and fins, typically in the spring when prevalence reached nearly 40% in one of the sampled lakes. Transmission electron microscopic examination of affected tissues revealed abundant nuclear and cytoplasmic virus particles displaying herpesvirus morphology. Degenerate PCR targeting a conserved region of the DNA polymerase gene of large DNA viruses amplified a 520 bp product in 5 of 5 affected perch skin samples tested. Phylogenetic analysis of concatenated partial DNA polymerase and terminase (exon 2) gene sequences produced a well-supported tree grouping the European perch herpesvirus with alloherpesviruses infecting acipenserid, esocid, ictalurid, and salmonid fishes. The phenetic analysis of the European perch herpesvirus partial DNA polymerase and terminase nucleotide gene sequences ranged from 34.6 to 63.9% and 39.6 to 59.6% to other alloherpesviruses, respectively. These data support the European perch herpesvirus as a new alloherpesvirus, and we propose the formal species designation of Percid herpesvirus 2 (PeHV2) to be considered for approval by the International Committee on Taxonomy of Viruses.

Investigations on aviadenoviruses isolated from turkey flocks in Germany

ABSTRACTDuring routine diagnosis in 2012, 69 samples of diseased turkey breeding and fattening flocks in Germany were examined for infection with aviadenoviruses by virus isolation using primary chicken embryo liver cells. In total, 21 aviadenovirus isolates, identified by a group-specific indirect immunofluorescence test, were obtained from 19 flocks. In almost all cases, molecular typing of these isolates based on partial hexon gene sequences revealed the presence of different types of turkey aviadenoviruses (TAdVs), including species Turkey aviadenovirus B (TAdV-B) with at least two different genotypes, as well as the species Turkey aviadenovirus C (TAdV-C) and Turkey aviadenovirus D (TAdV-D). Further analysis of DNA-dependent DNA polymerase gene sequences confirmed the classification of selected TAdV-C and TAdV-D isolates. Based on the results obtained for both genes, we suggest that TAdV-2, in addition to TAdV-4, belongs to the species TAdV-C. In contrast, amplification of the DNA polymerase gene fragment of nearly all investigated TAdV-B isolates failed due to unknown reasons. The results of sequence and phylogenetic analysis support the previously proposed classification of TAdVs into three different species and demonstrated how widely spread these viruses are in German turkey flocks. Analysis of case histories revealed a wide range of clinical and pathological changes; however an apparent link between types and disease conditions was not identified.

Genetic characterization of esocid herpesvirus 1 (EsHV1).

Blue spot disease, believed to be caused by esocid herpesvirus 1 (EsHV1), has been observed in wild northern pike Esox lucius in a number of cold-water locations, including the northern USA, Canada, and Ireland. In the spring of 2014, a northern pike was caught in Wisconsin displaying the characteristic bluish-white circular plaques on the dorsum and fins. Microscopic examination of hematoxylin and eosin-stained sections of the proliferative cutaneous lesions revealed a focally extensive abundance of panepidermal, megalocytic keratinocytes with karyomegaly. Enlarged nuclei stained basophilic, and an abundance of coarse eosinophilic granules were observed in the expanded cytoplasm. Transmission electron microscopy revealed aggregates of enveloped virus particles with electron-dense, hexagonal nucleocapsids surrounded by a uniformly staining ellipsoidal tegument layer within cytoplasmic vacuoles of megalocytic epidermal cells. More than 7000 bp of the EsHV1 genome were sequenced from infected skin tissues. Phylogenetic and phenetic analyses, based on the partial DNA-dependent DNA polymerase and terminase gene sequences, revealed EsHV1 forms a novel branch within the family Alloherpesviridae as the sister group to the clade that includes members of the genera Ictalurivirus and Salmonivirus. The gross, microscopic, and ultrastructural lesions reported in our study were identical to previous reports of blue spot disease in northern pike; however, here we provide the first molecular evidence supporting EsHV1 as a new species in the family Alloherpesviridae.

New lipid envelope-containing dsDNA virus isolates infecting Micromonas pusilla reveal a separate phylogenetic group

Viral infection of phytoplankton has major implications for biochemical and energy cycles, community dynamics, and microbial evolution in the marine environment. The non-bloom forming picoplankter Micromonas pusilla, a significant component of the plankton community worldwide, is known to be susceptible to infection by both dsDNA and dsRNA viruses. Logically, comprehensive knowledge of the ecology of M. pusilla requires a better understanding of the diversity and infection mechanisms of their viruses. Here, we investigated 19 new M. pusilla-specific viruses (MpVs) isolated from different locations and years. We performed partial characterization of those MpVs including structural characteristics, genome size, phylogenetic analysis based on partial DNA polymerase gene sequences, host range, and stability at different temperatures and upon exposure to chloroform. Combined, these characteristics allowed classification of the MpVs into 2 groups. Exposure to chloroform led to loss of infectivity by all MpVs in one group, which suggests the presence of an outer lipid envelope. In addition, all except one of the members in that group formed a monophylogenetic clade that was distinct from all other MpV isolates. The distinctive characteristics of the 2 MpV groups suggest different infection strategies, which may have important implications for the ecology of both host and virus populations in the environment. Knowledge gained from our study adds value to the MpV isolates as a scientific resource as it will aid in developing and testing in the laboratory new hypotheses about the ecological and biogeochemical implications of M. pusilla viral infection in the environment.

African great apes are naturally infected with roseoloviruses closely related to human herpesvirus 7.

Primates are naturally infected with herpesviruses. During the last 15 years, the search for homologues of human herpesviruses in nonhuman primates allowed the identification of numerous viruses belonging to the different herpesvirus subfamilies and genera. No simian homologue of human herpesvirus 7 (HHV7) has been reported to date. To investigate the putative existence of HHV7-like viruses in African great apes, we applied the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) program-mediated PCR strategy to blood DNA samples from the four common chimpanzee subspecies (Pan troglodytes verus, P. t. ellioti, P. t. troglodytes, and P. t. schweinfurthii), pygmy chimpanzees (Pan paniscus), as well as lowland gorillas (Gorilla gorilla gorilla). This study led to the discovery of a novel roseolovirus close to HHV7 in each of these nonhuman primate species and subspecies. Generation of the partial glycoprotein B (1,111-bp) and full-length DNA polymerase (3,036/3,042-bp) gene sequences allowed the deciphering of their evolutionary relationships. Phylogenetic analyses revealed that HHV7 and its African great ape homologues formed well-supported monophyletic lineages whose topological resemblance to the host phylogeny is suggestive of virus-host codivergence. Notably, the evolutionary branching points that separate HHV7 from African great ape herpesvirus 7 are remarkably congruent with the dates of divergence of their hosts. Our study shows that African great apes are hosts of human herpesvirus homologues, including HHV7 homologues, and that the latter, like other DNA viruses that establish persistent infections, have cospeciated with their hosts. Human herpesviruses are known to possess simian homologues. However, surprisingly, none has been identified to date for human herpesvirus 7 (HHV7). This study is the first to describe simian homologues of HHV7. The extensive search performed on almost all African great ape species and subspecies, i.e., common chimpanzees of the four subspecies, bonobos, and lowland gorillas, has allowed characterization of a specific virus in each. Genetic characterization of the partial glycoprotein B and full-length DNA polymerase gene sequences, followed by their phylogenetic analysis and estimation of divergence times, has shed light on the evolutionary relationships of these viruses. In this respect, we conclusively demonstrate the cospeciation between these new viruses and their hosts and report cases of cross-species transmission between two common chimpanzee subspecies in both directions.

Partial characterization of new adenoviruses found in lizards.

In the years 2011-2012, a consensus nested polymerase chain reaction was used for the detection of adenovirus (AdV) infection in reptiles. During this screening, three new AdVs were detected. One of these viruses was detected in three lizards from a group of green striped tree dragons (Japalura splendida). Another was detected in a green anole (Anolis carolinensis). A third virus was detected in a Jackson's chameleon (Chamaeleo jacksonii). Analysis of a portion of the DNA-dependent DNA polymerase genes of each of these viruses revealed that they all were different from one another and from all previously described reptilian AdVs. Phylogenetic analysis of the partial DNA polymerase gene sequence showed that all newly detected viruses clustered within the genus Atadenovirus. This is the first description of AdVs in these lizard species.

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.

Identification and characterization of a novel lymphocystis disease virus isolate from cultured grouper in China

Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.

Detection and Partial Characterization of an Atadenovirus in a Common Agama (Agama agama)

A common agama (Agama agama) with clinical signs of dehydration, anorexia, and an abscess in the oral cavity was presented for medical examination. Bacteriological testing of an oral swab demonstrated the presence of Proteus vulgaris and Morganella morganii (Proteus morganii). A fecal examination showed oocysts of Choleoeimeria sp. and pinworm eggs. The animal died four days after examination. Samples from organs (liver, kidney, heart, and intestine) were collected for virological testing. An adenovirus was detected by polymerase chain reaction testing in the intestine. Analyses of the partial DNA-dependent–DNA-polymerase gene sequence of this adenovirus showed that the detected adenovirus clustered with members of the genus Atadenovirus.

Partial sequence of the DNA-dependent DNA polymerase gene of fowl adenoviruses: A reference panel for a general diagnostic PCR in poultry

Adenoviruses are frequent infectious agents in different poultry species. The traditional, serological typing of new isolates by virus neutralisation tests is now in transition to be replaced by PCR and sequencing. The first PCRs, recommended for the detection of adenoviruses, had been designed to target the gene of the major capsid protein, the hexon. In birds, members of three different genera of the family Adenoviridae may occur. Accordingly, three specific hexon PCRs had to be elaborated for the detection of adenoviruses in poultry. A significantly more sensitive PCR, targeting the viral DNA-dependent DNA polymerase gene, has been described recently. This method proved to be an efficient alternative for the general detection of adenoviruses irrespective of their genus affiliation. Fowl adenoviruses (FAdVs), isolated from chicken to date, comprise twelve serotypes classified into five virus species (FAdV-A to E). The polymerase gene sequence has been determined yet only from three FAdV types representing three species. In the present work, the panel of polymerase gene sequences was completed with those of the rest of FAdVs. The newly determined sequences will facilitate the identification of new FAdV isolates as an existing species or as a putative new FAdV. Once the polymerase sequence is known, more specific PCRs for the amplification of the hexon and other genes can be designed and performed according to the preliminary species classification.

Investigation of ornamental fish entering the EU for the presence of ranaviruses

A survey was performed on ornamental fish imported into the EU to detect viral agents belonging to the genus Ranavirus. The objective was to gain knowledge of the potential for these systemic iridoviruses to gain entry into the EU via international trade in ornamental fish. A total of 208 pooled samples, representing 753 individual fish, were tested. The samples included 13 orders and 37 families, originating from different countries and continents. Tissues from fish that died during or just after transport were collected and examined by standard virological techniques in epithelioma papulosum cyprini cells, by transmission electron microscopy and by PCR for the detection of the major capsid protein and DNA polymerase gene sequences of ranaviruses. Virus was isolated from nine fish species but ranavirus was not identified in those samples. The results suggest that ranaviruses are not highly prevalent in ornamental fish imported into the EU.