Development of subfamily-based consensus PCR assays for the detection of human and animal herpesviruses.

Consensus PCR assays that can be used to sensitively detect several herpesvirus (HV) species across the different subfamilies were developed in this study. Primers containing degenerate bases were designed to amplify regions of the DNA polymerase (DPOL) gene of alpha- and gamma-HVs, and the glycoprotein B (gB) gene of beta-HVs in a singleplex, non-nested touchdown PCR format. The singleplex touchdown consensus PCR (STC-PCR) was used to amplify the DNA of eight human and 24 animal HVs. The assay was able to detect the lowest DNA dilution of 10-5 for alpha-HVs and 10-3 for beta- and gamma-HVs. In comparison, lowest detection limits of 10-5, 10-3, and 10-2 were obtained for alpha-, beta-, and gamma-HVs respectively when a nested PCR was used. The findings in this study suggest that the STC-PCR assays can be employed for the molecular surveys and clinical detection of novel and known HVs.

Detection and prevalence of adenoviruses from free-ranging black howler monkeys (Alouatta pigra).

Adenoviruses are important pathogens known to infect vertebrate hosts, including a wide range of primates. Despite its importance, data on the diversity of these viruses in non-human primates living in their natural habitat remain scarce. In this study, we conducted a surveillance of adenoviral infection in wild black howler monkeys from two protected natural areas in Mexico. This was achieved by analyzing 67 fecal samples using a nested PCR that targets the adenovirus DNA polymerase gene. Adenoviral DNA was detected in 12 samples from both study sites, with an overall prevalence of 17.9%. The amplified DNA sequences shared 100% nucleotide identity and phylogenetic analyses revealed that the haplotype detected was novel, and clustered with Platyrrhini mastadenovirus A, which was previously described in captive New World monkeys. Our data, along with the previous evidence, confirm that monkeys native to the Americas are the original hosts of these adenoviruses.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6-10) and temperatures (4-45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.

Case report: Diagnosis and autogenous vaccine treatment of herpesvirus in a green turtle (Chelonia mydas) in Santa Marta, Colombia.

This study reports the first case of fibropapillomatosis (FP) in the green turtle Chelonia mydas that has been successfully diagnosed and treated in Colombia. Worldwide, FP has reached epizootic proportions as it has been reported in marine turtles of tropical and subtropical waters, and in severe cases, it reduces the probability of survival. Treatment has been elusive as multiple surgical excisions are needed due to tumor recurrence. In this case, one green turtle with multiple tumors was diagnosed by histopathology and molecular detection of the chelonid herpesvirus 5 (ChHV5) by means of amplification and sequencing of the DNA polymerase (DNApol) gene. Two separate treatments that consisted of autogenous vaccines and surgical excisions were applied; the first one had a partial success as one out of the tumors treated reappeared after 3 months post-treatment. Treatment 2 consisted of an autogenous vaccine enriched with adjuvants and applied at increasing doses, after which, the tumor significatively decreased in size and was surgically removed. At the end of the 6 months follow-up period, no tumor recurrence was observed, and the turtle was in apparent optimal health conditions. These findings, although limited, suggest a possible treatment that might help to contain this epizootic problem.

Quantitative analysis of herpes simplex virus-1 transcript in suspected viral keratitis corneal buttons and its clinical significance.

Purpose: The evaluation of Herpes Simplex virus-1 (HSV-1) transcript by different investigative methods (qPCR, PCR and IHC) in corneal buttons from suspected viral keratitis patients and the comparison of results with histopathological findings and clinical diagnosis. Methods: Sixty corneal buttons, 30 suspected viral keratitis, and 30 controls (keratoconus and bullous keratopathy) obtained after primary penetrating keratoplasty, were included in the study. All the corneal buttons were subjected to reverse transcriptase quantitative PCR (qPCR) for the detection of latency-associated transcript (LAT) gene, conventional PCR for polymerase (pol) gene, and immunohistochemistry (IHC) for HSV-1 antigen respectively. After obtaining baseline preoperative clinical data, all the patients were followed up for three years. The results obtained were correlated with clinicopathological features and follow-up data. Results: Of the 30 suspected viral keratitis patients there were 6 females and 24 males with mean age 46.5 ± 24.62 years (3-80 yrs). There was a marked male preponderance (80%). HSV-1 LAT transcript was detected in 23% (7/30) corneal buttons by qPCR, HSV-1 DNA in 6.7% (2/30) and HSV-1 antigen in 30% (9/30) cases by conventional PCR and IHC respectively. A statistically significant association was found between qPCR and DNA PCR (P = 0.04). All the 30 control corneas were negative for HSV-1 LAT gene, DNA and antigen. Conclusion: Detection of HSV-1 LAT transcript by qPCR may be superior to HSV-1 DNA PCR (conventional) and IHC, which has low sensitivity. However, the utility of HSV-1 LAT mRNA analysis as a diagnostic modality by qPCR needs to be validated on a larger patient cohort.

Isolation of equid alphaherpesvirus 3 from a horse in Iceland with equine coital exanthema.

Equine coital exanthema (ECE) caused by equid alphaherpesvirus 3 (EHV-3) is a contagious venereal disease. It is characterized by the formation of papules, vesicles, pustules and ulcers on the external genitals of both mares and stallions. The Icelandic horse is the only breed in Iceland and has lived isolated in the country for over 1000 years. Three types of equine herpesviruses (EHV) have been found in Iceland, EHV-4, EHV-2 and EHV-5, while EHV-1 has never been detected. Symptoms resembling ECE have previous been observed in horses in Iceland, arousing suspicion of EHV-3 infection, but this has never been confirmed using virological methods. Samples were collected from a mare with papules on the vulva and inoculated in primary equine kidney cells. Cytopathic effects developed as rounded cells and syncytial formation. Polymerase chain reaction and sequencing of the partial glycoprotein G and DNA polymerase genes identified the isolated virus as EHV-3. On the basis of the findings, EHV-3 infection was verified for the first time in the native Icelandic horse population.

Three nuclear protein-coding genes corroborate a recent phylogenomic model of the Branchiopoda (Crustacea) and provide estimates of the divergence times of the major branchiopodan taxa.

The class Branchiopoda (Crustacea) shows great diversity in morphology and lifestyle among its constituent higher-level taxa: Anostraca, Notostraca, Laevicaudata, Spinicaudata, Cyclestherida and Cladocera. The phylogenetic relationships among these taxa have long been controversial. We sequenced three orthologous nuclear genes that encode the catalytic subunit of DNA polymerase delta and the largest and second-largest subunits of RNA polymerase II in the expectation that the amino acid sequences encoded by these genes might be effective in clarifying branchiopod phylogeny and estimating the times of divergence of the major branchiopodan taxa. The results of phylogenetic analyses based on these amino acid sequences support the monophyly of Branchiopoda and provide strong molecular evidence in support of the following phylogenetic relationships: (Anostraca, (Notostraca, (Laevicaudata, (Spinicaudata, (Cyclestherida, Cladocera))))). Within Cladocera, comparison of the nucleotide sequences of these same genes shows Ctenopoda to be the sister group of Haplopoda + Anomopoda. Three statistical tests based on the present amino acid sequence data-the approximately unbiased test, Kishino-Hasegawa test and weighted Shimodaira-Hasegawa test-tend to refute most of the previous molecular phylogenetic studies on Branchiopoda, which have placed Notostraca differently than here; however, our results corroborate those of one recent phylogenomic study, thus confirming the effectiveness of these three genes to investigate relationships among branchiopod higher taxa. Divergence time estimates calibrated on the basis of fossil evidence suggest that the first divergence of extant branchiopods occurred about 534 Ma during the early Cambrian period and that diversification within the extant branchiopod lineages started in or after the late Permian.

Molecular Phylogeny of an Avipoxvirus Isolated from Red-Flanked Blue Robin in China.

We first report avipoxvirus (APV) infection and an isolate named APV/03/2016 from a red-flanked blue robin (Tarsiger cyanurus) captured at Songhua Lake Scenic Area in Jilin City (Jilin Province, China) on March 24, 2016. The partial sequence of the 4b core protein gene and DNA polymerase gene of APV/03/2016 suggests that the virus belongs to the subclade B1 cluster of clade B (canarypox virus). The BLAST results showed the highest similarity of the two genes with the Pacific shearwater-isolated strain SWPV-2 (KX857215), canarypox virus strain D98-11133 (GQ487567), canarypox virus strain ATCC VR-111 (AY318871), avipoxvirus Mississippi isolate P89 (KC018048), and avipoxvirus Wisconsin isolate P92 (KC018051). The results indicate that APV/03/2016 is a canarypox-like virus. These findings demonstrate the continuous emergence of new APV hosts such as red-flanked blue robins and suggest that monitoring of APV circulation and evolution should be strengthened for T. cyanurus conservation.

Filogenia molecular de un Avipoxvirus aislado de ruiseñor coliazul en China. Se reporta por primera vez la infección por poxvirus aviar (APV) y un aislamiento denominado APV/03/2016 obtenido de un ruiseñor coliazul (Tarsiger cyanurus) capturado en el área escénica del Lago Songhua en la ciudad de Jilin (provincia de Jilin, China) el 24 de marzo de 2016. La secuencia parcial del gene de la proteína central 4b y el gene de la polimerasa de ADN del virus APV/03/2016 sugiere que el virus pertenece al subclado B1 del clado B (virus de la viruela del canario). Los resultados de la búsqueda mediante BLAST mostraron la mayor similitud de los dos genes con la cepa aislada de aves pelágicas del Pacífico (KX857215), virus de la viruela del canario cepa de virus D98-11133 (GQ487567), cepa de virus de la viruela del canario ATCC VR-111 (AY318871), aislamiento de avipoxvirus de Mississippi P89 (KC018048), y el aislamiento P89 de avipoxvirus en Wisconsin (KC018051). Los resultados indican que el virus APV/03/2016 es un virus similar al de la viruela del canario. Estos hallazgos demuestran la aparición continua de nuevos hospedadores de poxvirus aviares como el ruiseñor coliazul y sugieren que el monitoreo de la circulación y evolución de poxvirus aviares debería fortalecerse para la conservación del T. cyanurus.

Novel groups of cyanobacterial podovirus DNA polymerase (pol) genes exist in paddy waters in northeast China.

In this study, we surveyed cyanopodovirus DNA polymerase (pol) sequences in paddy waters using the culture-independent PCR and Sanger sequencing methods. Four paddy waters generated from a pot experiment with different soil types collected from op E: n paddy fields in northeast China were used in this study. A total of 438 DNA pol clones were identified as cyanopodoviruses. The clones from the paddy waters formed nine unique groups of cyanopodoviruses either exclusively or with clones from East Lake in China (subclusters α-1 to α-8 and cluster ß). None of the clones from open oceans or coastal waters fell into these unique groups. Additionally, the distribution proportions of the clones into different cyanopodovirus groups varied among paddy water samples, which suggested that the cyanopodovirus compositions were spatially distributed in the paddy fields. The comparison of clone libraries in different studies indicated that the diversity of cyanopodoviruses in paddy waters was comparable to the diversity in the open oceans but was less than the diversity in the coastal estuary of Chesapeake Bay. Non-metric multidimensional scaling analysis indicated that the cyanopodovirus communities in paddy waters were similar to those in lake freshwater but distinct from the communities in marine and coastal waters.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers.

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.

Unique distribution of cyanobacterial podoviruses and their potential hosts in a paddy field of northeast China.

We first surveyed the DNA polymerase (pol) gene of cyanopodoviruses and the 16S-23S rRNA gene internal transcribed spacer (ITS) region of picocyanobacteria in a paddy field of northeast China. A total of 49 DNA pol clones and 76 ITS clones were obtained. The blast search results showed that all DNA pol clones and nearly 50% of the ITS clones had up to 76% and 50% identity/similarity to known sequences, respectively. Phylogenetic analyses showed that the DNA pol clones were narrowly distributed in the phylogenetic tree, and two new subclusters of cyanopodoviruses (PG-Pol-I and PG-Pol-II) specific to paddy field were discovered. In contrast, the distribution of ITS clones was very broad, and seven paddy-specific groups of picocyanobacteria (PG-Picocya-I-VII) were identified. In general, novel groups of cyanopodoviruses and picocyanobacteria were observed in this study, suggesting that coevolution between cyanopodoviruses and their hosts occurs in the paddy field.

Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers

Partial nucleotide sequences of ORF72 (glycoprotein D, gD), ORF64 (infected cell protein 4, ICP4) and ORF30 (DNA polymerase) genes were compared with corresponding sequences of EHV-1 reference strains to characterize the molecular variability of Brazilian strains. Virus isolation assays were applied to 74 samples including visceral tissue, total blood, cerebrospinal fluid (CSF) and nasal swabs of specimens from a total of 64 animals. Only one CSF sample (Iso07/05 strain) was positive by virus isolation in cell culture. EHV-1 Iso07/05 neurologic strain and two abortion visceral tissues samples (Iso11/06 and Iso33/06) were PCR-positive for ORF33 (glycoprotein B, gB) gene of EHV-1. A sequence analysis of the ORF72, ORF64 and ORF30 genes from three EHV-1 archival strains (A3/97, A4/72, A9/92) and three clinical samples (Iso07/05, Iso11/06 and Iso33/06) suggested that among Brazilian EHV-1 strains, the amplified region of the gD gene sequence is highly conserved. Additionally, the analysis of ICP4 gene showed high nucleotide and amino acid identities when compared with genotype P strains, suggesting that the EHV-1 Brazilian strains belonged to the same group. All the EHV-1 Brazilian strains were classified as non-neuropathogenic variants (N752) based on the ORF30 analysis. These findings indicate a high conservation of the gD-, ICP4- and ORF30-encoding sequences. Different pathotypes of the EHV-1 strain might share identical genes with no specific markers, and tissue tropism is not completely dependent on the gD envelope, immediate-early ICP4 and DNA polymerase proteins.