Differential and defective transcription of koala retrovirus indicates the complexity of host and virus evolution.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of 'southern' (south Australian) and 'northern' (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of 'southern' (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, 'RecKoRV' variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

Con: The Length of Adult Cardiothoracic Anesthesiology Fellowship Training Should Not Be Extended Beyond One Year.

Fellowship training in adult cardiothoracic anesthesiology (ACTA) is a one-year postgraduate experience with formal accreditation by the Accreditation Council for Graduate Medical Education. ACTA is a competitive and evolving subspeciality. With expanding knowledge, clinical roles and technical skills required of the modern cardiothoracic anesthesiologists, the optimal structure and duration of the fellowship training are worth considering. This manuscript provides supporting rationale for fellowship training in ACTA to remain one year in duration. The expanding responsibilities of the cardiothoracic anesthesiologist and strategies to best train the future of the subspecialty within the current training structure are discussed. It also briefly examines the history and current status of the fellowship training, reviews considerations for increasing fellowship duration, and highlights personal and financial considerations during the training.

Aortic Stenosis and Noncardiac Surgery in the Era of Transcatheter Aortic Valve Replacement.

Aortic stenosis (AS) is the most common form of valvular heart disease in the elderly. As this patient population continues to increase, anesthesiologists more often will encounter these patients in the perioperative setting for noncardiac surgical procedures. Cardiac risk during noncardiac surgery in the patient with AS appears to have decreased significantly compared with previous reports that shaped current practice guidelines for perioperative management. In addition, these guidelines preceded the publication of current data supporting the continually expanding role for transcatheter aortic valve replacement in the treatment of AS. In this review, the authors highlight the most recent evidence suggesting that an update of these guidelines is warranted. In addition, the accumulating evidence supporting the role for transcatheter aortic valve replacement to treat AS since the publication of guidelines is reviewed.

A novel Australian flying-fox retrovirus shares an evolutionary ancestor with Koala, Gibbon and Melomys gamma-retroviruses.

A novel gamma-retroviral sequence (7912 bp), inclusive of both partial 5' and 3' long terminal repeat regions, was identified from the brain of a black flying-fox (Pteropus alecto), Queensland, Australia. The sequence was distinct from other retroviral sequences identified in bats and showed greater identity to Koala, Gibbon ape leukaemia, Melomys burtoni and Woolly monkey retroviruses, forming their own phylogenetic clade. This finding suggests that these retroviruses may have an unknown common ancestor and that further investigation into the diversity of gamma-retroviruses in Australian Pteropus species may elucidate their evolutionary origins.

Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population.

Koala populations are in serious decline across many areas of mainland Australia, with infectious disease a contributing factor. Koala retrovirus (KoRV) is a gammaretrovirus present in most wild koala populations and captive colonies. Five subtypes of KoRV (A to E) have been identified based on amino acid sequence divergence in a hypervariable region of the receptor binding domain of the envelope protein. However, analysis of viral genetic diversity has been conducted primarily on KoRV in captive koalas housed in zoos in Japan, the United States, and Germany. Wild koalas within Australia have not been comparably assessed. Here we report a detailed analysis of KoRV genetic diversity in samples collected from 18 wild koalas from southeast Queensland. By employing deep sequencing we identified 108 novel KoRV envelope sequences and determined their phylogenetic diversity. Genetic diversity in KoRV was abundant and fell into three major groups; two comprised the previously identified subtypes A and B, while the third contained the remaining hypervariable region subtypes (C, D, and E) as well as four hypervariable region subtypes that we newly define here (F, G, H, and I). In addition to the ubiquitous presence of KoRV-A, which may represent an exclusively endogenous variant, subtypes B, D, and F were found to be at high prevalence, while subtypes G, H, and I were present in a smaller number of animals. IMPORTANCE: Koala retrovirus (KoRV) is thought to be a significant contributor to koala disease and population decline across mainland Australia. This study is the first to determine KoRV subtype prevalence among a wild koala population, and it significantly expands the total number of KoRV sequences available, providing a more precise picture of genetic diversity. This understanding of KoRV subtype prevalence and genetic diversity will be important for conservation efforts attempting to limit the spread of KoRV. Furthermore, KoRV is one of the only retroviruses shown to exist in both endogenous (transmitted vertically to offspring in the germ line DNA) and exogenous (horizontally transmitted between infected individuals) forms, a division of fundamental evolutionary importance.

Highly Pathogenic Avian Influenza (H5N1) Virus in Feathers.

H5N1 highly pathogenic avian influenza (HPAI) virus causes high mortality of infected birds, with infection in multiple organs, including in feathers. Feathers have been proposed as samples for diagnosis of HPAI infection in birds, and this study is part of a broader investigation validating the use of feathers for diagnostic purposes. To understand and characterize the morphological basis for feather infection, sections from 7 different skin tracts of ducks and chickens infected with 3 different clades of H5N1 HPAI virus from Indonesia and Vietnam were examined histologically. Results showed that in ducks, lesions and viral antigen were mainly detected in the epidermis of feathers and follicles, whereas in chickens, they were mostly found in the dermis of these structures. Abundant viral antigen was found in nearly all the feathers examined from chickens, and there was no apparent difference between virus isolates or skin tracts in the proportion of feathers that were antigen positive. By immunohistochemistry, the majority of feathers from most skin tracts from ducks infected with a Vietnamese H5N1 HPAI virus contained abundant levels of viral antigen, while few feathers were antigen positive from ducks infected with 2 Indonesian viruses. These results support and inform the use of feathers for diagnostic detection of H5N1 HPAI virus in birds.

Assessing the risk of Nipah virus establishment in Australian flying-foxes.

Nipah virus (NiV) is a recently emerged zoonotic virus that causes severe disease in humans. The reservoir hosts for NiV, bats of the genus Pteropus (known as flying-foxes) are found across the Asia-Pacific including Australia. While NiV has not been detected in Australia, evidence for NiV infection has been found in flying-foxes in some of Australia's closest neighbours. A qualitative risk assessment was undertaken to assess the risk of NiV establishing in Australian flying-foxes through flying-fox movements from nearby regions. Events surrounding the emergence of new diseases are typically uncertain and in this study an expert opinion workshop was used to address gaps in knowledge. Given the difficulties in combining expert opinion, five different combination methods were analysed to assess their influence on the risk outcome. Under the baseline scenario where the median was used to combine opinions, the risk was estimated to be very low. However, this risk increased when the mean and linear opinion pooling combination methods were used. This assessment highlights the effects that different methods for combining expert opinion have on final risk estimates and the caution needed when interpreting these outcomes given the high degree of uncertainty in expert opinion. This work has provided a flexible model framework for assessing the risk of NiV establishment in Australian flying-foxes through bat movements which can be updated when new data become available.

Incidence and risk factors for H5 highly pathogenic avian influenza infection in flocks of apparently clinically healthy ducks.

A prospective longitudinal study was conducted on 96 smallholder duck farms in Indonesia over a period of 14 months in 2007 and 2008 to monitor bird- and flock-level incidence rates of H5 highly pathogenic avian influenza (HPAI) infection in duck flocks, and to identify risk factors associated with these flocks becoming H5 seropositive. Flocks that scavenged around neighbouring houses within the village were at increased risk of developing H5 antibodies, as were flocks from which carcases of birds that died during the 2 months between visits were consumed by the family. Duck flock confinement overnight on the farm and sudden deaths of birds between visits were associated with lower risk of the flock developing H5 antibodies. Scavenging around neighbouring houses and non-confinement overnight are likely to be causal risk factors for infection. With this study we have provided insights into farm-level risk factors of HPAI virus introduction into duck flocks. Preventive messages based on these risk factors should be included in HPAI awareness programmes.

Pathogens associated with pleuritic pig lungs at an abattoir in Queensland Australia.

OBJECTIVE: Pleurisy in pigs has economic impacts in the production stage and at slaughter. This study sought to establish if some micro-organisms can be found in high numbers in lungs with pleurisy by assessing batches of pigs at an abattoir in Queensland Australia. DESIGN: Samples of lung (including trachea/bronchus and lymph nodes) from a maximum of 5 pleurisy affected pigs were collected from 46 batches of pigs representing 46 Queensland farms. PROCEDURE: Pleurisy-affected lung areas were cultured by traditional bacteriological methods and bacteria quantified by plate scores. Additionally, tracheal or bronchial swabs and apical lobe fluid were tested for Mycoplasma hyopneumoniae DNA and the superior tracheobronchial lymph nodes were tested for porcine circovirus type 2 DNA by polymerase chain reaction (PCR). All apparently significant bacteria were identified via PCR or sequencing. Typing was undertaken on some of the bacterial isolates. RESULTS: The most prevalent pathogens were M. hyopneumoniae, Streptococcus suis and Porcine Circovirus type 2, being found in 34, 38 and 31 batches, respectively. Other bacteria found were Actinobacillus species (29 batches), Pasteurella multocida (24 batches), Mycoplasma flocculare (9 batches), Actinobacillus pleuropneumoniae (7 batches), Mycoplasma hyorhinis (4 batches), Bisgaard Taxon 10 (1 batch), Glaesserella parasuis (1 batch), Streptococcus minor (1 batch) and Streptococcus porcinus (1 batch). Most batches had more than one bacterial species. CONCLUSION: The high percentage of batches infected with S. suis (83%), M. hyopneumoniae (74%) and PCV2 (70%) and clustering by a batch of these pathogens, as well as the presence of many secondary pathogens, suggests synergy between these organisms may have resulted in pleurisy.

Genetic analysis of porcine circovirus type 2 (PCV2) in Queensland, Australia.

OBJECTIVE: To determine the current porcine circovirus type 2 (PCV2) genotypes circulating in pigs in Queensland (QLD). METHODS: The PCV2 infection status of pigs was determined by real-time PCR testing of 210 lymph nodes and 30 serum samples derived from 45 QLD farms. PCV2-positive samples from 22 pigs from 15 farms were subjected to conventional polymerase chain reaction (PCR) and sequencing of the full PCV2 genome. Phylogenetic analysis of 17 of these sequences in relation to published PCV2 sequences was then performed, and the genotypes were compared. RESULTS: PCV2 DNA was detected in 95 lymph nodes and 15 serum samples. Phylogenetic analysis of 17 PCV2 sequences demonstrated that seven belonged to genotype PCV2b, two to PCV2d, one to PCV2f and seven to an "intermediate group" that clustered with PCV2d on the full genome analysis. CONCLUSION: This work confirms earlier studies reporting the presence of PCV2b in Australia. It is the first study to report that PCV2d and PCV2f are also present in this country. PCV2d is currently a fast-spreading genotype globally, with reported high virulence. The potential implications of these findings with respect to pathogenicity and vaccine efficacy require further investigation.

Pathological Findings in Koala Retrovirus-positive Koalas (Phascolarctos cinereus) from Northern and Southern Australia.

Koala retrovirus (KoRV) infection shows differences in prevalence and load between northern and southern Australian koala populations; however, the effect of this on diseases such as lymphoma and chlamydial disease is unclear. This study compared clinicopathological findings, haematology and splenic lymphoid area of KoRV-positive koalas from northern (Queensland [Qld], n = 67) and southern (South Australia [SA], n = 92) populations in order to provide further insight into KoRV pathogenesis. Blood was collected for routine haematology and for measurement of KoRV proviral load by quantitative polymerase chain reaction (qPCR). Plasma samples were assessed for KoRV viral load by reverse transcriptase qPCR and conjunctival and cloacal swabs were collected for measurement of the load of Chlamydia pecorum (qPCR). During necropsy examination, spleen was collected for lymphoid area analysis. Lymphoma was morphologically similar between the populations and occurred in koalas with the highest KoRV proviral and viral loads. Severe ocular chlamydial disease was observed in both populations, but urinary tract disease was more severe in Qld, despite similar C. pecorum loads. No associations between KoRV and chlamydial disease severity or load were observed, except in SA where viral load correlated positively with chlamydial disease severity. In both populations, proviral and viral loads correlated positively with lymphocyte and metarubricyte counts and correlated negatively with erythrocyte and neutrophil counts. Splenic lymphoid area was correlated positively with viral load. This study has shown further evidence for KoRV-induced oncogenesis and highlighted that lymphocytes and splenic lymphoid tissue may be key sites for KoRV replication. However, KoRV infection appears to be highly complex and continued investigation is required to fully understand its pathogenesis.

Biology and evolution of the endogenous koala retrovirus.

Although endogenous retroviruses are ubiquitous features of all mammalian genomes, the process of initial germ line invasion and subsequent inactivation from a pathogenic element has not yet been observed in a wild species. Koala retrovirus (KoRV) provides a unique opportunity to study this process of endogenisation in action as it still appears to be spreading through the koala population. Ongoing expression of the endogenous sequence and consequent high levels of viraemia have been linked to neoplasia and immunosuppression in koalas. This apparently recent invader of the koala genome shares a remarkably close sequence relationship with the pathogenic exogenous Gibbon ape leukaemia virus (GALV), and comparative analyses of KoRV and GALVare helping to shed light on how retroviruses in general adapt to a relatively benign or at least less pathogenic existence within a new host genome. (Part of a multi-author review).

Mortality rates adjusted for unobserved deaths and associations with Newcastle disease virus serology among unvaccinated village chickens in Myanmar.

Village chickens are an important livestock for many rural families in Myanmar and other developing countries. Village chickens are kept under free-ranging conditions, with confinement only at night. Therefore, it is likely that some deaths are not observed by farmers. We conducted a longitudinal study from November 2003 until May 2004 to describe temporal patterns of mortality of village chickens in 10 villages in Myanmar. Field veterinarians first identified the numbers of birds in all chicken-owning households in each village. We then selected 307 households randomly with stratification by flock size. Each study household was then visited once monthly at which time questionnaires were completed recording current flock structure and numbers of hatchings, mortalities, sales and birds consumed since the previous visit. In addition, sera were collected from a sample of adult birds and growers. Depending on month and age group of chicken, from 71 to 231 (out of 290-307) households had discrepancies in the counts of birds. For chicks, at least one-quarter of the households had unobserved losses of at least 5 chicks per household (maximum 66 chicks); unobserved losses were less for growers and adult chickens. The median month-specific, village-specific mortality rates per 1000 bird-days at risk (counting missing birds as deaths) ranged from 0.8 to 1.7 for adults, from 0.4 to 4.7 for growers and from 8.0 to 16.5 for chicks. Across all birds, the prevalence of protective titres against Newcastle disease virus was 79% (95% confidence interval 74, 84); higher prevalences of protective titres were associated with reduced mortality rates in the following months.

Shelter-housed cats show no evidence of faecal shedding of canine parvovirus DNA.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are deoxyriboncucleic acid (DNA) viruses in the taxon Carnivore protoparvovirus 1. Exposure of cats to either CPV or FPV results in productive infection and faecal shedding of virus. Asymptomatic shedding of CPVs by one-third of shelter-housed cats in a UK study suggests that cats may be an important reservoir for parvoviral disease in dogs. The aim of this cross-sectional study was to determine the prevalence of faecal shedding of CPVs in asymptomatic shelter-housed cats in Australia. Faecal samples (n=218) were collected from cats housed in three shelters receiving both cats and dogs, in Queensland and NSW. Molecular testing for Carnivore protoparvovirus 1 DNA was performed by polymerase chain reaction (PCR) amplification followed by DNA sequencing of the VP2 region to differentiate CPV from FPV. Carnivore protoparvovirus 1 DNA was detected in only four (1.8%, 95% confidence interval 0.49-4.53%) faecal samples from a single shelter. Sequencing identified all four positive samples as FPV. Faecal shedding of CPV by shelter-cats was not detected in this study. While the potential for cross-species transmission of CPV between cats and dogs is high, this study found no evidence of a role for cats in maintaining CPV in cat and dog populations through faecal shedding in the regions tested.

Identification of stable reference genes for quantitative PCR in koalas.

To better understand host and immune response to diseases, gene expression studies require identification of reference genes with stable expression for accurate normalisation. This study describes the identification and testing of reference genes with stable expression profiles in koala lymph node tissues across two genetically distinct koala populations. From the 25 most stable genes identified in transcriptome analysis, 11 genes were selected for verification using reverse transcription quantitative PCR, in addition to the commonly used ACTB and GAPDH genes. The expression data were analysed using stable genes statistical software - geNorm, BestKeeper, NormFinder, the comparative ΔCt method and RefFinder. All 13 genes showed relative stability in expression in koala lymph node tissues, however Tmem97 and Hmg20a were identified as the most stable genes across the two koala populations.

Genetic analysis of canine parvovirus from dogs in Australia.

OBJECTIVE: To determine the genetic variants of canine parvovirus-2 (CPV) present in domestic dogs in Australia and to investigate 26 cases of apparent vaccine failure. DESIGN: Thirty-three samples of faeces or intestinal tissues and 16 cell culture virus isolates collected over a period from 1980 to 2005 from five Australian states were analysed. Procedure DNA was extracted from the samples and a 1975 bp fragment of the VP1/2 gene of CPV was amplified by polymerase chain reaction (PCR) and sequenced. Sequences were compared to published strains of CPV-2, CPV-2a, CPV-2b and CPV-2c. RESULTS: Forty-one of 43 PCR-positive samples contained CPV-2a viruses. One sample collected in 2002 from a pup in northern NSW contained a CPV-2b virus. One sample that had been included in the study as a CPV-antigen negative control sample contained a CPV-2 virus. CONCLUSION: CPV-2a remains the predominant genetic variant of CPV in dogs in Australia and has not been replaced by CPV-2b or CPV-2c as in many other countries. The vaccine failures investigated in the study were likely caused not by genetic variation of field viruses but by maternal antibody interference in the response of pups to vaccination.