Degradation and remobilization of endogenous retroviruses by recombination during the earliest stages of a germ-line invasion.

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded.

Detection of porcine circovirus type 2 (PCV2) in the Philippines and the complexity of PCV2-associated disease diagnosis.

Porcine circovirus type 2 (PCV2), an important pig viral pathogen, can cause porcine circovirus-associated disease (PCVAD), resulting in economic losses associated with decreased growth and mortalities. The diagnosis of PCVAD is complex requiring clinical, pathological and virological approaches. This study assessed PCV2 infection using histopathology and immunohistochemistry (IHC) on tissue samples and quantitative polymerase chain reaction (qPCR) on serum samples from 47 grower-finisher pigs allocated in three clinical groups in the Philippines. Typical PCV2 histopathological lesions were observed in mediastinal lymph nodes (MLN) of eight of 47 pigs. Lymphoid depletion was seen in all eight pigs and granulomatous inflammation in one of these pigs. Four of these eight pigs were PCV2 positive by both IHC and qPCR. IHC revealed PCV2 antigen in 8 pigs in at least one of the following tissues: MLN (5/8), spleen (3/8), tonsils (4/8) and lungs (5/8). PCV2 antigen was observed in 3/8 MLN with lymphoid depletion and in one MLN with depletion and granulomatous inflammation. The qPCR test showed that 33 sera had a non-detectable level, twelve had < 106 and two had > 106 PCV2 DNA copies/ml serum. One pig with lymphoid depletion had > 106 PCV2 DNA copies/ml serum, and another pig without MLN lesions also had > 106 PCV2 DNA copies/ml serum. These findings suggest that PCVAD is present in the Philippines and confirm the challenges of PCVAD diagnosis as different patterns of results were obtained from the different tests.

Genetic diversity of Koala retrovirus env gene subtypes: insights into northern and southern koala populations.

Koala retrovirus (KoRV) is a recently endogenized retrovirus associated with neoplasia and immunosuppression in koala populations. The virus is known to display sequence variability and to be present at varying prevalence in different populations, with animals in southern Australia displaying lower prevalence and viral loads than northern animals. This study used a PCR and next-generation sequencing strategy to examine the diversity of the KoRV env gene in both proviral DNA and viral RNA forms in two distinct populations representative of the 'northern' and 'southern' koala genotypes. The current study demonstrated that the full range of KoRV subtypes is present across both populations, and in both healthy and sick animals. KoRV-A was the predominant proviral subtype in both populations, but there was marked diversity of DNA and RNA subtypes within individuals. Many of the northern animals displayed a higher RNA viral diversity than evident in their proviral DNA, indicating relatively higher replication efficiency of non-KoRV-A subtypes. The southern animals displayed a lower absolute copy number of KoRV than the northern animals as reported previously and a higher preponderance of KoRV-A in individual animals. These discrepancies in viral replication and diversity remain unexplained but may indicate relative protection of the southern population from KoRV replication due to either viral or host factors and may represent an important protective effect for the host in KoRV's ongoing entry into the koala genome.

An Australian Newcastle Disease Virus With a Virulent Fusion Protein Cleavage Site Produces Minimal Pathogenicity in Chickens.

Newcastle disease is an important disease of poultry caused by virulent strains of Newcastle disease virus (NDV). During the 1998 to 2002 outbreaks of Newcastle disease in Australia, it was observed that the mild clinical signs seen in some chickens infected with NDV did not correlate with the viruses' virulent fusion protein cleavage site motifs or standard pathogenicity indices. The pathogenicity of 2 Australian NDV isolates was evaluated in experimentally challenged chickens based on clinical evaluation, histopathology, immunohistochemistry, and molecular techniques. One of these virus isolates, Meredith/02, was shown to induce only very mild clinical signs with no mortalities in an experimental setting, in contrast to the velogenic Herts 33/56 and Texas GB isolates. This minimal pathogenicity was associated with decreased virus replication and antigen distribution in tissues. This demonstrates that the Australian Meredith/02 NDV, despite possessing a virulent fusion protein cleavage site, did not display a velogenic phenotype.

Identification of non-essential loci within the Meleagrid herpesvirus 1 genome.

BACKGROUND: Meleagrid herpesvirus 1 (MeHV-1) infectious bacterial artificial chromosomes (iBACs) are ideal vectors for the development of recombinant vaccines for the poultry industry. However, the full potential of iBACS as vectors can only be realised after thorough genetic characterisation, including identification of those genetic locations that are non-essential for virus replication. Generally, transposition has proven to be a highly effective strategy for rapid and efficient mutagenesis of iBAC clones. The current study describes the characterisation of 34 MeHV-1 mutants containing transposon insertions within the pMeHV1-C18 iBAC genome. METHODS: Tn5 and MuA transposition methods were used to generate a library of 76 MeHV-1 insertion mutants. The capacity of each mutant to facilitate the recovery of infectious MeHV-1 was determined by the transfection of clone DNA into chicken embryo fibroblasts. RESULTS: Attempts to recover infectious virus from the modified clones identified 14 genetic locations that were essential for MeHV-1 replication in cell culture. Infectious MeHV-1 was recovered from the remaining 14 intragenic insertion mutants and six intergenic insertion mutants, suggesting that the respective insertion locations are non-essential for MeHV-1 replication in cell culture. CONCLUSIONS: The essential and non-essential designations for those MeHV-1 genes characterised in this study were generally in agreement with previous reports for other herpesviruses homologues. However, the requirement for the mardivirus-specific genes LORF4A and LORF5 are reported for the first time. These findings will help direct future work on the development of recombinant poultry vaccines using MeHV-1 as a vector by identifying potential transgene insertion sites within the viral genome.

Genomic deletions and mutations resulting in the loss of eight genes reduce the in vivo replication capacity of Meleagrid herpesvirus 1.

Meleagrid herpesvirus 1 (MeHV-1 or turkey herpesvirus) has been widely used as a vaccine in commercial poultry. Initially, these vaccine applications were for the prevention of Marek's disease resulting from Gallid herpesvirus 2 infections, while more recently MeHV-1 has been used as recombinant vector for other poultry infections. The construction of herpesvirus infectious clones that permit propagation and manipulation of the viral genome in bacterial hosts has advanced the studies of herpesviral genetics. The current study reports the construction of five MeHV-1 infectious clones. The in vitro properties of viruses recovered from these clones were indistinguishable from the parental MeHV-1. In contrast, the rescued MeHV-1 viruses were significantly attenuated when used in vivo. Complete sequencing of the infectious clones identified the absence of two regions of the MeHV-1 genome compared to the MeHV-1 reference sequence. These analyses determined the rescued viruses have seven genes, UL43, UL44, UL45, UL56, HVT071, sorf3 and US2 either partially or completely deleted. In addition, single nucleotide polymorphisms were identified in all clones compared with the MeHV-1 reference sequence. As a consequence of one of the polymorphisms identified in the UL13 gene, four of the rescued viruses were predicted to encode a serine/threonine protein kinase lacking two of three domains required for activity. Thus four of the recovered viruses have a total of eight missing or defective genes. The implications of these findings in the context of herpesvirus biology and infectious clone construction are discussed.

The Meleagrid herpesvirus 1 genome is partially resistant to transposition.

The propagation of herpesvirus genomes as infectious bacterial artificial chromosomes (iBAC) has enabled the application of highly efficient strategies to investigate gene function across the genome. One of these strategies, transposition, has been used successfully on a number of herpesvirus iBACs to generate libraries of gene disruption mutants. Gene deletion studies aimed at determining the dispensable gene repertoire of the Meleagrid herpesvirus 1 (MeHV-1) genome to enhance the utility of this virus as a vaccine vector have been conducted in this report. A MeHV-1 iBAC was used in combination with the Tn5 and MuA transposition systems in an attempt to generate MeHV-1 gene interruption libraries. However, these studies demonstrated that Tn5 transposition events into the MeHV-1 genome occurred at unexpectedly low frequencies. Furthermore, characterization of genomic locations of the rare Tn5 transposon insertion events indicated a nonrandom distribution within the viral genome, with seven of the 24 insertions occurring within the gene encoding infected cell protein 4. Although insertion events with the MuA system occurred at higher frequency compared with the Tn5 system, fewer insertion events were generated than has previously been reported with this system. The characterization and distribution of these MeHV-1 iBAC transposed mutants is discussed at both the nucleotide and genomic level, and the properties of the MeHV-1 genome that could influence transposition frequency are discussed.

Characteristics of two duck farming systems in the Mekong Delta of Viet Nam: stationary flocks and moving flocks, and their potential relevance to the spread of highly pathogenic avian influenza.

Ducks are considered to play a major role in the spread of highly pathogenic avian influenza (HPAI) H5N1 in Viet Nam, but detailed information on their management is limited. We distinguished two different systems (1) stationary duck flocks that are not commonly driven to rice fields beyond village boundaries and that are confined overnight on farms and (2) moving duck flocks that are intentionally driven to rice fields beyond village boundaries, that are not returning to home farms for extended periods and that are housed overnight in temporary enclosures in rice paddies. A total of 115 stationary and 22 moving flock farmers were interviewed in 2007 in the Mekong Delta of Viet Nam. Moving duck flocks are larger than stationary flocks, which is indicative of their more commercial production. Moving flock farmers apparently are more aware of HPAI risks than stationary flock farmers, as their flocks are more likely fully vaccinated and have less contact with chickens during scavenging. On the other hand, the spread of HPAI virus between birds might be promoted by moving duck flocks as they repeatedly use transport vehicles and numerous rice paddies for scavenging and are often visited by hatchery owners in the field for purchasing duck eggs. In addition, long distances travelled by moving duck flocks might also result in widespread dissemination of HPAI virus. Further studies are necessary to describe HPAI prevalence and travel patterns of moving duck flocks and to explore the moving duck flock network in detail.

Presence of Multiple Herpesvirus Variants in Australian Flying Foxes (Pteropus spp.).

Herpesviruses have been detected in bat species from several countries, with a limited number of studies examining herpesviruses in Pteropus spp. (flying foxes) and no investigation of herpesviruses in Australian flying foxes. We examined the presence and prevalence of herpesviruses in the four mainland Australian flying fox species. A nested PCR targeting highly conserved amino acid motifs in the DNA polymerase (DPOL) gene of herpesviruses was used to analyze 564 samples collected from 514 individual Pteropus scapulatus, Pteropus poliocephalus, Pteropus alecto, and Pteropus conspicillatus. The prevalence of herpesvirus DNA in blood, urine, oral, and fecal swabs from the four species was 17% in P. scapulatus, 11% in P. poliocephalus, 10% in P. alecto, and 9% in P. conspicillatus (31% in P. conspicillatus spleen tissue). Five putative novel herpesviruses were detected. Following PCR amplicon sequence analysis, four of the herpesviruses grouped phylogenetically with the gammaherpesviruses, with nucleotide identities between 79% and 90% to gammaherpesviruses from Asian megabats. A betaherpesvirus was detected in P. scapulatus with 99% nucleotide identity to the partial DPOL gene sequence of an Indonesian fruit bat betaherpesvirus. This study lays the foundation for future epidemiology research of herpesviruses in Australian Pteropus spp. and adds to the discussion of hypotheses surrounding the evolutionary epidemiology of bat-borne viruses on a global scale.

Cleft Palate Syndrome in the Endangered Spectacled Flying Fox (Pteropus conspicillatus): Implications for Conservation and Comparative Research.

Cleft palate syndrome, first observed in the spectacled flying fox population in 1998, has produced sporadic neonatal mortality events over the past two decades, with an estimated incidence of up to 1/1000 births per year. This study presents a rudimentary characterisation of the syndrome, presenting gross pathology of syndromic signs upon visual inspection, a histological examination of palate malformations, and syndrome incidence data representing the past two decades. The syndrome presents with a range of signs, primarily congenital palate malformations ranging from a pinhole cleft to a complete hard and soft palate deficit, resulting in the death or abandonment of neonates shortly after birth. The congenital palate malformations are often associated with claw deformities, wiry facial hair, and in some instances, muscle weakness and neurological signs. The natural occurrence of the lethal congenital orofacial birth defects in the spectacled flying fox presents a unique opportunity for the investigation of putative aetiologies, drawing parallels between bat and other mammalian cleft palate risk factors. Further syndrome investigation has the potential to deliver both biodiversity conservation and comparative veterinary and biomedical outcomes.

Genome Reference Assembly for Bottlenecked Southern Australian Koalas.

Koala populations show marked differences in inbreeding levels and in the presence or absence of the endogenous Koala retrovirus (KoRV). These genetic differences among populations may lead to severe disease impacts threatening koala population viability. In addition, the recent colonization of the koala genome by KoRV provides a unique opportunity to study the process of retroviral adaptation to vertebrate genomes and the impact this has on speciation, genome structure, and function. The genome build described here is from an animal from the bottlenecked Southern population free of endogenous and exogenous KoRV. It provides a more contiguous genome build than the previous koala reference derived from an animal from a more outbred Northern population and is the first koala genome from a KoRV polymerase-free animal.

Retroviral invasion of the koala genome.

Endogenous retroviruses are a common ancestral feature of mammalian genomes with most having been inactivated over time through mutation and deletion. A group of more intact endogenous retroviruses are considered to have entered the genomes of some species more recently, through infection by exogenous viruses, but this event has never been directly proved. We have previously reported koala retrovirus (KoRV) to be a functional virus that is associated with neoplasia. Here we show that KoRV also shows features of a recently inserted endogenous retrovirus that is vertically transmitted. The finding that some isolated koala populations have not yet incorporated KoRV into their genomes, combined with its high level of activity and variability in individual koalas, suggests that KoRV is a virus in transition between an exogenous and endogenous element. This ongoing dynamic interaction with a wild species provides an exciting opportunity to study the process and consequences of retroviral endogenization in action, and is an attractive model for studying the evolutionary event in which a retrovirus invades a mammalian genome.

Comparison of serological assays for detecting antibodies in ducks exposed to H5 subtype avian influenza virus.

BACKGROUND: Chicken red blood cells (RBCs) are commonly used in hemagglutination inhibition (HI) tests to measure hemagglutinating antibodies against influenza viruses. The use of horse RBCs in the HI test can reportedly increase its sensitivity when testing human sera for avian influenza antibodies. This study aims to compare the proportion of positives detected and the agreement between two HI tests using either chicken or horse red blood cells for antibody detection in sera of ducks experimentally infected or naturally exposed to Indonesian H5 subtype avian influenza virus. In addition, comparison with a virus neutralisation (VN) test was conducted with the experimental sera. RESULTS: In the experimental study, the proportion of HI antibody-positive ducks increased slightly, from 0.57 when using chicken RBCs to 0.60 when using horse RBCs. The HI tests indicated almost perfect agreement (kappa = 0.86) when results were dichotomised (titre ≥ 4 log2), and substantial agreement (weighted kappa = 0.80) for log titres. Overall agreements between the two HI tests were greater than between either of the HI tests and the VN test. The use of horse RBCs also identified a higher proportion of antibody positives in field duck sera (0.08, compared to chicken RBCs 0.02), with also almost perfect agreements for dichotomized results (Prevalence and bias adjusted Kappa (PABAK) = 0.88) and for log titres (weighted PABAK = 0.93), respectively. Factors that might explain observed differences in the proportion of antibody-positive ducks and in the agreements between HI tests are discussed. CONCLUSION: In conclusion, we identified a good agreement between HI tests. However, when horse RBCs were used, a higher proportion of sera was positive (titre ≥ 4 log2) than using chicken RBCs, especially during the early response against H5N1 virus. The HRBC-HI might be more responsive in identifying early H5N1 HPAI serological response and could be a recommended assay for avian influenza sero-surveillance in both wild and domestic birds.

A molecular and antigenic survey of H5N1 highly pathogenic avian influenza virus isolates from smallholder duck farms in Central Java, Indonesia during 2007-2008.

BACKGROUND: Indonesia is one of the countries most severely affected by H5N1 highly pathogenic avian influenza (HPAI) virus in terms of poultry and human health. However, there is little information on the diversity of H5N1 viruses circulating in backyard farms, where chickens and ducks often intermingle. In this study, H5N1 virus infection occurring in 96 smallholder duck farms in central Java, Indonesia from 2007-2008 was investigated and the molecular and antigenic characteristics of H5N1 viruses isolated from these farms were analysed. RESULTS: All 84 characterised viruses belonged to H5N1 clade 2.1 with three virus sublineages being identified: clade 2.1.1 (1), clade 2.1.3 (80), and IDN/6/05-like viruses (3) that did not belong to any of the present clades. All three clades were found in ducks, while only clade 2.1.3 was isolated from chickens. There were no significant amino acid mutations of the hemagglutinin (HA) and neuraminidase (NA) sites of the viruses, including the receptor binding, glycosylation, antigenic and catalytic sites and NA inhibitor targets. All the viruses had polybasic amino acids at the HA cleavage site. No evidence of major antigenic variants was detected. Based on the HA gene, identical virus variants could be found on different farms across the study sites and multiple genetic variants could be isolated from HPAI outbreaks simultaneously or at different time points from single farms. HPAI virus was isolated from both ducks and chickens; however, the proportion of surviving duck cases was considerably higher than in chickens. CONCLUSIONS: The 2.1.3 clade was the most common lineage found in this study. All the viruses had sequence characteristic of HPAI, but negligible variations in other recognized amino acids at the HA and NA proteins which determine virus phenotypes. Multiple genetic variants appeared to be circulating simultaneously within poultry communities. The high proportion of live duck cases compared to chickens over the study period suggests that ducks are more likely to survive infection and they may better suit the role of long-term maintenance host for H5N1. As some viruses were isolated from dead birds, there was no clear correlation between genetic variations and pathogenicity of these viruses.

Transport of Moving Duck Flocks in Indonesia and Vietnam: Management Practices That Potentially Impact Avian Pathogen Dissemination.

Highly pathogenic avian influenza (HPAI) virus is endemic in Indonesia and Vietnam, where "moving" duck production is commonly practiced. Questionnaire surveys were conducted with transporters of "moving" duck flocks in Indonesia (N = 55) and Vietnam (N = 43). The main purpose of transportation was to transport duck flocks between rice paddies used for scavenging. Trucks were commonly utilized for transport in both countries (Indonesia: 98.2%, 54/55; Vietnam: 37.2%, 16/43), while boats were only used in Vietnam (62.8%, 27/43). Transporters in Vietnam moved larger flocks and traveled over longer distances. Deaths of ducks due to diseases were reported in both countries (Indonesia: 16.4%, 9/55; Vietnam: 4.7%, 2/43; p = 0.11). Throwing away of carcasses was the primary method of disposal of dead birds in Indonesia (60.0%, 33/55), but was not practiced in Vietnam (p < 0.001), while more transporters in Vietnam (34.9%, 15/43) buried carcasses compared to Indonesia (6.8%, 4/55; p = 0.001). Consumption of carcasses (20.9%, 9/43), sale of dead ducks (14.0%, 6/43) and processing of ducks for fish feed (9.3%, 4/43) was conducted in Vietnam, but not in Indonesia. Vehicles were predominantly cleaned in rivers and stored outside in Vietnam, while cleaning and storage was usually conducted in houses/garages in Indonesia. In conclusion, we identified management practices that potentially impact transmission of avian pathogens, such as HPAI virus. In Indonesia, unsafe management practices were related to multipurpose usage of transport vehicles and disposal of birds in the environment, while in Vietnam, they were related to the mixing of birds during transport, the processing of dead carcasses and the storage and cleaning of transport vehicles.

Development of a Luminex microbead-based serotyping assay for Glaesserella parasuis.

Glaesserella parasuis consists of 15 serovars with some of them highly virulent and some of them avirulent. As killed vaccines do not provide crossprotection across serovars, serotyping is of importance. Serotyping, previously done by gel diffusion, is now done by multiplex PCR followed by electrophoresis. Accurately differentiating 15 serovars by electrophoresis is problematic. To overcome this problem, a Luminex microbead-based multiplex assay was used to differentiate the serovars. The assay consisted of a multiplex PCR assay followed by hybridisation to microbeads which were then analysed on a Luminex machine. The newly developed assay was compared to the multiplex serotyping PCR and the gel diffusion/indirect haemagglutination assay (GD/IHA). The microbead-based assay worked very well for the 15 reference strains but when used on the 74 Australian field strains displayed some problems. The main problems were with the eight out of nine serovar 4 field isolates and the five serovar 7 and three serovar 14 field isolates. While the microbead-based assay could differentiate between the serovar 5 and 12 reference strains, which the serovar multiplex PCR could not, all four field isolates identified by GD/IHA as serovar 12 were identified as serovar 5 by the microbead-based assay. Serovar 4 has been noted to have a high diversity especially among strains from different countries. Our work clearly shows that the diversity of strains at both the national and the international level has to be taken into account when developing diagnostic assays.

Canine parvovirus is shed infrequently by cats without diarrhoea in multi-cat environments.

Whether subclinical shedding of canine parvovirus (CPV) by cats might contribute to the epidemiology of canine CPV infections, particularly in facilities housing both cats and dogs, requires clarification. Conflicting results are reported to date. Using conventional PCR (cPCR) to amplify the VP2 gene, shedding of the CPV variants (CPV-2a, 2b, 2c) by healthy cats in multi-cat environments was reportedly common in Europe but rare in Australia. The aim of this study was to determine whether low-level faecal CPV shedding occurs in multi-cat environments in Australia and Italy using a TaqMan real-time PCR to detect Carnivore protoparvovirus 1 (CPV and feline parvovirus, FPV) DNA, and minor-groove binder probe real-time PCR assay to differentiate FPV and CPV types and to characterize CPV variants. In total, 741 non-diarrhoeic faecal samples from shelters in Australia (n = 263) and from shelters or cat colonies in Italy (n = 478) were tested. Overall, Carnivore protoparvovirus 1 DNA was detected in 49 of 741 (6.61 %) samples. Differentiation was possible for 31 positive samples. FPV was most common among positive samples (28/31, 90.3 %). CPV was detected in 4/31 samples (12.9 %) including CPV-2a in one sample, CPV-2b in another and co-infections of FPV/CPV-2b and CPV-2a/CPV-2b in the remaining two samples. A high rate of subclinical FPV infection was detected in one shelter during an outbreak of feline panleukopenia, during which 21 of 22 asymptomatic cats (95.5 %) sampled were shedding FPV. Faecal shedding of CPV by cats in multi-cat environments is uncommon suggesting that domestic cats are not significant reservoirs of CPV.

Analysis of canine parvoviruses circulating in Australia reveals predominance of variant 2b and identifies feline parvovirus-like mutations in the capsid proteins.

Canine parvovirus (CPV) is a major enteric pathogen of dogs worldwide that emerged in the late 1970s from a feline parvovirus (FPV)-like ancestral virus. Shortly after its emergence, variant CPVs acquired amino acid (aa) mutations in key capsid residues, associated with biological and/or antigenic changes. This study aimed to identify and analyse CPV variants and their capsid mutations amongst Australian dogs, to gain insights into the evolution of CPV in Australia and to investigate relationships between the disease and vaccination status of dogs from which viruses were detected. CPV VP2 sequences were amplified from 79 faecal samples collected from dogs with parvoviral enteritis at 20 veterinary practices in five Australian states. The median age at diagnosis was 4 months (range 1-96 months). Only 3.7% of dogs with vaccination histories had completed recommended vaccination schedules, while 49% were incompletely vaccinated and 47.2% were unvaccinated. For the first time, CPV-2b has emerged as the dominant antigenic CPV variant circulating in dogs with parvoviral enteritis in Australia, comprising 54.4% of viruses, while CPV-2a and CPV-2 comprised 43.1% and 2.5%, respectively. The antigenic variant CPV-2c was not identified. Analysis of translated VP2 sequences revealed a vast repertoire of amino acid (aa) mutations. Several Australian CPV strains displayed signatures in the VP2 protein typical of Asian CPVs, suggesting possible introduction of CPV strains from Asia, and/or CPV circulation between Asia and Australia. Canine parvoviruses were identified containing aa residues typical of FPV at key capsid (VP2) positions, representing reverse mutations or residual mutations retained from CPV-2 during adaptation from an FPV-like ancestor, suggesting that evolutionary intermediates between CPV-2 and FPV are circulating in the field. Similarly, intermediates between CPV-2a-like viruses and CPV-2 were also identified. These findings help inform a better understanding of the evolution of CPV in dogs.

Latent class analysis identifies multimorbidity patterns in pigs with respiratory disease.

Respiratory disease is one of the major causes of losses to the pig industry worldwide. The pig subsector is the largest component of the livestock sector in the Philippines. Using lung scoring, this study aimed to estimate the prevalence of thoracic lesions in slaughter-age pigs in two provinces in the Philippines (Batangas and Albay) and define classes for respiratory health of pigs characterised by different patterns of thoracic lesions. A total of 260 pigs from Batangas and 300 pigs from Albay from either commercial or backyard farm types were included in this cross-sectional study. Lungs were scored for cranio-ventral pneumonia (0-55) and pleurisy (0-3). Presence or absence of pericarditis as well as focal dorso-caudal pneumonia were recorded. Latent class analyses considering four indicator variables, and province and farm type as covariates were used to explore different patterns of thoracic lesions across the study populations. Using a threshold of ≥7, the prevalence of a high lung score was 51.9% (95% confidence interval [CI]: 42.3-61.4%) and 13.7% (95% CI: 8.1-22.2%) in Batangas and Albay, respectively. Similarly, the prevalence of a pleurisy score of ≥1 was 56.9% (95% CI: 37.5-74.4%) and 5.0% (95% CI: 2.9-8.4%), pericarditis 24.6% (95%CI: 10.1-48.6%) and 1.7% (95%CI: 0.3-6.7%) and focal dorso-caudal pneumonia lesions 7.7% (95% CI: 3.7-15.5%) and 0% (97.5% one-sided CI: 0-1.2%), respectively. Latent class analyses identified four classes based on lung score, pleurisy score and the presence/absence of pericarditis: "healthy", "mild respiratory disease", "moderate pneumonia", and "multi-lesion". The relative frequency of these classes differed with province and farm type. Most pigs from Albay were "healthy", whereas in Batangas most pigs from commercial farms were "multi-lesion" and those from backyard farms were in the "mild respiratory disease" class. This study has provided baseline data on thoracic lesions in slaughter-age pigs for the provinces of Batangas and Albay in the Philippines. Targeting farms and areas where "multi-lesion pigs" are most common and further research to identify risk factors for particular classes should maximize impact of future control measures. The latent class analysis approach used could be applied more widely and could add value to analysis of multi-morbidity data collected routinely as part of ongoing monitoring schemes.

Scavenging ducks and transmission of highly pathogenic avian influenza, Java, Indonesia.

In Java, Indonesia, during March 2007-March 2008, 96 farms with scavenging ducks that were not vaccinated against highly pathogenic avian influenza (HPAI) were monitored bimonthly. Bird-level (prevalence among individual birds) H5 seroprevalence was 2.6% for ducks and 0.5% for chickens in contact with ducks. At least 1 seropositive bird was detected during 19.5% and 2.0% of duck- and chicken-flock visits, respectively. Duck flocks were 12.4x more likely than chicken flocks to have seropositive birds. During 21.4% of farm visits,