Chao-Ting Xiao

Global molecular genetic analysis of porcine circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and reveals a rapid increase of PCV2d

The oldest porcine circovirus type 2 (PCV2) sequence dates back to 1962 and is among several hundreds of publicly available PCV2 sequences. Despite this resource, few studies have investigated the global genetic diversity of PCV2. To evaluate the phylogenetic relationship of PCV2 strains, 1680 PCV2 open reading frame 2 (ORF2) sequences were compared and analysed by methods of neighbour-joining, maximum-likelihood, Bayesian inference and network analysis. Four distinct clades were consistently identified and included PCV2a, PCV2b, PCV2c and PCV2d; the p-distance between PCV2d and PCV2b was 0.055±0.008, larger than the PCV2 genotype-definition cut-off of 0.035, supporting PCV2d as an independent genotype. Among the 1680 sequences, 278-285 (16.5-17 %) were classified as PCV2a, 1007-1058 (59.9-63 %) as PCV2b, three (0.2 %) as PCV2c and 322-323 (19.2 %) as PCV2d, with the remaining 12-78 sequences (0.7-4.6 %) classified as intermediate clades or strains by the various methods. Classification of strains to genotypes differed based on the number of sequences used for the analysis, indicating that sample size is important when determining classification and assessing PCV2 trends and shifts. PCV2d was initially identified in 1999 in samples collected in Switzerland, now appears to be widespread in China and has been present in North America since 2012. During 2012-2013, 37 % of all investigated PCV2 sequences from US pigs were classified as PCV2d and overall data analysis suggests an ongoing genotype shift from PCV2b towards PCV2d. The present analyses indicate that PCV2d emerged approximately 20 years ago.

Emergence of a novel mutant PCV2b variant associated with clinical PCVAD in two vaccinated pig farms in the U.S. concurrently infected with PPV2

Porcine circovirus (PCV) type 2b (PCV2b) emerged in North America in 2005-2006. During May of 2012, PCVAD occurred in 10-18-week-old pigs in two farms within a production system that routinely vaccinated against PCV2. Both farms received replacement gilts from the same multiplier. A mutant PCV2b strain not previously present in North America was identified. The strain was found to be 99.9% identical to a recently described mutant PCV2 isolate reported in China in 2010 and thought to be more virulent than classical PCV2a or PCV2b strains. It is possible that the current PCV2a-based commercial vaccines are not fully protective against this new strain. In addition, emerging porcine parvovirus type 2 (PPV2) was detected in 55% of the serum samples (73/132), perhaps implying that PPV2 could be a cofactor in cases of PCVAD.

Complete genome sequence of a novel porcine circovirus type 2b variant present in cases of vaccine failures in the United States.

ABSTRACT Two genomes of a new porcine circovirus type 2 (PCV2) strain associated with cases of perceived failure of PCV2 vaccination were sequenced and analyzed. Based on the genome, this is the first report of this mutant of PCV2b in the United States. The genomic knowledge of this mutant PCV2b will improve understanding of the epidemiology of PCV and potentially inform the development of new and more effective vaccines for PCV2.

Porcine Epidemic Diarrhea Virus RNA Present in Commercial Spray-Dried Porcine Plasma Is Not Infectious to Naïve Pigs

Porcine epidemic diarrhea virus emerged in North America in April 2013 and has since been identified in 30 U.S. States, Canada and Mexico. The rapid spread of PEDV has raised concerns about the role of feed and particularly pork-by-product components such as spray-dried porcine plasma (SDPP) in PEDV transmission. The aim of this study was to determine the infectivity of PEDV RNA present in commercial SDPP. Specifically, 40 3-week-old PEDV naïve pigs were randomly assigned to one of five treatment groups. At day post inoculation (dpi) 0, NEG-CONTROL pigs were sham-inoculated, PEDV-CONTROL pigs received cell culture propagated PEDV, and SDPP-CONTROL pigs were switched to a diet with 5% SDPP containing 5.1±0.1 log10 PEDV RNA copies/g. To evaluate a potential positive effect of anti-PEDV antibodies in SDPP on PEDV challenge, four days prior to PEDV challenge the pigs in the SDPP-PEDV group were switched to and remained on a 5% SDPP diet through dpi 28. Another group, EGG-PEDV, was orally administered a commercial egg-derived liquid PEDV globulin product from dpi -4 through 6. All PEDV-CONTROL pigs began shedding PEDV in feces by dpi 3 and seroconverted between dpi 7 and 14, whereas pigs in NEG-CONTROL and SDPP-CONTROL groups remained PEDV RNA negative and did not seroconvert to PEDV for the study duration. This indicates no evidence of infectivity of the PEDV RNA in the SDPP lot utilized. Furthermore, under the study conditions SDPP or egg-derived liquid PEDV globulin addition did not significantly alter PEDV-shedding or overall disease course after experimental challenge.

Identification and characterization of novel porcine astroviruses (PAstVs) with high prevalence and frequent co-infection of individual pigs with multiple PAstV types

Many astrovirus (AstV) species are associated with enteric disease, although extraintestinal manifestations in mammalian and avian hosts have also been described. In this study, the prevalence rates of porcine AstV types 1-5 (PAstV1-PAstV5) were investigated using faecal samples from 509 pigs of which 488 (95.9%) came from farms with a history of diarrhoea. All of the five known PAstV types were found to circulate in pigs in the USA, and co-infection of a single pig with two or more PAstV types was frequently observed. A high overall prevalence of 64.0% (326/509) of PAstV RNA-positive samples was detected, with 97.2% (317/326) of the PAstV RNA-positive pigs infected with PAstV4. Further genomic sequencing and characterization of the selected isolates revealed low sequence identities (49.2-89.0%) with known PAstV strains, indicating novel types or genotypes of PAstV2, PAstV4 and PAstV5. Some new features of the genomes of the PAstVs were also discovered. The first complete genome of a PAstV3 isolate was obtained and showed identities of 50.5-55.3% with mink AstV and the novel human AstVs compared with 38.4-42.7% with other PAstV types. Phylogenetic analysis revealed that PAstV1, PAstV2 and PAstV3 were more closely related to AstVs from humans and other animals than to each other, indicating past cross-species transmission and the zoonotic potential of these PAstVs.

The spray-drying process is sufficient to inactivate infectious porcine epidemic diarrhea virus in plasma.

Abstract Porcine epidemic diarrhea virus (PEDV) is considered an emergent pathogen associated with high economic losses in many pig rearing areas. Recently it has been suggested that PEDV could be transmitted to naïve pig populations through inclusion of spray-dried porcine plasma (SDPP) into the nursery diet which led to a ban of SDPP in several areas in North America and Europe. To determine the effect of spray-drying on PEDV infectivity, 3-week-old pigs were intragastrically inoculated with (1) raw porcine plasma spiked with PEDV (RAW-PEDV-CONTROL), (2) porcine plasma spiked with PEDV and then spray dried (SD-PEDV-CONTROL), (3) raw plasma from PEDV infected pigs (RAW-SICK), (4) spray-dried plasma from PEDV infected pigs (SD-SICK), or (5) spray-dried plasma from PEDV negative pigs (SD-NEG-CONTROL). For the spray-drying process, a tabletop spray-dryer with industry-like settings for inlet and outlet temperatures was used. In the RAW-PEDV-CONTROL group, PEDV RNA was present in feces at day post infection (dpi) 3 and the pigs seroconverted by dpi 14. In contrast, PEDV RNA in feces was not detected in any of the pigs in the other groups including the SD-PEDV-CONTROL group and none of the pigs had seroconverted by termination of the project at dpi 28. This work provides direct evidence that the experimental spray-drying process used in this study was effective in inactivating infectious PEDV in the plasma. Additionally, plasma collected from PEDV infected pigs at peak disease did not contain infectious PEDV. These findings suggest that the risk for PEDV transmission through commercially produced SDPP is minimal.

The prevalence of Torque teno sus virus (TTSuV) is common and increases with the age of growing pigs in the United States

Infection with the Torque teno sus virus (TTSuV) is believed to be common yet limited information is available on the epidemiology of TTSuV. The objectives of this study were to develop novel and improve existing diagnostic methods for TTSuV infection and to investigate the prevalence of TTSuV species 1 (TTSuV1) and 2 (TTSuV2) in the USA. Three hundred and four blood or fetal thoracic fluid samples were collected from pigs on 40 US farms in 12 States. Samples were collected from fetuses and in pre-suckle neonates (n=73), suckling pigs (1-20 days of age; n=27), nursery pigs (21-55 days of age; n=60), finisher pigs (8-25 weeks of age; n=90) and adults (>25 weeks of age; n=54). Samples were tested by a new quantitative differential real-time PCR for TTSuV1 and TTSuV2 DNA and by ELISA for detection of anti-TTSuV2-antibodies. The prevalence of TTSuV1 DNA ranged from 8.2% (fetuses and neonates) to 81% (finisher pigs) and the prevalence of TTSuV2 DNA ranged from 3.7% (suckling pigs) to 67% (finisher pigs). Evidence of fetal TTSuV infection was minimal. Mixed infection of TTSuV1 and TTSuV2 was seen in 6.7% of the nursery pigs, 52.2% of the finisher pigs, and 22.2% of the mature pigs. The prevalence of TTSuV1 was higher than that of TTSuV2. Anti-TTSuV2 antibodies were not detected in the fetuses and neonates and the seroprevalence of TTSuV2 was between 3.8% and 100% in growing pigs. The results of this study indicate that vertical transmission may not be a main route of TTSuV transmission in pigs in the USA.

Identification of recently described porcine parvoviruses in archived North American samples from 1996 and association with porcine circovirus associated disease.

The association of porcine circovirus (PCV) type 2 and porcine parvovirus (PPV) type 1 as a cause of porcine circovirus associated disease (PCVAD) is well established. The objective of this study was to investigate the prevalence rates of classical PPV1 and recently recognized PPV2-5 in serum and lung samples from pigs and farms with known PCV2 status. A total of 586 serum samples and 164 lung homogenates collected from 1996 to 2013 in the USA and Canada were utilized. All samples were tested for PPV1-5 and PCV2. PCV2 was detected in 27.7% (162/586) and PPV in 48.8% (286/586) of the serum samples, whereas 78.7% (129/164) of the lung tissues were positive for PCV2 and 56.7% (93/164) were positive for PPV. Overall, PPV2 had the highest prevalence rates in sera (35.2%) and tissues (42.7%). Concurrent infection of PCV2 and PPV occurred in 14.3% (84/586) of the serum samples and in 49.4% (81/164) of the tissue samples. Moreover, the prevalence of PPV1 or PPV2 DNA was significantly higher in tissues containing high amounts of PCV2 DNA compared to non-PCVAD cases. The frequency of concurrent PPV/PCV2 infection was higher for PCVAD herds compared to negative or subclinically infected herds. PPV2, PPV3 and PPV4 were all identified in samples collected in 1998 and PPV5 was first identified in 2006. The obtained findings indicate that similar to PCV2, PPVs are widespread in North American pigs. Nevertheless, diagnostic investigations into PCVAD cases should give more consideration to the role of PPV1 and PPV2 as contributing cofactors.

A commercial vaccine based on PCV2a and an experimental vaccine based on a variant mPCV2b are both effective in protecting pigs against challenge with a 2013 U.S. variant mPCV2b strain

During 2012 and 2013, an apparent increase in porcine circovirus associated disease occurred in the USA. A variant PCV2b strain designated mPCV2b was recovered from many of these cases. This raised concerns of a decrease in efficacy of commercially available PCV2 vaccines. The objective of this study was to compare the ability of a commercial PCV2a-based vaccine and an experimental mPCV2b-based vaccine to control mPCV2b-associated disease, lesions, and viremia in a challenge model. Twenty-six caesarian-derived, colostrum-deprived pigs were randomly assigned to one of four groups: (1) vaccinated with a commercial PCV2a-based vaccine and challenged (PCV2a-VAC; n=7), (2) vaccinated with an experimental mPCV2b-based vaccine and challenged (mPCV2b; n=7), (3) sham-vaccinated with saline and challenged (positive controls; n=7), and (4) sham-vaccinated with saline without challenge (negative controls; n=5). Vaccination was done on D0 and D14, challenge was done on D28 using a tissue homogenate containing PRRSV and mPCV2b and the experiment was terminated on D49. Among the challenged pigs, 47.6% (10/21) developed severe clinical disease and either died or had to be humanely euthanized between D39 and D48 (11-20 days after challenge). PCV2 viremia was almost completely absent in the vaccinated groups regardless of vaccine type except for two PCV2a-vaccinated pigs which had detectable PCV2 DNA levels on individual days after challenge. Microscopic lesions typical of PCV2 infection were limited to the positive control group which developed mild-to-severe lesions associated with low-to-abundant PCV2 antigen. Under the conditions of this study, PCV2 vaccines regardless of PCV2 type were effective against mPCV2b challenge.

Molecular evolutionary genetic analysis of emerging parvoviruses identified in pigs.

Parvoviruses infect a wide variety of vertebrates and arthropods and are associated with various clinical manifestations. Due to the advent of new sequence-independent PCR methods and high-throughput sequencing, several novel members of parvoviruses within the subfamily Parvovirinae were recently described. Several of these viruses do not fit in the current classification and others now have confusing or contradictory nomenclature because two or more names were used for similar or identical groups of parvoviruses or identical names were used for distinct virus groups. In this study, recently described vertebrate parvoviruses with emphasis on those identified in pigs were classified through phylogenetic analyses based on the sequences of their complete or near complete genomes, open reading frame (ORF) 1 (non-structural protein, NS1), ORF2 (capsid protein, VP1), and ORF3 (nuclear phosphoprotein, NP1) genes by using Bayesian Markov chain Monte Carlo (MCMC), Maximum Likelihood (ML) and Neighbor-Joining (NJ) methods. Among all available vertebrate parvovirus sequences, eight distinct clades were identified, corresponding to the five well established genera Parvovirus, Erythrovirus, Denpendovirus, Amdovirus and Bocavirus. Moreover, three novel clades were identified and tentatively designated as PARV4-like virus, novel clade 1 and novel clade 2. Parvoviruses in pigs were found to be distributed across four different clades including Parvovirus, Bocavirus, PARV4-like virus and the novel clade 2. All pig parvoviruses identified to date were organized based on the current analysis. The present analysis will assist to clarify the nomenclature of parvoviruses in pigs and facilitate future uniform assignment of names for new parvoviruses within the subfamily Parvovirinae.