Emergence of novel circoviruses in humans and pigs and their possible importance for xenotransplantation and blood transfusions.

BACKGROUND: As sequencing is becoming more broadly available, virus discovery continues. Small DNA viruses contribute to up to 60% of the overall virus load in pigs. Porcine circoviruses (PCVs) are small DNA viruses with a single-stranded circular genome. They are common in pig breeds and have not been properly addressed for their potential risk in xenotransplantation. Whereas PCV1 is non-pathogenic in pigs, PCV2 has been associated with various disease manifestations. Recently two new circoviruses have been described, PCV3 and PCV4. While PCV4 is currently present mainly in Asia, PCV3 is widely distributed, and has been identified in commercial pigs, wild boars, and pigs generated for xenotransplantation. In one case PCV3 was transmitted by pigs to baboons via heart transplantation. PCV3 pathogenicity in pigs was controversial initially, however, the virus was found to be associated with porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, and multisystemic inflammation. Inoculation studies with PCV3 infectious clones confirmed that PCV3 is pathogenic. Most importantly, recently discovered human circoviruses (CV) are closely related to PCV3. METHODS: Literature was evaluated and summarized. A dendrogram of existing circoviruses in pigs, humans, and other animal species was created and assessed at the species level. RESULTS: We found that human circoviruses can be divided into three species, human CV1, CV2, and CV3. Human CV2 and CV3 are closest to PCV3. CONCLUSIONS: Circoviruses are ubiquitous. This communication should create awareness of PCV3 and the newly discovered human circoviruses, which may be a problem for blood transfusions and xenotransplantation in immune suppressed individuals.

Failure to experimentally infect 10 days-old piglets with a cell culture-propagated infectious stock of a classical genotype 1a porcine epidemic diarrhea virus.

Introduction: Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs of all ages. PEDV can be grouped into G1 (classical strains) and G2 (variant strains) based on sequence differences in the spike gene. Although several pathogenesis studies using contemporary strains of PEDV have been conducted to date, there is limited information on the pathogenesis of historical PEDV strains in contemporary pigs. This study aimed to investigate the clinical disease course of 10 days-old pigs infected with a classical European G1a PEDV strain from the 1980s which was last passaged in pigs in 1994. Methods: Sequencing results confirmed that the virus inoculum was a PEDV strain closely related to the prototype CV777 strain. The PEDV stock was serially passaged three times in Vero cells, and the P3 infectious virus stock was used to inoculate the pigs. A total of 40 pigs were inoculated using the oral route. Results: Pigs showed no enteric disease signs, and PEDV shedding was not detected for 44 days post-inoculation (dpi). At necropsy at 3 (5 pigs) or 7 dpi (5 pigs), no lesions were observed in intestinal sections, which were negative for PEDV antigen by immunohistochemistry. In addition, no IgG or IgA PEDV-specific antibodies in serum or fecal samples for 35 dpi further indicates a lack of infection. Titration of the leftover thawed and refrozen PEDV virus stock inoculum showed that the virus stock retained its infectivity in Vero cell culture and the porcine small intestine enterocytes cell line IPEC-J2. Discussion: The reasons for the loss of infectivity in pigs are unknown. In conclusion, we showed that a classical G1a PEDV strain successfully propagated in cell cultures could not orally infect 40 piglets.

Isolation and characterization of a novel goose ovaritis-associated cluster 3 Tembusu virus.

Tembusu virus (TMUV) is a member of the genus Flavivirus in the family Flaviviridae. Currently, TMUV was classified into 4 distinct clusters, with cluster 2 strains widely distributed in duck and goose populations in Asia, causing significant economic losses to the producing industries. In this study, a novel TMUV strain TMUV/goose/CHN/2019/HNU-NX2 (HNU-NX2-2019) was isolated and characterized from geese with ovaritis from Hunan province, China. Phylogenetic analyses of genome and the E gene indicated the present TMUV could be grouped into the newly defined TMUV cluster 3. The genome of HNU-NX2-2019 showed the highest identities of 98.1% to 98.2% to the cluster 3 TMUVs newly identified in 2020 and 2021 from chickens with a severe egg-drop syndrome from Guangdong, Guangxi and Shandong provinces of China, which were all showing a close relation to a mosquito-origin TMUV (KT607936) identified in 2012. Further experiments confirmed HNU-NX2-2019 could grow well in chicken fibroblast cell line DF-1 and in SPF chicken embryos, with titers varied from 107.3 to 108.8 viral genomic copies per mL in the culture solutions. A pilot virus challenge study in 3-day-old chicks demonstrated that this virus could efficiently infect chicks with virus distributed in the brains, small intestines and other visceral organs, with titers varied from 105.4 to 106.7viral genomic copies per gram of the tissues. Furthermore, HNU-NX2-2019 can induce specific antibody in ducklings but with no obvious disease and virus shedding, and on necropsy no TMUV was detected in the tissues in the present study. This is the first report to identify a novel cluster 3 TUMV from goose, and further demonstrated this goose TMUV strain could infect chicken efficiently but not in ducklings under the present experimental conditions, which highlighted intensive attentions may be paid to this novel mosquito-origin cluster 3 TMUV.

Identification and characterization of a novel avian nephritis virus variant in chickens with enteritis in Hunan province, China.

Avian nephritis virus (ANV) infection is associated with diarrhea, uricosis, stunting, tubulonephrosis, interstitial nephritis, and mortality of chicken flocks, leading to economic losses in the poultry industry. In this study, an ANV strain designated as HNU-ANV-ML-2020 was identified in tissue samples collected from chickens with severe enteritis on a poultry farm in Hunan province, China, and analyzed. The genome of HNU-ANV-ML-2020 is 6943 nucleotides in length. It showed the highest sequence identity (88.1%) to ANV strain CHN/GXJL815/2017 (MN732559) from Guangxi province, China, while it showed less than 86% identity to other astrovirus (AstV) genome sequences available in the GenBank database. The capsid protein of this virus showed the highest sequence identity to ANV strains HQ330482 and HQ330498 from the UK (81.2% and 81.06%, respectively), while it showed only 73.9% identity to MN732559 and less than 80% identity to the capsid proteins of other AstVs available in GenBank. Further phylogenetic analysis demonstrated that HNU-ANV-ML-2020 belongs to group 4, together with ANV strains identified in Australia, Brazil, the UK, and the Netherlands. Furthermore, ANV strains identified in chickens in China were found to be separated into four distinct groups/genotypes, indicating substantial genetic divergence and a complex circulation pattern in China. The virus characterized in the present study is a novel ANV variant identified for the first time in Hunan province, China.

Identification and characterization of a novel circovirus species in domestic laying ducks designated as duck circovirus 3 (DuCV3) from Hunan province, China.

Duck circovirus (DuCV) is an immunosuppressive virus and can induce immunosuppression increasing rates of infection caused by other pathogens, which has resulted in gross economic losses in poultry industries. The genome sizes of DuCVs varied from 1987 to 1996 nucleotides (nt). Up to date two DuCV genotypes/lineages, DuCV1 and DuCV2, have been defined, with genome identities of around 83% between each other. In this study, a novel duck circovirus having a genome size of 1755 nt was identified from laying ducks with a disease of egg production declining or abrogation from Hunan province, China. Two major open reading frames (ORFs) were identified, with ORF1 (882 nt) and ORF2 (738 nt) encoding replicase (Rep) and capsid protein (Cap), respectively. Its genome showed the highest identities of 62.3-63.7% and 66.3%- 67.8% to the known genomes of DuCVs and goose circoviruses (GoCVs) available in GenBank, respectively, while it showed less than 50% identities to the genomes of other circoviruses. The amino acid sequence of capsid of this virus showed the highest identities of 45.4%- 47.3% and 42.9%- 44.5% to capsids of the known DuCVs and GoCVs, respectively, while it showed less than 27% identity to the capsid proteins of other circoviruses available in GenBank. Further phylogenetic analyses based on genomes, amino acid sequences of Rep and Cap proteins demonstrated that the present duck circovirus was clustered in a separate clade, distinct from other DuCVs and GoCVs, corroborating it is a distinct novel circovirus species in ducks, tentatively designated as duck cirovirus 3 (DuCV3). The clinical significance and pathogenesis of this virus needs further investigation.

Current knowledge on epidemiology and evolution of novel porcine circovirus 4.

Porcine circovirus type 4 (PCV4) is a newly emerging virus, with both PCV4 genomic DNA and specific antibodies detected in swine herds in several provinces in China and South Korea. Although the virus was first identified in 2019 in Hunan, China, retrospective research suggests that serum samples collected as early as 2008 were positive for PCV4 antibody. Infections with only PCV4 or co-infections with other pathogens have been associated with several clinical manifestations, but its pathogenesis remains to be determined. The purpose of this review was the following: (1) to characterize PCV4 epidemiology by assessing evolutionary dynamics and genetic diversity of PCV4 strains circulating in swine herds; (2) to reconstruct a computerized 3D model to analyze PCV4 Cap properties; (3) and to summarize the current evidence of PCV4-associated clinical-pathological manifestations. The origin of PCV4 is apparently distinct from other PCV, based on analysis of phylogenetic trees. Of note, PCV4 shares an ancient common ancestor with mink circoviruses. Furthermore, the amino acid residue at position 27 of the PCV4 Cap is a key benchmark to distinguish PCV4a (27S) from PCV4b (27 N), based on PCV4 strains currently available, and variation of this residue may alter Cap antigenicity. In addition, the capsid surface of PCV4 has characteristics of increased polar residues, compared to PCV2, which raises the possibility that PCV4 may target negatively charged host receptors to promote virus infection. Further studies are required, including virus isolation and culture, and more detailed characterization of molecular epidemiology and genetic diversity of PCV4 in swine herds.

Identification and genomic characterization of a novel porcine CRESS DNA virus from a pig suffering from diarrhea in China.

The circular replication-associated protein (Rep)-encoding ssDNA (CRESS-DNA) viruses show high diversity and have a very wide range of hosts, including all three domains of cellular life. In the present study, a novel CRESS DNA virus, provisionally named "kirkovirus HNU-XX-2020" was discovered in a growing pig with watery diarrhea. The virus has a circular genome of 2961 nucleotides (nt) and three major putative open reading frames (ORFs), encoding a Rep protein (327 amino acids), a capsid protein (175 amino acids), and one protein (209 amino acids) of unknown function. The genome showed the highest sequence similarity (68.6% identity) to the genome of porcine circo-like virus 51 (JF713719), which was identified in pig faeces, and it showed very limited sequence similarity (less than 40% identity) to other virus genomes. Further phylogenetic analysis suggested that it could be a novel member in the proposed family "Kirkoviridae".

Prevalence and Genetic Analysis of Porcine Circovirus 3 in China From 2019 to 2020.

Porcine circovirus type 3 (PCV3), a virus belonging to the Circoviridae family, is considered to be associated with respiratory and neurological signs, cardiac and multisystemic inflammation, reproductive failure, and porcine dermatitis and nephropathy syndrome-like disease in pigs (Sus scrofa). In this study, epidemiological and serological investigations of PCV3 in clinically healthy pigs from different regions of China were performed. Overall, 42.87% (1,101/2,568) of pigs were positive for PCV3 Cap antibody via indirect enzyme-linked immunosorbent assay, with a higher prevalence of PCV3 in multiparous sows (62.22%, 881/1,416) and fattening pigs (28.96%, 159/549) than in suckling piglets (8.96%, 32/357) and nursery pigs (11.79%, 29/246). Of the 2,568 samples, 255 were further tested for PCV3 DNA using real-time polymerase chain reaction, and 63.14% of these were positive, with nearly half having <10 virus copies. The PCV3 DNA and antibody positivity rates were high in the pig serum samples; however, the virus titers and antibody levels were both low, indicating that the humoral immune response of PCV3-infected pigs was weak or lagging, and persistent or repeated infections could occur. Additionally, the complete genomes of 23 PCV3 strains were sequenced and analyzed, which showed nucleotide identities of 98.5~100.0%, 98.6~100.0%, and 99.2~100.0% in the complete genome, open reading frame (ORF)2, and ORF1 sequences, respectively, and amino acid identities of 96.7~100.0% and 99.3~100.0% in the capsid and replicase proteins, respectively. Phylogenetic analysis based on ORF2 nucleotide sequences indicated that the PCV3 strains obtained in the present study could be classified into three sub-clades, with most strains clustered into clade 3c, indicating that PCV3c is the dominant subtype in the regions of China investigated. In general, the present study revealed a high prevalence and high genetic divergence of PCV3 among Chinese pig herds, and indicated that the potential effect of PCV3 on the pig industry may be a concern.

The seroprevalence of the newly identified porcine circovirus type 4 in China investigated by an enzymed-linked immunosorbent assay.

Porcine circovirus 4 (PCV4) is the fourth porcine circovirus newly identified in China, and it could be detected in diseased and healthy pigs. To date, the prevalence of PCV4 DNA in pig herds has been investigated in many provinces from both China and Korea, with positive rates varied from 3.28% to 25.4% in samples from different regions. However, up to now no serological data have been reported to evaluate the prevalence of PCV4 in pig herds. In this study, an indirect anti-PCV4 IgG enzyme-linked immunosorbent assay (ELISA) based on replicase protein (Rep) was developed and utilized to investigate the seroprevalence of PCV4 in pig herds of China. A total of 1790 swine serum samples from 17 provinces of China were tested including samples confirmed positive for PCV4 DNA. There was no cross-reactivity of this ELISA with PCV1, PCV2 and PCV3. PCV4 Rep antibodies have been detected in serum samples from 16 out of 17 provinces in China. The PCV4 overall seroprevalence was 43.97%, with the highest of 67.8% been detected in sows, followed by fattening and suckling pigs with positive rates of 35.0% and 14.56%, respectively, and the lowest of 12.61% been detected in nursery pigs. Moreover, from the present data, the earliest positive sample could be retrieved to at least 2008. The present study provides an overall seroprevalence of PCV4 in China, and is helpful to understand the prevalence of PCV4 in the pig herds since it was discovered.

Isolation and evolutionary analyses of gout-associated goose astrovirus causing disease in experimentally infected chickens.

Astroviruses are a common cause of gastroenteritis in humans and animals. They are also associated with extraintestinal infections, including hepatitis in ducklings, nephritis in chickens, as well as fatal meningitis and encephalitis in humans and other mammals. Since 2014, outbreaks of disease characterized by visceral gout and swelling of kidneys have been reported in goslings and ducklings in China, with the causative agent revealed to be a novel avian astrovirus designated goose astrovirus (GoAstV). In the present study, this novel gout-associated GoAstV was identified in diseased goslings from 2 farms in Hunan province, China. Three genomes were successfully sequenced and analyzed and were shown to have high identities of 99.7 to 99.8% between each other, with some specific amino acid alterations revealed in open reading frame 2 when compared with other gout-associated GoAstVs. Two strains were further efficiently isolated in the DF-1 chicken fibroblast cell line with high virus titers of 1011 viral genomic copies per mL of culture media. A pilot virus challenge study using GoAstV in chickens demonstrated that this virus can cause clinical visceral gout in chickens, indicating its ability to cross the species barrier. Based on the phylogenetic analyses of capsid sequences, the identified GoAstVs were proposed to be classified into 2 genotypes, GoAstV1 and GoAstV2, and the novel gout-associated GoAstVs were all clustered in GoAstV2. Further Bayesian inference analyses indicated a nucleotide substitution rate of 1.46 × 10-3 substitutions/site/year for avian astrovirus based on open reading frame 2 sequences, and the time to the most recent common ancestor of GoAstVs was estimated to be around 2011. This is the first report to confirm GoAstV can infect chickens while also providing an estimation of the evolutionary rates of Avastroviruses.

Porcine Astrovirus Type 5-Associated Enteritis in Pigs.

Astroviruses (AstVs) are emerging, potentially zoonotic pathogens, associated with enteric and neurological signs in various animal species. In pigs, five lineages have been identified, porcine AstVs (PoAstVs) 1-5. We now report PoAstV5-associated atrophic enteritis in a colostrum-deprived (CD) pig and a similar disease in field cases of enteritis. Four newborn CD piglets were housed under biosafety level 2 conditions and monitored for the presence of PoAstV RNA in rectal swabs and tissues. PoAstV4 RNA was detected in low numbers (0.9-5.3 log10 genomic copies/rectal swab) in rectal swabs from all of these pigs while all animals were negative for PoAstV1, PoAstV2, PoAstV3 and PoAstV5. At 19 and 20 days of age, high levels of PoAstV5 RNA (7.4-7.5 log10 genomic copies/rectal swab) were detected in one pig, which had developed enteritis. At necropsy 1 day after the first evidence of PoAstV5 shedding, this pig was strongly positive for PoAstV5 RNA in most tissues with strong immunolabelling of PoAstV5 in enterocytes. Gene sequencing confirmed PoAstV5 infection. A retrospective investigation of PoAstVs 3, 4 and 5 in archival tissues from field cases of post-weaning enteritis identified high PoAstV4 or PoAstV5 antigen levels in intestinal tissues. These pigs were often concurrently infected with porcine rotavirus. Our findings suggest that PoAstV5 may contribute to the pathogenesis of enteritis in young pigs.

Porcine circoviruses: current status, knowledge gaps and challenges.

Circoviruses (CV) include some of the smallest viruses known. They were named after their circularly arranged single-stranded DNA genome with a gene encoding a conserved replicase protein on the sense strand. Circoviruses are widely distributed in mammals, fish, avian species and even insects. In pigs, four different CVs have been identified and named with consecutive numbers based on the order of their discovery: Porcine circovirus 1 (PCV1), Porcine circovirus 2 (PCV2), Porcine circovirus 3 (PCV3) and most recently Porcine circovirus 4 (PCV4). PCVs are ubiquitous in global pig populations and uninfected herds are rarely found. It is generally accepted that PCV1 is non-pathogenic. In contrast, PCV2 is considered an important, economically challenging pathogen on a global scale with comprehensive vaccination schemes in place. The role of PCV3 is still controversial several years after its discovery. Propagation of PCV3 appears to be challenging and only one successful experimental infection model has been published to date. Similarly to PCV2, PCV3 is widespread and found in many pigs regardless of their health history, including high health herds. PCV4 has only recently been discovered and further information on this virus is required to understand its potential impact. This review summarizes current knowledge on CVs in pigs and aims to contrast and compare known facts on PCVs.

Novel circovirus species identified in farmed pigs designated as Porcine circovirus 4, Hunan province, China.

In pigs, three circovirus species within the genus Circovirus have been identified so far, including the non-pathogenic Porcine circovirus 1 (PCV1), the pathogenic Porcine circovirus 2 (PCV2) and the recently identified Porcine circovirus 3 (PCV3). In April 2019, a new circovirus with a distinct relationship to other circoviruses was identified in several pigs with severe clinical disease in Hunan province, China. The size of the viral genome, tentatively designated as porcine circovirus type 4 (PCV4), is 1,770 nucleotides (nt). PCV4 shows the highest genomic identity to mink circovirus (66.9%) and has identities of 43.2%-51.5% to the other PCV genomes. Two major genes, a replicase (Rep) gene spanning 891 nt and a capsid (Cap) gene spanning 687 nt, were predicted. Furthermore, a TaqMan® real-time polymerase chain reaction (PCR) targeting the replicase gene was developed to investigate the prevalence of PCV4 in 187 clinical samples from Hunan province, China. The results revealed an overall PCV4 prevalence of 12.8%, with the highest positive rates in nasal swabs (28.5%, 6/21) followed by serum samples (13.4%, 11/82). The clinical significance and pathogenesis of this virus needs further investigation.

Genomic characterization of a novel astrovirus identified in Amur tigers from a zoo in China.

Astroviruses (AstVs) have a very wide range of hosts and are associated with enteric and extra-enteric disease in mammals and birds. Cross-species transmission of AstVs has been observed frequently. In the present study, the genome of a novel astrovirus from Amur tigers (Panthera tigris) from a zoo in China was characterized and was found to have the typical genomic features of other mammal AstVs. It showed the highest nucleotide sequence similarity (46.1-87.3% identity) to AstVs from cats, indicating a close phylogenetic relationship and possible cross-species transmission between them. To our knowledge, this is the first identification and characterization of AstV from tigers, and this virus is the third astrovirus identified in hosts of the family Felidae. The results of this study will be helpful for understanding the origin, genetic diversity, and cross-species transmission of AstV.

Homologous recombination shapes the genetic diversity of African swine fever viruses.

The African swine fever virus (ASFV) has severely influenced the swine industry of the world. Currently, there is no effective vaccine or drugs against the ASFV. How to effectively control the virus is challenging. In this study, we have analyzed all the publicly available ASFV genomes and demonstrated that there was a large genetic diversity of ASFV genomes. Interestingly, the genetic diversity was mainly caused by extensive genomic insertions and/or deletions (indels) instead of the point mutations. Further analyses showed that the indels may be attributed much to the homologous recombination, as supported by significant associations between the occurrence of extensive recombination events and the indels in the ASFV genomes. Besides, the homologous recombination also led to changes of gene content of ASFVs. Finally, repeated elements of dozens of nucleotides in length were observed to widely distribute and cluster in the adjacent positions of ASFV genomes, which may facilitate the occurrence of homologous recombination. This work highlighted the importance of homologous recombination in shaping the genetic diversity of the ASFVs, and could help understand the evolution of the virus.

High genetic diversity and recombination events of porcine astrovirus strains identified from ill and asymptomatic pigs in 2017, Hunan Province, China.

Astroviruses (AstV) are associated with enteric and systemic disease in mammals and birds. Astroviruses have received increased attention recently as they have been found to be associated with sporadic neurologic disease in mammals including humans. In pigs, porcine astrovirus (PoAstV) can be widely detected and has been grouped in five genotypes (PoAstV1 to PoAstV5). In the present study, we detected multiple PoAstVs in serum samples, nasal swabs, and fecal swabs collected from pigs suffering from respiratory disease or diarrhea but also from asymptomatic pigs, indicating a wide tissue tropism of the identified PoAstV genotypes. Coinfection of different genotypes in the same pig was commonly observed, and within an individual pig a high genetic diversity was observed for viruses belonging to the same PoAstV genotype. Two complete genomes of PoAstV2-WG-R2/2017 and PoAstV4-WG-R2/2017 were successfully obtained and characterized, with genome sizes of 6396 and 6643 nucleotides, respectively. The PoAstV2-WG-R2/2017 genome showed identities of 67.2-77.4% to other known PoAstV2 genomes, and the PoAstV4-WG-R2/2017 genome showed identities of 72.8-80.5% to other known PoAstV4 genomes. The predicted spike domain of open reading frame 2 (ORF2) of these strains showed the highest genetic heterogeneity, with amino acid identities of 13.7-70.9% for PoAstV2-WG-R2/2017 to other known PoAstV2 strains, and identities of 24.4-63.3% for the PoAstV4-WG-R2/2017 to other known PoAstV4 strains. Possible recombination events were identified in each of the two sequences. Two subclades of PoAstV2 and three subclades of PoAstV4 were defined in the present analyses. The obtained data provide further evidence for extraintestinal infectivity of PoAstVs, and confirmed the high genetic diversity of PoAstVs and the coinfection potential of different PoAstV types in a single pig.

A commercial porcine circovirus (PCV) type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naïve pigs under experimental conditions.

Porcine circovirus type 2 (PCV2) vaccination has been effective in protecting pigs from clinical disease and today is used extensively. Recent studies in vaccinated populations indicate a major PCV2 genotype shift from the predominant PCV2 genotype 2b towards 2d. The aims of this study were to determine the ability of the commercial inactivated PCV2a vaccine Circovac® to protect pigs against experimental challenge with a 2013 PCV2d strain and prevent transmission. Thirty-eight pigs were randomly divided into four groups with 9-10 pigs per group: NEG (sham-vaccinated, sham-challenged), VAC (PCV2a-vaccinated, sham-challenged), VAC+CHAL (PCV2a-vaccinated and PCV2d-challenged), and CHAL (sham-vaccinated, PCV2d-challenged). Vaccination was done at 3weeks of age using Circovac® according to label instructions. The CHAL and VAC+CHAL groups were challenged with PCV2d at 7weeks of age and all pigs were necropsied 21days post-challenge (dpc). The VAC-CHAL pigs seroconverted to PCV2 by 21days post vaccination (dpv). At PCV2d challenge on 28dpv, 3/9 VAC and 1/9 VAC+CHAL pigs were seropositive. NEG pigs remained seronegative for the duration of the study. Vaccination significantly reduced PCV2d viremia (VAC+CHAL) at dpc 14 and 21, PCV2d fecal shedding at dpc 14 and 21 and PCV2d nasal shedding at dpc 7, 14 and 21 compared to CHAL pigs. Vaccination significantly reduced mean PCV2 antigen load in lymph nodes in VAC+CHAL pigs compared to CHAL pigs. When pooled serum or feces collected from VAC+CHAL and CHAL pigs at dpc 21 were used to expose single-housed PCV2 naïve pigs, a pooled fecal sample from CHAL pigs contained infectious PCV2 whereas this was not the case for VAC+CHAL pigs suggesting reduction of PCV2d transmission by vaccination. Under the study conditions, the PCV2a-based vaccine was effective in reducing PCV2d viremia, tissue loads, shedding and transmission indicating that PCV2a vaccination should be effective in PCV2d-infected herds.

First identification of porcine parvovirus 6 in Poland.

Porcine parvovirus type 1 is a major causative agent of swine reproductive failure. During the past decade, several new parvoviruses have been discovered in pigs. Porcine parvovirus type 6 (PPV6), recently identified, has been reported in pigs in China and in the USA while the PPV6 status in the European pig population remains undetermined. In the present study, PPV6 DNA was identified in serum samples collected from domestic pigs in Poland. In investigated herds, the prevalence of PPV6 was 14.9 % (15/101 samples). Sequencing was conducted, and 11 nearly complete PPV6 genomes were obtained. Phylogenetic analysis indicated that PPV6 sequences cluster into four distinct groups, and the Polish PPV6 strains from three individual farms were present in three of these four groups. In addition, the Polish PPV6 strain P15-1 was identified as a putative recombination of an ORF1 from US stains and an ORF2 from Chinese strains. This is the first identification of PPV6 in Europe, and this finding will encourage future epidemiological studies on parvoviruses in European pigs.

Identification and characterization of multiple porcine astrovirus genotypes in Hunan province, China.

Astroviruses (AstVs) can infect a variety of hosts, including mammalian and avian species, and are commonly associated with enteric infections. Recently, mammalian AstVs have been linked to extra-intestinal manifestations, including neurologic disorders in humans, cattle and minks, demonstrating zoonotic potential. So far, five porcine AstV (PAstV) genotypes have been identified, with PAstV1, PAstV2, PAstV3 and PAstV5 implicated in cross-species transmission. Our knowledge about PAstV epidemiology in China is still limited. In this study, two duplex differential RT-PCR assays were developed to investigate the distribution and prevalence of PAstV1, PAstV2, PAstV4 and PAstV5. Two hundred eighteen samples were collected from 33 farms and pigs with known diarrhea status in nine regions of Hunan province in China. Specifically, 126 small intestines, 51 fecal swabs, 20 lungs, 19 spleens and two kidneys were obtained. PAstVs were detected in all nine regions and in 81.8% (27/33) of the pig farms investigated. The overall prevalence of PAstV was 46.3% (101/218), with PAstV5 as the predominant type, with a positive rate of 24.8% (54/218). The prevalence of PAstV4, PAstV1 and PAstV2 was 16.1% (35/218), 14.7% (32/218) and 10.1% (22/218), respectively. Besides being present in intestines and fecal swabs, PAstV RNA was also detected in lungs, spleens and kidneys. Sequencing revealed a high level of genetic divergence within each genotype, and a higher positive rate of PAstV5 was associated with pigs with diarrhea compared to pigs without diarrhea. This study revealed for the first time that PAstV4 is circulating in China, and that PAstV5 is the dominant genotype in pig herds in Hunan province in China.

PCV2d-2 is the predominant type of PCV2 DNA in pig samples collected in the U.S. during 2014-2016.

Porcine circovirus type 2 (PCV2) vaccination was introduced in the US in 2006 and since has been adopted by most pig producers. While porcine circovirus associated disease (PCVAD) outbreaks are now relatively uncommon in the US, PCV2 remains a concern which is emphasized by increasing numbers of PCR and sequencing requests for PCV2. In the present study, randomly selected lung tissues from 586 pigs submitted in 2015 were tested for presence of PCV2 DNA. Positive samples were further characterized by sequencing and combined with available PCV2 open-reading-frame (ORF) 2 sequences from the client data base of the Iowa State University Veterinary Diagnostic Laboratory. The prevalence of PCV2 in the randomly selected lung tissues was 23% (135/586) with 11.3% PCV2a, 29% PCV2b and 71.8% for PCV2d subgroup PCV2d-2. A total of 455 ORF2 sequences obtained from 2014 through 2016 were analyzed and PCV2d accounted for 66.7% of the 2014 sequences, 71.8% of the 2015 sequences, and 72% of the 2016 sequences. Interestingly, only 1.9% (9/455) of the sequences belonged to the recently identified PCV2e genotype. The present data indicates that despite an almost 100% PCV2 vaccine coverage in the US, PCV2 DNA can still be detected in almost 1 of 4 randomly selected pig tissues. PCV2d-2 is now the predominant genotype in the USA suggesting that PCV2d-2 may have some advantage over PCV2a and PCV2b in its ability to replicate in pigs under vaccination pressure.