Molecular detection and genetic characteristics of a novel porcine circovirus (porcine circovirus 4) and porcine reproductive and respiratory syndrome virus in Shaanxi and Henan Provinces of China

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) are economically important pathogens in swine, and pigs with dual infections of PCV2 and PRRSV consistently have more severe clinical symptoms and interstitial pneumonia. However, the synergistic pathogenesis mechanism induced by PRRSV and PCV2 co-infection has not yet been illuminated. Therefore, the aim of this study was to characterize the kinetic changes of immune regulatory molecules, inflammatory factors and immune checkpoint molecules in porcine alveolar macrophages (PAMs) in individuals infected or co-infected with PRRSV and/or PCV2. The experiment was divided into six groups: a negative control group (mock, no infected virus), a group infected with PCV2 alone (PCV2), a group infected with PRRSV alone (PRRSV), a PCV2-PRRSV co-infected group (PCV2-PRRSV inoculated with PCV2, followed by PRRSV 12 h later), a PRRSV-PCV2 co-infected group (PRRSV-PCV2 inoculated with PRRSV, followed by PCV2 12 h later) and a PCV2 + PRRSV co-infected group (PCV2 + PRRSV, inoculated with PCV2 and PRRSV at the same time). Then, PAM samples from the different infection groups and the mock group were collected at 6, 12, 24, 36 and 48 h post-infection (hpi) to detect the viral loads of PCV2 and PRRSV and the relative quantification of immune regulatory molecules, inflammatory factors and immune checkpoint molecules. The results indicated that PCV2 and PRRSV co-infection, regardless of the order of infection, had no effect on promoting PCV2 replication, while PRRSV and PCV2 co-infection was able to promote PRRSV replication. The immune regulatory molecules (IFN-α and IFN-γ) were significantly down-regulated, while inflammatory factors (TNF-α, IL-1β, IL-10 and TGF-β) and immune checkpoint molecules (PD-1, LAG-3, CTLA-4 and TIM-3) were significantly up-regulated in the PRRSV and PCV2 co-infection groups, especially in PAMs with PCV2 inoculation first followed by PRRSV. The dynamic changes in the aforementioned immune molecules were associated with a high viral load, immunosuppression and cell exhaustion, which may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions by dual infection with PCV2 and PRRSV in PAMs.

To the Editor: Since April 2006, a highly pathogenic disease caused by unknown agents and characterized by high fever and a high proportion of deaths in pigs of all ages, emerged in some swine farms in Jiangxi Province, People’s Republic of China. The morbidity rate was 50%–100% and mortality rate was 20%–100%. In the next several months, the disease spread rapidly to most provinces of China. In almost all affected swine herds, the following clinical signs were observed: high and continuous fever, anorexia, red discolorations in the bodies, and blue ears; in the late phase of the disease, diarrhea and other clinical signs might be seen due to the secondary infections. Clinical samples (from lungs, kidneys, liver, and lymph nodes) were collected from animals in different provinces and sent for laboratory diagnosis. DNA and RNA were extracted from the tissue homogenate and PCR or reverse transcription–PCR (RT-PCR) was conducted to detect porcine reproductive and respiratory syndrome virus (PRRSV), classic swine fever virus, porcine circovirus, and pseudorabies virus, respectively (1). In clinical samples, only PRRSV was found to be the dominant virus (48 of 50 samples were PRRSV positive). PRRSVs were then isolated successfully on MARC-145 cells with an obvious cytopathologic effect, characterized by cell congregation, contraction, and brushing off at passage 2; immunofluorescence assay using PRRSV NP-, M- and GP5-specific monoclonal antibodies confirmed that the isolated viruses were PRRSV (2,3). Full-length genomic sequencing of 1 of the isolates (HuN4 strain) showed extensive amino acid (aa) mutations in GP5 protein and 2 deletions in Nsp2, 1 aa deletion at 482, and 29 aa deletions at 533–561, compared with the previous Chinese isolates CH-1a and BJ-4. The newly isolated PRRSV was used to examine the pathogenicity in 60-day-old PRRSV-free piglets, under closed and biosafety (P2) conditions. Each of the piglets (N = 5) received intranasally 105.0 50% tissue culture infecting dose of the isolated virus propagated in MARC-145 cells (4,5). The animals were kept in separate rooms throughout the experiment. Clinical observations of respiratory signs, behavior, rectal temperature, and coughing were recorded daily. Blood samples were collected every 2 days and tested for PRRSV-specific antibodies by ELISA (6,7). Tissue samples (from heart, lungs, kidneys, spleen, and lymph nodes) from all animals that died during the experiment were collected and detected by histopathologic examination (8) and virus isolation. Results showed that the clinical manifestations of all pigs were similar to those that appeared in the field investigation (including high and continuous fever, anorexia, red discolorations in the bodies, and blue ears). The specific antibodies to PRRSV were detected at 8 days postinfection, and the high antibody level lasted until the animal’s death, and all infected pigs died at either 7, 8, 12, 16, or 21 days postinoculation, respectively. Furthermore, viruses reisolated from the dead pigs showed an identical homology with the inoculated PRRSV in genes coding for GP5 and partial Nsp2 (2,535–3,307 nt). The results showed that the emerging PRRSV, characterized by deletions in Nsp2, is highly pathogenic to pigs. To investigate whether the emerging PRRSV was the causative agent of the pandemic diseases on swine farms, an extensive virus survey was conducted. More than 48 samples collected from different swine farms in12 provinces were found to be PRRSV positive by RT-PCR, based on open reading frame (ORF) 5 and Nsp2 (Figure). Sequence analysis of ORF5 and partial Nsp2 showed that these PRRSVs are highly homologous to each other (98.5%–100% for GP5; 98.2%–100% for Nsp2) and share the same deletions at the same positions of Nsp2 gene with HuN4 strain. Sequence comparison of ORF5 indicated that the HuN4 strain shares 93%, 86%, and 88% nucleotide identities with CH-1a (Chinese isolate), BJ-4 (Chinese isolate), and VR2332 (American isolate), respectively. All the newly isolated PRRSVs belong to the North American type. Figure Geographic distribution of porcine reproductive and respiratory syndrome viruses (PRRSVs) examined in the study. Shaded areas indicate the provinces where the PRRSVs characterized by deletions in Nsp2 were detected. Although the cause of the emerging pandemic disease of pigs with a high proportion of deaths in 2006 is unknown, we found high correlation between PRRSV isolation rate and the diseased pigs. The regression test in its natural animal showed that the newly isolated PRRSV was much more virulent than earlier PRRSV isolates. Also, sequence analysis demonstrated a substantial diversity from the PRRSVs isolated during 1996–2005. Further study is needed to answer the question: What role did the newly isolated PRRSV play in the 2006 outbreaks on many of the swine farms in China?

Postweaning multisystemic wasting syndrome (PMWS) is a disease of nursery and fattening pigs characterized by growth retardation, paleness of the skin, dyspnea, and increased mortality rates. Porcine circovirus 2 (PCV2) has been demonstrated to be the cause of PMWS. However, other factors are needed for full development of the syndrome, and porcine reproductive and respiratory syndrome virus (PRRSV) infection has been suggested to be one of them. Twenty-four conventional 5-week-old pigs were distributed in four groups: control (n = 5), PRRSV inoculated (n = 5), PCV2 inoculated (n = 7), and PRRSV and PCV2 inoculated (n = 7). The two groups inoculated with PRRSV showed growth retardation. Pigs inoculated with both PRRSV and PCV2 had increased rectal temperature. One of these pigs developed wasting, had severe respiratory distress, and died. The most important microscopic lesion in pigs inoculated with PCV2 was lymphocyte depletion with histiocytic infiltration of the lymphoid organs, more severe and in a wider range of tissues in doubly inoculated pigs. Interstitial pneumonia was observed in the three inoculated groups. PCV2 nucleic acid was found by in situ hybridization in larger amounts and in a wider range of lymphoid tissues in PRRSV- and PCV2-inoculated than in PCV2-inoculated pigs. TaqMan PCR was performed to quantify the PCV2 loads in serum during the experiment. PCV2 loads were higher in doubly inoculated pigs than in pigs inoculated with PCV2 alone. These findings indicate that severe disease can be reproduced in conventional 5-week-old pigs by inoculation of PRRSV and PCV2. Moreover, these results support the hypothesis that PRRSV infection enhances PCV2 replication.

Samples were obtained on four occasions from three closed pig farms for the purposes of polymerase chain reaction (PCR) tests to detect the Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus 2 (PCV-2). No clinical signs of the disease were observed on those farms. Sera for PRRSV detection and whole blood, nasal swabs, and feces for PCV-2 detection were collected from 313 pigs in 5 age groups: nursery pigs; pigs aged from 1 to 2 months, from 3 to 4 months, and from 5 to 6 months; and sows. Breakdown detection by month is as follows: in August, PRRSV in 1 and PCV-2 in 24 of 72 head; in November, PRRSV in 20 and PCV-2 in 60 of 120 head; in March, PRRSV in 0 and PCV-2 in 9 of 67 head; in June, PRRSV in 3 and PCV-2 in 27 of 72 head. Total percentages: PRRSV 24 head (7.7%) and PCV-2 120 head (38.3%). Frequency of PRRSV and PCV-2-positive pigs increased after the nursery stage. PRRSV frequency peaked among pigs aged from 1 to 2 months. PCV-2 frequency peaked among pigs aged from 3 to 4 months. After those stages, frequencies of pigs positive for PRRS and PCV-2 decreased to reach low levels in grown sows. These results suggest that the frequency of pigs positive to PRRSV and PCV-2 varies seasonally and that, on the farms studied, these infections develop rapidly after weaning.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen that poses a serious threat to the global pig industry. Sichuan Province is one of the largest pig breeding provinces in China. There is a lack of reports on the continuous surveillance and systematic analysis of prevalent strains of PRRSV in Sichuan Province in recent years. To fill this gap, a total of 539 samples were collected from 13 breeding regions in Sichuan during 2012-2020. The detection result showed that the positive rate of PRRSV was 52.32% (282/539). The ORF5s and Nsp2 were obtained and further analyzed, with Chinese reference strains downloaded from the GenBank. Phylogenetic analysis showed that the PRRSV strains sequenced in this study belonged to PRRSV-1 and PRRSV-2 (lineage 1, 3, 5 and 8). In total, 168 PRRSV-2 strains were selected for ORF5 analyses, and these strains were classified into sub-lineage 8.7 (HP-PRRSV), sub-lineage 5.1 (classical PRRSV), sub-lineage 1.8 (NADC30-like), sub-lineage 1.5 (NADC34-like) and sub-lineage 3.5 (QYYZ-like), accounting for 60.71% (102/168), 11.31% (19/168), 18.45% (31/168), 2.97% (5/168) and 6.55% (11/168) of the total analyzed strains, respectively. The Nsp2 of identified PRRSV strains exhibited a nucleotide identity of 44.5-100%, and an amino acid identity of 46.82-100%. The ORF5 of the identified PRRSV strains exhibited a nucleotide identity of 81.3-100%, and an amino acid identity of 78.5-100%. A sequence analysis of ORF5 revealed that the mutation sites of GP5 were mainly concentrated in HVR1 and HVR2 and the virulence sites. In summary, the HP-PRRSV, NADC30-like PRRSV, Classic-PRRSV, QYYZ-like PRRSV, NADC34-like PRRSV and PRRSV-1 strains exist simultaneously in pigs in Sichuan. NADC30-like PRRSV was gradually becoming the most prevalent genotype currently in Sichuan province. This study suggested that PRRSV strains in Sichuan were undergoing genomic divergence.

Epidemiological and genetic characteristics of porcine reproductive and respiratory syndrome virus circulating in central and South China in 2016

The purpose of this case report is to describe for the first time concurrent porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV-2) infections in a commercial farrow-to-finish pig farm in Albania, as well as the phylogenetical analysis of isolated PRRSV strain. The present study reports on a farrow-to-finish commercial pig farm, located in South Albania. In a percentage of about 40% of weaners in each batch (60-70 piglets per batch), clinical signs, including fever, severe respiratory signs, wasting, jaundice, rough hairy coat, palpable inguinal lymphadenopathy, and high mortality rate, were performed. The clinical signs of sows included sporadic premature farrowings (22%), with increased number of stillbirth (3.3%) and weak piglets (4.1%) based on the record system of the farm. Blood samples were obtained from 8 sows (4 lactating and 4 dry-period sows), 25 piglets of 5 different batches (5 at 15-20 days, 5 at 40 days, 5 at 50 days, 5 of 60 days, and 5 of 70 days), and 5 finishers of 130-150 days of age. Moreover, tissue samples were collected from five weaners at 20-70 days of age. Histopathological examination of lung and lymph node sections revealed findings compatible with PRRSV and PCV-2 infection. Pigs between 15 and 130-140 days of age were positive for type 1 (European) PRRSV and pigs between 50 and 130-140 days of age were positive for PCV-2. Blood serum samples were tested by real-time polymerase chain reaction (PCR) for PCV-2 and one real-time reverse transcription-PCR-positive sample was selected for subsequent complete ORF5 (Gp5) gene sequencing. The results of this case report confirm the detection of PRRSV and PCV-2 concurrent infection in an Albanian farrow-to-finish pig farm. The full-length ORF5 sequence of the detected PRRSV strain (named "Mursi/AL/15") was successfully determined, revealing high nucleotide identity with other type 1 European isolates.

Co-infection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circoviruses (PCVs) is commonly observed under field conditions and elicits more severe diseases than any singular infection. In this study, the co-infection of PRRSV, PCV2 and PCV3 was analyzed in tissue samples collected from 150 pigs from April 2016 to April 2018. PRRSV, PCV2 and PCV3 was detected in 55 (36.67%), 43 (28.67%) and 3 (2%) of 150 pigs respectively. Remarkably, one lung sample (SD17-36) collected from a diseased pig was co-infected with PRRSV, PCV2 and PCV3. The complete genomes of SD17-36 viruses of PRRSV, PCV2 and PCV3 were determined, which belong to the subgroups of NADC30-like PRRSV, PCV2d and PCV3a respectively. Sequence comparison showed that PRRSV SD17-36 isolate contains a N33 deletion in GP5. Animal challenge study showed that the novel NADC30-like PRRSV SD17-36 isolate is low pathogenic. Our results indicate that the co-infection of PRRSV and PCVs might cause diseases even when PRRSV plays a limited role in the pathogenicity of the co-infection.

To investigate the prevalence and evolution of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) at commercial fattening pig farms, a total of 1397 clinical samples were collected from a single fattening cycle at seven pig farms in five provinces of China from 2020 to 2021. The RT‒PCR results revealed that PRRSV was present on all seven farms, and the percentage of PRRSV-positive individuals was 17.54-53.33%. A total of 344 partial NSP2 gene sequences and 334 complete ORF5 gene sequences were obtained from the positive samples. The statistical results showed that PRRSV-2 was present on all seven commercial fattening farms, and PRRSV-1 was present on only one commercial fattening farm. A total of six PRRSV-2 subtypes were detected, and five of the seven farms had two or more PRRSV-2 subtypes. L1.8 (L1C) PRRSV was the dominant epidemic strain on five of the seven pig farms. Sequence analysis of L1.8 (L1C) PRRSV from different commercial fattening pig farms revealed that its consistency across farms varied substantially. The amino acid alignment results demonstrated that there were 131 aa discontinuous deletions in NSP2 between different L1.8 (L1C) PRRSV strains and that the GP5 mutation in L1.8 (L1C) PRRSV was mainly concentrated in the peptide signal region and T-cell epitopes. Selection pressure analysis of GP5 revealed that the use of the PRRSV MLV vaccine had no significant episodic diversifying effect on L1.8 (L1C) PRRSV. PRRSV infection is common at commercial fattening pig farms in China, and the percentage of positive individuals is high. There are multiple PRRSV subtypes of infection at commercial fattening pig farms in China. L1.8 (L1C) is the main circulating PRRSV strain on commercial fattening pig farms. L1.8 (L1C) PRRSV detected at different commercial fattening pig farms exhibited substantial differences in consistency but similar molecular characteristics. The pressure on the GP5 of L1.8 (L1C) PRRSV may not be directly related to the use of the vaccines.

Simultaneous detection of classical PRRSV, highly pathogenic PRRSV and NADC30-like PRRSV by TaqMan probe real-time PCR

Simultaneous detection of porcine reproductive and respiratory syndrome virus and porcine circovirus 3 by SYBR Green І-based duplex real-time PCR

Simple SummaryAbortions are a significant contributor to economic losses in the swine breeding industry. Identifying the factors responsible for abortion outbreaks is crucial for optimizing farm management and implementing preventive measures, though this can be challenging due to their often multifactorial nature. In this study, we retrospectively examined the infectious agents associated with abortion outbreaks from 2011 to 2021 in northern Italy. The most frequently detected pathogens in fetal samples were porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus-3 (PCV3), and PCV2, while Chlamydia spp., porcine parvovirus (PPV), and Leptospira spp. were less common. PRRSV prevalence fluctuated yearly without a clear trend, whereas PCV2 showed a slight decline and PCV3 increased over the study period. Our findings suggest a general decrease in abortion outbreaks between 2011 and 2021. PRRSV, PCV2, and PCV3 were commonly detected in aborted fetuses, while pathogens like Chlamydia spp. and Leptospira spp. had a limited impact, possibly due to improved on-farm hygiene and biosecurity measures.The present study retrospectively analyzed the infectious agents associated with 829 abortion outbreaks occurring from 2011 to 2021 in northern Italy. Foetuses were subjected to necropsies, and organ samples were analyzed by direct PCR to screen for six swine pathogens. In 42.0% of the examined outbreaks, at least one infectious agent was found. Porcine reproductive and respiratory syndrome virus (PRRSV) (24.9%) and porcine circovirus-2 (PCV2) (11.5%) were the most frequently detected among the known abortion-inducing pathogens. Chlamydia spp. (5.6%), porcine parvovirus (PPV) (4.0%), and Leptospira spp. (2.6%) were less common. Although its role in swine reproductive disorders is still unclear, PCV3 was detected in 19.6% of the cases. Coinfections were detected in 25.0% of positive outbreaks, and the most frequent coinfection was represented by PRRSV and PCV2 (32.2%), followed by PRRSV and PCV3 (23%). PCV2 prevalence showed a slight but consistent reduction during the study period, while PCV3 increased in frequency. Our data suggest an overall reduction in abortion outbreaks during the study period. PRRSV was confirmed as the main abortion agent detected in the examined area, while PCV2 prevalence showed a decline. Conversely, PCV3 detection has been increasing, supporting its potential role as an abortion agent. Our results highlight the importance of implementing a consistent and standardized sampling procedure, as well as a thorough diagnostic protocol, to reduce the incidence of inconclusive diagnoses.

Because transmission of porcine reproductive and respiratory syndrome virus (PRRSV) can occur through boar semen, it is important to identify persistently infected boars. However, even for boars given the same PRRSV strain and dose, variability in the duration of viral shedding in semen has been observed, suggesting that host factors are involved in PRRSV persistence. To determine whether there are host genetic factors, particularly litter and breed differences related to the persistence of PRRSV, 3 litters from 3 purebred swine breeds were used for this study. It was also determined whether PRRSV could be detected for a longer period of time in serum, semen, or peripheral blood mononuclear cells (PBMC) and if PRRSV could still be detected in tissues after these antemortem specimens were PRRSV negative for a minimum of 2-3 weeks. Three Hampshire, 3 Yorkshire, and 2 Landrace PRRSV-naive boars were obtained and inoculated intranasally with a wild-type PRRSV isolate (SD-23983). All boars within each breed were from the same litter, and litters were within 9 days of age. Serum and PBMC were collected twice weekly from each boar and analyzed for the presence of PRRSV by virus isolation and the polymerase chain reaction (PCR). Serum was also used to obtain virus neutralization titers and enzyme-linked immunosorbent assay S/P values. Semen was collected twice weekly from 7 of 8 boars and analyzed by PCR. After all specimens were PRRSV negative for a minimum of 2-3 weeks, each boar was euthanized, and 21 tissues plus saliva, serum, feces, and urine were collected. All postmortem specimens were evaluated by virus isolation. Specimens that were PRRSV negative by virus isolation were then evaluated by PCR. The mean number of days (+/-SD) for the duration of PRRSV shedding in semen was 51+/-26.9 days, 7.5+/-4.9 days, and 28.3+/-17.5 days for Landrace, Yorkshire, and Hampshire boars, respectively. Because of small sample sizes and large SDs, the differences in duration of PRRSV shedding in semen between breeds were not considered significant. However, the trend suggested that Yorkshire boars were more resistant to PRRSV shedding in semen than were Landrace boars, requiring further investigation using a larger numbers of boars. PRRSV was detected for a longer period in semen than in serum or PBMC in 4 of 7 boars. Viremia could be detected for a longer period in serum than in PBMC in 6 of 8 boars. After a minimum of 2-3 weeks of PRRSV-negative serum, semen, and PBMC, PRRSV could still be detected in the tonsil of 3 of 8 boars by virus isolation, indicating that boars still harbor PRRSV within the tonsil even though antemortem specimens are PRRSV negative.

To document shedding of porcine reproductive and respiratory syndrome (PRRS) virus in mammary gland secretions of experimentally inoculated sows, to evaluate effects of vaccination during gestation on virus shedding during the subsequent lactation, and to evaluate shedding of PRRS virus in milk of sows in commercial herds. 6 sows seronegative for PRRS virus were used for experiment 1, and 2 sows were retained for experiment 2. For experiment 3, 202 sows in commercial herds were used. In experiment 1, 2 sows were inoculated with PRRS virus, 2 sows were vaccinated with modified-live PRRS virus vaccine, and 2 sows served as control pigs. Mammary gland secretions were assayed for PRRS virus. In experiment 2, pregnant vaccinated sows from experiment 1 were vaccinated with another modified-live PRRS virus vaccine. Mammary gland secretions were assayed in the same manner as for experiment 1. For experiment 3, milk collected from 202 sows in commercial herds was assayed for PRRS virus. In experiment 1, PRRS virus was detected in mammary gland secretions of both vaccinated and 1 of 2 virus-inoculated sows. In experiment 2, virus was not detected in samples from either vaccinated sow. In experiment 3, all samples yielded negative results. Naïve sows inoculated late in gestation shed PRRS virus in mammary secretions. Previous vaccination appeared to prevent shedding during the subsequent lactation. Results for samples obtained from sows in commercial herds suggested that virus shedding in mammary gland secretions of such sows is uncommon.

BackgroundIn spring 2015, an outbreak of porcine reproductive and respiratory syndrome (PRRS) struck Lower Austria caused by a PRRS virus (PRRSV) strain spreading rapidly among both previously PRRSV negative and vaccinated pig herds. This case report describes the first well-documented emergence of the PRRSV strain responsible for this outbreak.Case presentationA PRRSV seronegative piglet-producing farm in Lower Austria encountered losses in foetuses and suckling piglets of up to 90 %; clinical signs in sows and nursery piglets included fever and reduced feed intake. Additionally, high percentages of repeat breeders and losses of up to 40 % in nursery piglets occurred. An infection with PRRSV was suggested by the detection of antibodies by enzyme linked immunosorbent assay and confirmed by quantitative real time PCR. The underlying PRRSV strain, termed AUT15-33, was isolated by passage on porcine alveolar macrophages, partially sequenced (ORF2-7) and grouped as PRRSV-1, subtype 1. In phylogenetic analysis of the genome region coding for the structural proteins, ORF2-7, AUT15-33 clustered with Belgian strains but identities were as low as 88 %. In contrast, analysis of ORF7 sequences revealed a close relationship to Croatian strains from 2012 with an identity of 94 – 95 %.ConclusionsIn the year following the outbreak, the same PRRSV strain was identified repeatedly in different regions of Austria. It can be speculated that the new strain has novel advantageous properties.