A Systematic Investigation Unveils High Coinfection Status of Porcine Parvovirus Types 1 through 7 in China from 2016 to 2020

Porcine parvoviruses (PPVs) are prevalent in China associating with reproductive failure in swine. The coinfection of seven genotypes of PPVs (PPV1-7) might have synergistic effects on PPV1 associated SMEDI syndrome.

Inactivation of Enveloped Bovine Viral Diarrhea Virus and Non-Enveloped Porcine Parvovirus Using Low-Pressure Non-Thermal Plasma

As the worldwide population has been experiencing since 2020, viruses represent a serious threat to global well-being. To avoid viral transmission through surgery or medical examination, sterilization of medical material is needed. From emerging sterilization processes, the use of non-thermal plasma (NTP) arises as a promising technique to efficiently reduce microbial burden on medical devices, including new complex polymers as thermosensitive ones. Thus, we evaluated the antiviral efficacy of a low-pressure NTP process taking place in a sealed bag. For this purpose, two different plasmas, O2 100% plasma and Ar 80%–O2 20% plasma, were tested against two viruses: the bovine viral diarrhea virus and the porcine parvovirus, surrogates of human hepatitis C virus and human parvovirus B19, respectively. The efficacy of both NTP treatments on viral load can be detected after only five minutes. Moreover, the longer the NTP treatments last, the more the load decreases. The most effective load reduction was obtained with a 120-min O2 plasma treatment inducing a minimum of four-log viral load reduction. So, this process demonstrated strong virucidal capacity inside a sealed bag and represents a very interesting opportunity in the field of fragile medical devices sterilization or disinfection.

Viral fitness and antigenic determinants of porcine parvovirus at the amino acid level of the capsid protein

Since 2001, strains of porcine parvovirus (PPV), designated 27a -like strains, were observed in Europe, suggesting a predominance of these viruses over older strains. The reasons for the obvious evolutionary advantage are unknown. Here, a series of mutants containing amino acid replacements found in the predominant field strains were generated in a PPV-NADL2 background and their impact on replication efficiency and antibody binding activity was determined. Some amino acid substitutions observed in the 27a- like strains significantly increased viral fitness and decreased neutralization activity of sera raised against commercial vaccines and old virus strains (e.g. NADL2). These mutant viruses and a monoclonal antibody raised against a classical PPV strain defined an 27a-specific neutralizing epitope around amino acid 228 of the capsid protein VP2. Based on the analysis of the mutant viruses, it is hypothesized that the predominant factor for the global spread of the PPV-27a strain substitutions is an increased viral fitness of the 27a- like viruses, possibly supported by a partial immune selection. This is reminiscent to the evolution of canine parvovirus and worldwide replacement of the original virus by the so-called new antigenic types. Importance Porcine parvovirus is one of the most important causes of reproductive failure in swine. Recently, despite the continuous use of vaccines, “new” strains emerged, leading to the hypothesis that the emergence of new amino acid substitutions could be a viral adaptation to the immune response against the commercial vaccines. Our results indicate the amino acid substitutions observed in the 27a -like strains can modify viral fitness and antigenicity. However, an absolute immune escape was not evident.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/β Nuclear Import Pathway

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283–291 (designated NES2) and 602–608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256–274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1, importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1 and importin α/β-mediated transport by specific inhibitors (LMB, importazole and ivermectin) clearly blocked PPV replication. The mutant viruses of delete NESs or NLS motif of the NS1 by using reverse genetics could not be rescued, suggesting that NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited if blocking NS1 nuclear import or export. Importance PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (> 50 kDa), it cannot pass through the nuclear pore complex by diffusion alone, and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and its dependence on the CRM1 pathway for nuclear export demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

N-2-Hydroxypropyl Trimethyl Ammonium Chloride Chitosan as Adjuvant Enhances the Immunogenicity of a VP2 Subunit Vaccine against Porcine Parvovirus Infection in Sows

Porcine parvovirus (PPV) is the most important infectious agent causing infertility in pigs, which can be prevented by routine vaccination. Successful vaccination depends on the association with potent adjuvants that can enhance the immunogenicity of antigen and activate the immune system. Polysaccharide adjuvant has low toxicity and high safety, and they can enhance the humoral, cellular and mucosal immune responses. In the present study, we prepared the VP2 protein subunit vaccine against PPV (PPV/VP2/N-2-HACC) using water-soluble N-2-Hydroxypropyl trimethyl ammonium chloride chitosan (N-2-HACC) as the vaccine adjuvant, and the ability of the PPV/VP2/N-2-HACC to induce immune responses and protect sows from PPV infection was evaluated. In vivo immunization showed that the sows immunized with the PPV/VP2/N-2-HACC by intramuscular injection produced higher HI antibody levels and long-term immune protection compared with the other groups, while the subunit vaccine did not stimulate the proliferation of CD4+ and CD8+ T lymphocytes to trigger the secretion of higher levels of IL-2, IL-4, IFN-α, IFN-β, and IFN-γ, indicating that the PPV/VP2/N-2-HACC mainly induced humoral immunity rather than cellular immunity. PPV was not detected in the viscera of the sows immunized with the PPV/VP2/N-2-HACC, and the protective efficacy was 100%. Collectively, our findings suggested that the N-2-HACC was a potential candidate adjuvant, and the PPV/VP2/N-2-HACC had immense application value for the control of PPV.

Porcine Parvovirus 2 Is Predominantly Associated With Macrophages in Porcine Respiratory Disease Complex

Porcine respiratory disease complex (PRDC) is a significant source of morbidity and mortality, manifested by pneumonia of multiple etiologies, where a variety of pathogens and environment and management practices play a role in the disease. Porcine reproductive and respiratory syndrome virus (PRRSV), influenza A virus (IAV), and porcine circovirus 2 (PCV2) are well-established pathogens in PRDC. Porcine parvovirus 2 (PPV2) has been identified in both healthy and clinically diseased pigs at a high prevalence worldwide. Despite widespread circulation, the significance of PPV2 infection in PRDC and its association with other co-infections are unclear. Here, PPV2 was detected in the lung tissue in 39 of 100 (39%) PRDC-affected pigs by quantitative polymerase chain reaction (qPCR). Using in situ hybridization (ISH) in conjunction with tissue microarrays (TMA), PPV2 infection was localized in alveolar macrophages and other cells in the lungs with interstitial pneumonia in 28 of 99 (28.2%) samples. Viral load tended to correlate with the number of macrophages in the lungs. Assessment of the frequency, viral titers, and tissue distributions showed no association between infection of PPV2 and other major viral respiratory pathogens. In one-third of the PPV2-positive samples by qPCR, no other known viruses were identified by metagenomic sequencing. The genome sequences of PPV2 were 99.7% identical to the reference genomes. Although intensive intranuclear and intracytoplasmic signals of PPV2 were mainly detected in alveolar macrophages by ISH, no obvious virus replication was noted in in vitro cell culture. Together, these results suggest that PPV2 is associated, but may not be the sole causative agent, with PRDC, warranting the control and prevention of this underdiagnosed virus.

Exploring the Cause of Diarrhoea and Poor Growth in 8–11-Week-Old Pigs from an Australian Pig Herd Using Metagenomic Sequencing

Diarrhoea and poor growth among growing pigs is responsible for significant economic losses in pig herds globally and can have a wide range of possible aetiologies. Next generation sequencing (NGS) technologies are useful for the detection and characterisation of diverse groups of viruses and bacteria and can thereby provide a better understanding of complex interactions among microorganisms potentially causing clinical disease. Here, we used a metagenomics approach to identify and characterise the possible pathogens in colon and lung samples from pigs with diarrhoea and poor growth in an Australian pig herd. We identified and characterized a wide diversity of porcine viruses including RNA viruses, in particular several picornaviruses—porcine sapelovirus (PSV), enterovirus G (EV-G), and porcine teschovirus (PTV), and a porcine astrovirus (PAstV). Single stranded DNA viruses were also detected and included parvoviruses like porcine bocavirus (PBoV) and porcine parvovirus 2 (PPV2), porcine parvovirus 7 (PPV7), porcine bufa virus (PBuV), and porcine adeno-associated virus (AAV). We also detected single stranded circular DNA viruses such as porcine circovirus type 2 (PCV2) at very low abundance and torque teno sus viruses (TTSuVk2a and TTSuVk2b). Some of the viruses detected here may have had an evolutionary past including recombination events, which may be of importance and potential involvement in clinical disease in the pigs. In addition, our metagenomics data found evidence of the presence of the bacteria Lawsonia intracellularis, Brachyspira spp., and Campylobacter spp. that may, together with these viruses, have contributed to the development of clinical disease and poor growth.

Recombinase-Aided Amplification Coupled with Lateral Flow Dipstick for Efficient and Accurate Detection of Porcine Parvovirus

Porcine parvovirus (PPV) infection is the primary cause of SMEDI (stillbirth; mummification; embryonic death; infertility) syndrome, which is a global burden for the swine industry. Thus, it is crucial to establish a rapid and efficient detection method against PPV infection. In the present work, we developed a recombinase-aided amplification (RAA) assay, coupled with a lateral flow dipstick (LFD), to achieve an amplification of PPV DNA at 37 °C within 15 min. The detection limits of PPV RAA-LFD assay were 102 copies/μL recombinant plasmid pMD19-T-VP1, 6.38 × 10−7 ng/μL PPV DNA, and 10−1 TCID50/mL virus, respectively. This method was highly specific for PPV detection with no cross-reactivity for other swine pathogens. In contrast to polymerase chain reaction (PCR), the PPV RAA-LFD assay is more sensitive and cost-saving. Hence, the established PPV RAA-LFD assay provided an alternative for PPV detection, especially in resource-limited regions.