A Nanobody Targeting Viral Nonstructural Protein 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication.

Porcine reproductive and respiratory syndrome (PRRS) is of great concern to the swine industry due to pandemic outbreaks of the disease, current ineffective vaccinations, and a lack of efficient antiviral strategies. In our previous study, a PRRSV Nsp9-specific nanobody, Nb6, was successfully isolated, and the intracellularly expressed Nb6 could dramatically inhibit PRRSV replication in MARC-145 cells. However, despite its small size, the application of Nb6 protein in infected cells is greatly limited, as the protein itself cannot enter the cells physically. In this study, a trans-activating transduction (TAT) peptide was fused with Nb6 to promote protein entry into cells. TAT-Nb6 was expressed as an inclusion body in Escherichia coli, and indirect enzyme-linked immunosorbent assays and pulldown assays showed that E. coli-expressed TAT-Nb6 maintained the binding ability to E. coli-expressed or PRRSV-encoded Nsp9. We demonstrated that TAT delivered Nb6 into MARC-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner, and TAT-Nb6 efficiently inhibited the replication of several PRRSV genotype 2 strains as well as a genotype 1 strain. Using a yeast two-hybrid assay, Nb6 recognition sites were identified in the C-terminal part of Nsp9 and spanned two discontinuous regions (Nsp9aa454-551 and Nsp9aa599-646). Taken together, these results suggest that TAT-Nb6 can be developed as an antiviral drug for the inhibition of PRRSV replication and controlling PRRS disease.IMPORTANCE The pandemic outbreak of PRRS, which is caused by PRRSV, has greatly affected the swine industry. We still lack an efficient vaccine, and it is an immense challenge to control its infection. An intracellularly expressed Nsp9-specific nanobody, Nb6, has been shown to be able to inhibit PRRSV replication in MARC-145 cells. However, its application is limited, because Nb6 cannot physically enter cells. Here, we demonstrated that the cell-penetrating peptide TAT could deliver Nb6 into cultured cells. In addition, TAT-Nb6 fusion protein could suppress the replication of various PRRSV strains in MARC-145 cells and PAMs. These findings may provide a new approach for drug development to control PRRS.

Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus.

Hepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that 625DFCP628, 458PSRPF462, and 407EPTV410 peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design.IMPORTANCE Genotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studied in vitro and in vivo Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.

Porcine reproductive and respiratory syndrome virus inhibits MARC-145 proliferation via inducing apoptosis and G2/M arrest by activation of Chk/Cdc25C and p53/p21 pathway.

Porcine reproductive and respiratory syndrome virus(PRRSV) is an important immunosuppressive virus which can suppresses infected cells proliferation. In this work, we examined PRRSV ability to manipulate cell cycle progression of MARC-145 cells and explored the potential molecular mechanisms. The results showed that PRRSV infection imposed a growth-inhibitory effect on MARC-145 cells by inducing cell cycle arrest at G2/M phase. This arrest was due to the significant decrease of Cdc2-cyclinB1 complex activity in PRRSV-infected cells and the activity reduction was a result of Cdc2 Tyr15 phosphorylation and the accumulation of Cdc2 and cyclinB1 in the nucleus. Not only elevated Wee1 and Myt1 expression and inactivated Cdc25C, but also increase of p21 and 14-3-3σ in a p53-dependent manner caused the inhibitory Tyr15 phosphorylation of Cdc2. PRRSV infection also activated Chk1. Our data suggest PRRSV infection induces G2/M arrest via various molecular regulatory mechanisms. These results provide a new insights for PRRSV pathogenesis.

Effect of housing arrangement on fecal-oral transmission of avian hepatitis E virus in chicken flocks.

BACKGROUND: Avian hepatitis E virus (HEV) infection is common in chicken flocks in China, as currently no measures exist to prevent the spread of the disease. In this study, we analyzed the effect of caged versus cage-free housing arrangements on avian HEV transmission. First, 127 serum and 110 clinical fecal samples were collected from 4 chicken flocks including the two arrangements in Shaanxi Province, China and tested for HEV antibodies and/or virus. Concurrently, 36 specific-pathogen-free chickens were divided equally into four experimental living arrangement groups, designated cage-free (Inoculated), caged (Inoculated), cage-free (Negative) and caged (Negative) groups. In caged groups, three cages contained 3 chickens each. Three chickens each from cage-free (Inoculated) and caged (Inoculated) groups (one chicken of each cage) were inoculated by cutaneous ulnar vein with the same dose of avian HEV, respectively. The cage-free (Negative) and caged (Negative) groups served as negative control. Serum and fecal samples were collected at 1 to 7 weeks post-inoculation (wpi) and liver lesions were scored at 7 wpi. RESULTS: The results of serology showed that the avian HEV infection rate (54.10%) of the cage-free chickens was significantly higher than the one (12.12%) for caged chickens (P < 0.05). Also, the rate of detection of avian HEV RNA in the clinical fecal samples was significantly higher in the cage-free (22.80%, 13/57) than caged birds (5.66%, 3/53). Moreover, under experimental conditions, the infected number of uninoculated cage-free chickens (6) was significantly higher than the one for the uninoculated caged birds (2), as evidenced by seroconversion, fecal virus shedding, viremia and gross and microscopic liver lesions. CONCLUSIONS: These results suggest that reduction of contact with feces as seen in the caged arrangement of housing chickens can reduce avian HEV transmission. This study provides insights for prevention and control of avian HEV infection in chicken flocks.

Curcumin is a promising inhibitor of genotype 2 porcine reproductive and respiratory syndrome virus infection.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) could lead to pandemic diseases and huge financial losses to the swine industry worldwide. Curcumin, a natural compound, has been reported to serve as an entry inhibitor of hepatitis C virus, chikungunya virus and vesicular stomatitis virus. In this study, we investigated the potential effect of curcumin on early stages of PRRSV infection. RESULTS: Curcumin inhibited infection of Marc-145 cells and porcine alveolar macrophages (PAMs) by four different genotype 2 PRRSV strains, but had no effect on the levels of major PRRSV receptor proteins on Marc-145 cells and PAMs or on PRRSV binding to Marc-145 cells. However, curcumin did block two steps of the PRRSV infection process: virus internalization and virus-mediated cell fusion. CONCLUSIONS: Our results suggested that an inhibition of genotype 2 PRRSV infection by curcumin is virus strain-independent, and mainly inhibited by virus internalization and cell fusion mediated by virus. Collectively, these results demonstrate that curcumin holds promise as a new anti-PRRSV drug.

MicroRNA miR-24-3p promotes porcine reproductive and respiratory syndrome virus replication through suppression of heme oxygenase-1 expression.

UNLABELLED: Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important viruses affecting the swine industry worldwide. Our previous research showed that PRRSV downregulates the expression of heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, postinfection and that overexpression of HO-1 inhibits PRRSV replication. MicroRNAs regulate gene expression at the posttranscriptional level and have recently been demonstrated to play vital roles in pathogen-host interactions. The present study sought to determine whether microRNAs modulate HO-1 expression and, by doing so, regulate PRRSV replication. Using bioinformatic prediction and experimental verification, we demonstrate that HO-1 expression is regulated by miR-24-3p. A direct interaction between miR-24-3p and HO-1 mRNA was confirmed using a number of approaches. Overexpression of miR-24-3p significantly decreased HO-1 mRNA and protein levels. PRRSV infection induced miR-24-3p expression to facilitate viral replication. The suppressive effect of HO-1 induction by protoporphyrin IX cobalt chloride (CoPP; a classical inducer of HO-1 expression) on PRRSV replication in MARC-145 cells and primary porcine alveolar macrophages could also be reversed by overexpression of miR-24-3p. Collectively, these results suggested that miR-24-3p promotes PRRSV replication through suppression of HO-1 expression, which not only provides new insights into virus-host interactions during PRRSV infection but also suggests potential new antiviral strategies against PRRSV infection. IMPORTANCE: MicroRNAs (miRNAs) play vital roles in viral infections by regulating the expression of viral or host genes at the posttranscriptional level. Heme oxygenase-1 (HO-1), a pivotal cytoprotective enzyme, has antiviral activity for a number of viruses, such as Ebola virus, hepatitis C virus, human immunodeficiency virus, and our focus, PRRSV, which causes great economic losses each year in the swine industry worldwide. Here, we show that PRRSV infection induces host miRNA miR-24-3p expression and that miR-24-3p regulates HO-1 expression through both mRNA degradation and translation repression. Suppression of HO-1 expression by miR-24-3p facilitates PRRSV replication. This work lends credibility to the hypothesis that an arterivirus can manipulate cellular miRNAs to enhance virus replication by regulating antiviral responses following viral infection. Therefore, our findings provide new insights into the pathogenesis of PRRSV.

Seroprevalence of avian hepatitis E virus and avian leucosis virus subgroup J in chicken flocks with hepatitis syndrome, China.

BACKGROUND: From 2014 to 2015 in China, many broiler breeder and layer hen flocks exhibited a decrease in egg production and some chickens developed hepatitis syndrome including hepatomegaly, hepatic necrosis and hemorrhage. Avian hepatitis E virus (HEV) and avian leucosis virus subgroup J (ALV-J) both cause decreasing in egg production, hepatomegaly and hepatic hemorrhage in broiler breeder and layer hens. In the study, the seroprevalence of avian HEV and ALV-J in these flocks emerging the disease from Shandong and Shaanxi provinces were investigated. RESULTS: A total of 1995 serum samples were collected from 14 flocks with hepatitis syndrome in Shandong and Shaanxi provinces, China. Antibodies against avian HEV and ALV-J in these serum samples were detected using iELISAs. The seroprevalence of anti-avian HEV antibodies (35.09%) was significantly higher than that of anti-ALV-J antibodies (2.16%) (p = 0.00). Moreover, the 43 serum samples positive for anti-ALV-J antibodies were all also positive for anti-avian HEV antibodies. In a comparison of both provinces, Shandong chickens exhibited a significantly higher seroprevalence of anti-avian HEV antibodies (42.16%) than Shaanxi chickens (26%) (p = 0.00). In addition, the detection of avian HEV RNA and ALV-J cDNA in the liver samples from the flocks of two provinces also showed the same results of the seroprevalence. CONCLUSIONS: In the present study, the results showed that avian HEV infection is widely prevalent and ALV-J infection is endemic in the flocks with hepatitis syndrome from Shandong and Shaanxi provinces of China. These results suggested that avian HEV infection may be the major cause of increased egg drop and hepatitis syndrome observed during the last 2 years in China. These results should be useful to guide development of prevention and control measures to control the diseases within chicken flocks in China.

Identification of an antigenic domain in the N-terminal region of avian hepatitis E virus (HEV) capsid protein that is not common to swine and human HEVs.

The antigenic domains located in the C-terminal 268 amino acid residues of avian hepatitis E virus (HEV) capsid protein have been characterized. This region shares common epitopes with swine and human HEVs. However, epitopes in the N-terminal 338 amino acid residues have never been reported. In this study, an antigenic domain located between amino acids 23 and 85 was identified by indirect ELISA using the truncated recombinant capsid proteins as coating antigens and anti-avian HEV chicken sera as primary antibodies. In addition, this domain did not react with anti-swine and human HEV sera. These results indicated that the N-terminal 338 amino acid residues of avian HEV capsid protein do not share common epitopes with swine and human HEVs. This finding is important for our understanding of the antigenicity of the avian HEV capsid protein. Furthermore, it has important implications in the selection of viral antigens for serological diagnosis.

Lysosome-dependent p300/FOXP3 degradation and limits Treg cell functions and enhances targeted therapy against cancers.

p300 is one of several acetyltransferases that regulate FOXP3 acetylation and functions. Our recent studies have defined a complex set of histone acetyltransferase interactions which can lead to enhanced or repressed changes in FOXP3 function. We have explored the use of a natural p300 inhibitor, Garcinol, as a tool to understand mechanisms by which p300 regulates FOXP3 acetylation. In the presence of Garcinol, p300 appears to become disassociated from the FOXP3 complex and undergoes lysosome-dependent degradation. As a consequence of p300's physical absence, FOXP3 becomes less acetylated and eventually degraded, a process that cannot be rescued by the proteasome inhibitor MG132. p300 plays a complex role in FOXP3 acetylation, as it could also acetylate a subset of four Lys residues that repressively regulate total FOXP3 acetylation. Garcinol acts as a degradation device to reduce the suppressive activity of regulatory T cells (Treg) and to enhance the in vivo anti-tumor activity of a targeted therapeutic anti-p185(her2/neu) (ERBB2) antibody in MMTV-neu transgenics implanted with neu transformed breast tumor cells. Our studies provide the rationale for molecules that disrupt p300 stability to limit Treg functions in targeted therapies for cancers.

Smartphone recognition-based immune microparticles for rapid on-site visual data-sharing detection of Newcastle disease virus.

Since the last century, animal viruses have posed great threats to the health of humans and the farming industry. Therefore, virus control is of great urgency, and regular, timely, and accurate detection is essential to it. Here, we designed a rapid on-site visual data-sharing detection method for the Newcastle disease virus with smartphone recognition-based immune microparticles. The detection method we developed includes three major modules: preparation of virus detection vectors, sample detection, and smartphone image analysis with data upload. First, the hydrogel microparticles containing active carboxyl were manufactured, which coated nucleocapsid protein of NDV. Then, HRP enzyme-labeled anti-NP nanobody was used to compete with the NDV antibody in the serum for color reaction. Then the rough detection results were visible to the human eyes according to the different shades of color of the hydrogel microparticles. Next, the smartphone application was used to analyze the image to determine the accurate detection results according to the gray value of the hydrogel microparticles. Meanwhile, the result was automatically uploaded to the homemade cloud system. The total detection time was less than 50 min, even without trained personnel, which is shorter than conventional detection methods. According to experimental results, this detection method has high sensitivity and accuracy. And especially, it uploads the detection information via a cloud platform to realize data sharing, which plays an early warning function. We anticipate that this rapid on-site visual data-sharing detection method can promote the development of virus self-checking at home.

Isolation and Genetic Characterization of Parvoviruses From Dogs, Cats, Minks, and Raccoon Dogs in the Eastern Region of Shandong Province, China.

The eastern region of Shandong province, China, is an intensive economic mink and raccoon dog breeding area. To investigate the molecular variations of parvovirus in cats, dogs, minks, and raccoon dogs from this region, feline panleukopenia virus (FPV), canine parvovirus 2 (CPV-2), mink enteritis virus (MEV), and raccoon dog parvovirus (RDPV) were separately isolated and characterized from the respective animals with gastroenteritis. PCR amplification showed that there were 15/18 (83.3%), 9/13 (69.2%), 8/11 (72.7%), and 3/7 (42.9%) samples from the diseased animals separately positive for FPV, CPV-2, MEV, and RDPV. Of these, a total of six FPV, six MEV, four CPV-2, and three RDPV strains were successfully isolated using F81 cells. Next, the near-complete genomes of 19 parvovirus isolates were amplified and analyzed. The viral particle 2 (VP2) sequence alignment showed that they shared 97.2-100% nucleotide similarity. Phylogenetic analysis showed that the five FPV isolates were in the same branch, and an FPV isolate was closely related with MEV and RDPV isolates obtained in this study. These suggested that cross-species infection occurred in the Shandong region between the FPV, MEV, and RDPV. For the four CPV-2 isolates, three were antigenic variant strains CPV-2a, and the other was antigenic variant strain CPV-2c. Additionally, the mutations that had emerged in the VP2 amino acids of CPV-2 also occurred in the VP2 from the FPV, MEV, and RDPV isolates. This study suggested that the continuous evolution of the parvovirus may be accelerated in areas with a high density of economic animal trading/breeding, and controlling parvovirus infection in these animals remains a challenge.

A synthetic toll-like receptor 7 agonist inhibits porcine reproductive and respiratory syndrome virus replication in piglets.

Toll-like receptor 7 (TLR7) agonists have been shown to exert therapeutic effects against several viruses. However, antiviral potential of TLR7 agonist in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection has not been assessed in vivo. In our previous study, a synthetic TLR7 agonist, SZU101, was confirmed to inhibit PRRSV infection of porcine alveolar macrophages (PAMs). Here, antiviral effects of SZU101 were evaluated in PRRSV-challenged piglets based on assessments of rectal temperature, viremia, gross and microscopic lung lesions, PRRSV-specific antibodies, PRRSV-specific lymphocyte proliferation and serum IFN-ß level. Our results revealed that SZU101 treatment alleviated PRRSV-induced rectal temperature spikes, pulmonary pathologic changes, and serum viral load. Meanwhile, administration of SZU101 led to increased proliferation of PRRSV-specific lymphocytes and serum IFN-ß levels, but did not enhance PRRSV-specific antibody production. These results demonstrate that SZU101 has potential as a therapeutic treatment for PRRS.

LysGH15, a novel bacteriophage lysin, protects a murine bacteremia model efficiently against lethal methicillin-resistant Staphylococcus aureus infection.

Phage-coded lysin is an enzyme that destroys the cell walls of bacteria. Phage lysin could be an alternative to conventional antibiotic therapy against pathogens that are resistant to multiple antibiotics. In this study, a novel staphylococcal phage, GH15, was isolated, and the endogenous lytic enzyme (LysGH15) was expressed and purified. The lysin LysGH15 displayed a broad lytic spectrum; in vitro treatment killed a number of Staphylococcus aureus strains rapidly and completely, including methicillin-resistant S. aureus (MRSA). In animal experiments, a single intraperitoneal injection of LysGH15 (50 µg) administered 1 h after MRSA injections at double the minimum lethal dose was sufficient to protect mice (P < 0.01). Bacteremia in unprotected mice reached colony counts of about 10(7) CFU/ml within 3.5 h after challenge, whereas the mean colony count in lysin-protected mice was less than 10(4) CFU/ml (and ultimately became undetectable). These results indicate that LysGH15 can kill S. aureus in vitro and can protect mice efficiently from bacteremia in vivo. The phage lysin LysGH15 might be an alternative treatment strategy for infections caused by MRSA.

Major Vault Protein Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection in CRL2843CD163 Cell Lines and Primary Porcine Alveolar Macrophages.

Porcine reproductive and respiratory syndrome (PRRS), a significant viral infectious disease that commonly occurs among farmed pigs, leads to considerable economic losses to the swine industry worldwide. Major vault protein (MVP) is a host factor that induces type Ⅰ interferon (IFN) production. In this study, we evaluated the effect of MVP on PRRSV infection in CRL2843CD163 cell lines and porcine alveolar macrophages (PAMs). Our results showed that MVP expression was downregulated by PRRSV infection. Adenoviral overexpression of MVP inhibited PRRSV replication, whereas the siRNA knockdown of MVP promoted PRRSV replication. In addition, MVP knockdown has an adverse effect on the inhibitive role of MVP overexpression on PRRSV replication. Moreover, MVP could induce the expression of type Ⅰ IFNs and IFN-stimulated gene 15 (ISG15) in PRRSV-infected PAMs. Based on these results, MVP may be a potential molecular target of drugs for the effective prevention and treatment of PRRSV infection.

Identification and characterization of Th cell epitopes in MrkD adhesin of Klebsiella pneumoniae.

In this study, we identified the Th epitopes in MrkD of Klebsiella pneumoniae, an excellent vaccine candidate antigen. By using the RANKPEP prediction algorithm, we have identified and characterized three Th epitopes within the MrkD antigen, which can be recognized by CD4+ T cells from BALB/c (H-2(d)) mice. They were M(221-235), M(175-189), and M(264-278). These epitopes have important value for studying the immune response of K. pneumoniae infection and for designing effective vaccine against K. pneumoniae.

Biotinylated Single-Domain Antibody-Based Blocking ELISA for Detection of Antibodies Against Swine Influenza Virus.

BACKGROUND: Enzyme-linked immunosorbent assay (ELISA) is a common method for diagnosing swine influenza. However, the production of classical antibodies is both costly and time-consuming. As a promising alternative diagnostic tool, single-domain antibodies (sdAbs) offer the advantages of simpler and faster generation, good stability and solubility, and high affinity and specificity. METHODS: Phage display technology was used to isolate sdAbs against the SIV-NP protein from a camel VHH library. The sdAb5 was fused to the biotin acceptor peptide (BAP) and a His-Tag for its expression as monomeric and site-specific biotinylation in E.coli to develop an sdAb-based blocking ELISA (sdAb-ELISA). In the sdAb-ELISA, the anti-SIV antibodies from swine samples were used to block the binding between the biotinylated sdAb5 and SIV-NP protein coated on the ELISA plate. The specificity, sensitivity, and reproducibility of sdAb-ELISA were determined. In addition, consistency among sdAb-ELISA, commercial ELISA kit, and Western blot was evaluated. RESULTS: Six SIV-NP-specific sdAbs were isolated, among which sdAb5 was identified as a dominant sdAb with higher reactivity. The cut-off value of biotinylated sdAb5-based bELISA was determined to be 29.8%. Compared with the positive reference serum against five different types of swine viruses, the developed sdAb-ELISA showed 100% specificity. The detection limit of sdAb-ELISA was 1:160 in an anti-SIV positive reference serum, which is lower than that of the commercial ELISA kit (1:20). In 78 diluted anti-SIV positive serum (1:80), 21 and 42 samples were confirmed as positive by the commercial ELISA kit and sdAb-ELISA, respectively. The coefficients of variation of intra- and inter-assay were 1.79-4.57% and 5.54-9.98%, respectively. The sdAb-ELISA and commercial ELISA kit showed a consistency of 94.17% in clinical swine serum samples. Furthermore, the coincidence rate was 96.67% between the results detected by sdAb-ELISA and Western blot. CONCLUSION: A specific, sensitive, and reproducible sdAb-ELISA was successfully developed, which offers a new, promising method to detect anti-SIV antibodies in swine serum.

Experimental infection of rabbit with swine-derived hepatitis E virus genotype 4.

Hepatitis E virus (HEV) is a zoonotic virus that is capable of causing cross-species infection. Rabbits can be experimentally infected with human- and swine-derived HEV-4 in the species Orthohepevirus A, and avian-derived HEV-3 in the species Orthohepevirus B suggesting rabbits can serve as an animal model for zoonotic HEV infection study. However, these studies show that the infectivity of swine HEV isolates in rabbits is not consistent. In this study, the animal study was conducted by the experimental infection of rabbit with a swine-derived HEV-4 isolated in China (designated CHN-SD-sHEV) for 28 weeks post-inoculation (wpi) in compassion to that infected with a rabbit-derived HEV-3 (designated HEV-SX-rHEV). Two rabbits were euthanized every 2 wpi for pathological examinations. The results showed that rabbits infected with CHN-SD-sHEV had the viremia and virus fecal shedding from 1 wpi to 22 wpi and seroconverted from 10 to 28 wpi. Meanwhile, elevated ALT levels were detected at 2 wpi. Moreover, virus replication was confirmed by the detection of both positive- and negative-strand HEV RNAs in the livers and spleens. Diarrhea and hepatocellular lesions were also observed in some animals. In contrast, rabbits experimentally infected with CHN-SX-rHEV exhibited earlier seroconversion, viremia and virus fecal shedding and hepatocellular lesions. Taken together, our data demonstrate that in comparison to the previously reported cases, the swine-derived HEV-4 isolated in China could cross-species infect rabbit accompanied with prolonged virus fecal shedding and liver lesions.

Cross-species infection of mice by rabbit hepatitis E virus.

Rabbits are recognized as a zoonotic reservoir of hepatitis E virus (HEV) for transmission to humans and other zoonotic reservoirs such as swine. The purpose of this study was to assess the ability of rabbit HEV to cross the species barrier to infect mice and also the usefulness of this animal to study HEV transmission and pathogenesis. In this study, uninfected BALB/c mice were experimentally inoculated with rabbit HEV either via gavage or through contact-exposure with infected mice. Rabbit HEV propagation in mice was evaluated by studying fecal virus shedding, viremia, seroconversion and microscopic liver lesions. Rabbit HEV could be detected in all mice infected by gavage, but only in some contact-exposed mice, with some animals exhibiting fecal virus shedding, seroconversion or viremia (one mouse only). Compared with inoculated mice, anti-rabbit HEV antibody titers and viral copy numbers in fecal and serum samples were lower in contact-exposed mice. Infected mice mainly exhibited phlebitis, hepatocyte swelling and necrosis. Microscopic liver lesion scores for inoculated and contact-exposed infected mice were higher than scores for negative controls. This study therefore demonstrates that rabbit HEV could infect BALB/c mice both though inoculation via gavage and through contact-exposure.

Platycodin D Suppresses Type 2 Porcine Reproductive and Respiratory Syndrome Virus In Primary and Established Cell Lines.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a continuous threat to the pork industry as it continues to cause significant economic loss worldwide. Currently, vaccination strategies provide very limited protection against PRRSV transmission. Consequently, there is an urgent need to develop new antiviral strategies. Platycodin D (PD) is one of the major bioactive triterpenoid saponins derived from Platycodon grandiflorum, a traditional Chinese medicine used as an expectorant for pulmonary diseases and a remedy for respiratory disorders. Here, we demonstrate that PD exhibits potent activity against PRRSV infection in Marc-145 cells and primary porcine alveolar macrophages. PD exhibited broad-spectrum inhibitory activities in vitro against high pathogenic type 2 PRRSV GD-HD strain and GD-XH strain as well as classical CH-1a and VR2332 strains. PD at concentrations ranging 1⁻4 µM significantly inhibited PRRSV RNA synthesis, viral protein expression and progeny virus production in a dose-dependent manner. EC50 values of PD against four tested PRRSV strains infection in Marc-145 cells ranged from 0.74 to 1.76 µM. Mechanistically, PD inhibited PRRSV replication by directly interacting with virions therefore affecting multiple stages of the virus life cycle, including viral entry and progeny virus release. In addition, PD decreased PRRSV- and LPS-induced cytokine (IFN-α, IFN-ß, IL-1α, IL-6, IL-8 and TNF-α) production in PAMs. Altogether, our findings suggested that PD is a potent inhibitor of PPRSV infection in vitro. However, further in vivo studies are necessary to confirm PD as a potential novel and effective PPRSV inhibitor in swine.