Isolation and characterization of a new population of nasal surface macrophages and their susceptibility to PRRSV-1 subtype 1 (LV) and subtype 3 (Lena).

Sialoadhesin (Sn) and CD163 have been recognized as two important mediators for porcine reproductive and respiratory syndrome virus (PRRSV) in host macrophages. Recently, it has been demonstrated that the highly virulent Lena strain has a wider macrophage tropism than the low virulent LV strain in the nasal mucosa. Not only CD163+Sn+ macrophages are infected by Lena but also CD163+Sn- macrophages. This suggests that an alternative receptor exists for binding and internalization of PRRSV Lena in the CD163+Sn- macrophages. Further investigation to find the new entry receptor was hampered by the difficulty of isolating these macrophages from the nasal mucosa. In the present study, a new population of CD163+Sn- cells has been identified that is specifically localized in the nasal lamina propria and can be isolated by an intranasal digestion approach. Isolated nasal cells were characterized using specific cell markers and their susceptibility to two different PRRSV-1 strains (LV and Lena) was tested. Upon digestion, 3.2% (flow cytometry)-6.4% (confocal microscopy) of the nasal cells were identified as CD163+ and all (99.7%) of these CD163+ cells were Sn-. These CD163+Sn- cells, designated as "nasal surface macrophages", showed a 4.9 times higher susceptibility to the Lena strain than to the LV strain. Furthermore, the Lena-inoculated cell cultures showed an upregulation of CD163. These results showed that our new cell isolation system is ideal for the further functional and phenotypical analysis of the new population of nasal surface macrophages and further research on the molecular pathogenesis of PRRSV in the nose.

Equine herpesvirus 1 infection orchestrates the expression of chemokines in equine respiratory epithelial cells.

The ancestral equine herpesvirus 1 (EHV1), closely related to human herpes viruses, exploits leukocytes to reach its target organs, accordingly evading the immune surveillance system. Circulating EHV1 strains can be divided into abortigenic/neurovirulent, causing reproductive/neurological disorders. Neurovirulent EHV1 more efficiently recruits monocytic CD172a+ cells to the upper respiratory tract (URT), while abortigenic EHV1 tempers monocyte migration. Whether similar results could be expected for T lymphocytes is not known. Therefore, we questioned whether differences in T cell recruitment could be associated with variations in cell tropism between both EHV1 phenotypes, and which viral proteins might be involved. The expression of CXCL9 and CXCL10 was evaluated in abortigenic/neurovirulent EHV1-inoculated primary respiratory epithelial cells (ERECs). The bioactivity of chemokines was tested with a functional migration assay. Replication of neurovirulent EHV1 in the URT resulted in an enhanced expression/bioactivity of CXCL9 and CXCL10, compared to abortigenic EHV1. Interestingly, deletion of glycoprotein 2 resulted in an increased recruitment of both monocytic CD172a+ cells and T lymphocytes to the corresponding EREC supernatants. Our data reveal a novel function of EHV1-gp2, tempering leukocyte migration to the URT, further indicating a sophisticated virus-mediated orchestration of leukocyte recruitment to the URT.

Porcine rotavirus mainly infects primary porcine enterocytes at the basolateral surface.

Intestinal epithelium functions as a barrier to protect multicellular organisms from the outside world. It consists of epithelial cells closely connected by intercellular junctions, selective gates which control paracellular diffusion of solutes, ions and macromolecules across the epithelium and keep out pathogens. Rotavirus is one of the major enteric viruses causing severe diarrhea in humans and animals. It specifically infects the enterocytes on villi of small intestines. The polarity of rotavirus replication in their target enterocytes and the role of intestinal epithelial integrity were examined in the present study. Treatment with EGTA, a drug that chelates calcium and disrupts the intercellular junctions, (i) significantly enhanced the infection of rotavirus in primary enterocytes, (ii) increased the binding of rotavirus to enterocytes, but (iii) considerably blocked internalization of rotavirus. After internalization, rotavirus was resistant to EGTA treatment. To investigate the polarity of rotavirus infection, the primary enterocytes were cultured in a transwell system and infected with rotavirus at either the apical or the basolateral surface. Rotavirus preferentially infected enterocytes at the basolateral surface. Restriction of infection through apical inoculation was overcome by EGTA treatment. Overall, our findings demonstrate that integrity of the intestinal epithelium is crucial in the host's innate defense against rotavirus infection. In addition, the intercellular receptor is located basolaterally and disruption of intercellular junctions facilitates the binding of rotavirus to their receptor at the basolateral surface.

Gammaherpesvirus BoHV-4 infects bovine respiratory epithelial cells mainly at the basolateral side.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that is widespread in cattle. However, only a few studies about the pathogenesis of BoHV-4 primary infection have been reported. In the present study, ex vivo models with bovine nasal and tracheal mucosa explants were used to study the cellular BoHV-4-host interactions. Infection was observed in nasal but not in tracheal epithelial cells. To find a possible correlation between the integrity and restricted infection of the respiratory epithelium, both nasal mucosal and tracheal explants were treated with EGTA, a drug that disrupts the intercellular junctions, before inoculation. The infection was analyzed based on the number of plaques, plaque latitude and number of infected single cells, as determined by immunofluorescence. BoHV-4 infection in nasal mucosal explants was enhanced upon opening the tight junctions with EGTA. Infection in tracheal explants was only found after treatment with EGTA. In addition, primary bovine respiratory epithelial cells (BREC) were isolated, grown at the air-liquid interface and infected either at the apical or basolateral side by BoHV-4. The results showed that BoHV-4 preferentially bound to and entered BREC at the basolateral surfaces of both nasal and tracheal epithelial cells. The percentage of BoHV-4 infection was significantly increased both from nasal and tracheal epithelial cells after treatment with EGTA, which indicates that the BoHV-4 receptor is mainly located at the basolateral surface of these cells. Thus, our findings demonstrate that integrity of the respiratory epithelium is crucial in the host's innate defense against primary BoHV-4 infections.

Preferential use of Siglec-1 or Siglec-10 by type 1 and type 2 PRRSV strains to infect PK15S1-CD163 and PK15S10-CD163 cells.

Cellular entry mediators define whether the cell is permissive to PRRSV infection. Porcine sialoadhesin (pSn, Siglec-1) and CD163 are main entry mediators facilitating infection of porcine macrophages by PRRSV. Recently, Siglec-10 was demonstrated to be an alternative receptor for PRRSV. To examine if virulence and pathogenicity of PRRSV strains could be correlated with the use of different Siglecs, a PK15 cell line recombinantly expressing Siglec-1 and CD163 (PK15S1-CD163) and a PK15 cell line recombinantly expressing Siglec-10 and CD163 (PK15S10-CD163) were used to compare the virus replication of 7 genotype 1 subtype 1 strains (G1s1), 2 genotype 1 subtype 3 (G1s3) strains and 5 genotype 2 (G2) strains. Some strains (08VA (G1s1), 13V117 (G1s1), 17V035 (G1s1), VR2332 (G2)) were poor virus producers (<104 TCID50/mL), while other strains (07V063 (G1s1), 13V091 (G1s1), Su1-Bel (G1s3), MN-184 (G2), Korea17 (G2) and SDSU-73 (G2)) easily grew up to ≥106 TCID50/mL. PK15S10-CD163 cells exhibited a higher efficiency in virus production per infected cell than the PK15S1-CD163 cells. The G1s1 strains LV and 07V063 infected more cells in the PK15S1-CD163, whereas the 94V360 and 08VA strains preferred PK15S10-CD163. The highly virulent G1s3 strains Lena and Su1-Bel showed a strong preference for PK15S1-CD163. The G2 strains MN-184, SDSU-73, Korea17 had a much higher infection rate in PK15S10-CD163, while the reference strain VR2332 and the NADC30 strain had a slight preference for PK15S1-CD163. Differences in receptor use may influence the outcome of a PRRSV infection in pigs and explain in part the virulence/pathogenicity of PRRSV strains.

Molecular cloning of porcine Siglec-3, Siglec-5 and Siglec-10, and identification of Siglec-10 as an alternative receptor for porcine reproductive and respiratory syndrome virus (PRRSV).

In recent years, several entry mediators have been characterized for porcine reproductive and respiratory syndrome virus (PRRSV). Porcine sialoadhesin [pSn, also known as sialic acid-binding immunoglobulin-type lectin (Siglec-1)] and porcine CD163 (pCD163) have been identified as the most important host entry mediators that can fully coordinate PRRSV infection into macrophages. However, recent isolates have not only shown a tropism for sialoadhesin-positive cells, but also for sialoadhesin-negative cells. This observation might be partly explained by the existence of additional receptors that can support PRRSV binding and entry. In the search for new receptors, recently identified porcine Siglecs (Siglec-3, Siglec-5 and Siglec-10), members of the same family as sialoadhesin, were cloned and characterized. Only Siglec-10 was able to significantly improve PRRSV infection and production in a CD163-transfected cell line. Compared with sialoadhesin, Siglec-10 performed equally effectively as a receptor for PRRSV type 2 strain MN-184, but it was less capable of supporting infection with PRRSV type 1 strain LV (Lelystad virus). Siglec-10 was demonstrated to be involved in the endocytosis of PRRSV, confirming the important role of Siglec-10 in the entry process of PRRSV. In conclusion, it can be stated that PRRSV may use several Siglecs to enter macrophages, which may explain the strain differences in the pathogenesis.

Primary replication and invasion of the bovine gammaherpesvirus BoHV-4 in the genital mucosae.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus that is widespread in cattle. Ex vivo models with bovine genital tract mucosa explants were set up to study molecular/cellular BoHV-4-host interactions. Bovine posterior vagina, cervix and uterus body were collected from cows at two stages of the reproductive cycle for making mucosa explants. The BoHV-4 replication kinetics and characteristics within the three different mucosae of animals in the follicular and luteal phase were assessed by virus titration. The number of plaques, plaque latitude and number of infected cells were determined by immunofluorescence. BoHV-4 replicated in a productive way in all genital mucosal tissues. It infected single individual cells in both epithelium and lamina propria of the genital mucosae at 24 hours post-inoculation (hpi). Later, small BoHV-4 epithelial plaques were formed that did not spread through the basement membrane. 50% of the number of BoHV-4 infected cells were identified as cytokeratin+ and CD172a+ cells in the three parts of the genital tract at 24 hpi. Upon a direct injection of genital explants with BoHV-4, fibrocytes became infected, indicating that the unidentified 50% of the infected cells are most probably fibrocytes. In this study, in vivo-related in vitro genital tract models were successfully established and the early stage of the pathogenesis of a genital infection was clarified: BoHV-4 starts with a productive infection of epithelial cells in the reproductive tract, forming small foci followed by a non-productive infection of surveilling monocytic cells which help BoHV-4 to invade into deeper tissues.

Genetic variation, pathogenicity, and immunogenicity of highly pathogenic porcine reproductive and respiratory syndrome virus strain XH-GD at different passage levels.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases of swine worldwide. Immunization with an attenuated vaccine is considered an effective method for reducing the economic losses resulting from porcine reproductive and respiratory syndrome virus (PRRSV) infection. Several studies have shown that PRRSV can be attenuated by passage in Marc-145 cells, but it is still not clear whether this attenuation influences the immunogenicity of PRRSV and what the mechanism of attenuation is. In order to study the mechanism of attenuation and immunogenicity of highly pathogenic (HP) PRRSV, the HP-PRRSV strain XH-GD was serially 122 times passaged in Marc-145 cells. Genomic sequence comparisons were made at selected passages. To explore the differences in pathogenicity and immunogenicity at different passages, three passages (P5, P62 and P122) were selected for an animal challenge experiment, which showed that passage in Marc-145 cells resulted in attenuation of the virus. After 122 passages, 35 amino acid changes were observed in the structural proteins and non-structural proteins. The animal challenge experiment showed that pathogenicity decreased with increasing passage number. The N antibody level and specific neutralizing (SN) antibody titers also decreased with increasing passage number in the late stage of the animal experiment. This study indicated that the virulence of XH-GD was decreased by passage in Marc-145 cells and that overattenuation might influence the immunogenicity of virus. These results might contribute to our understanding of the mechanism of attenuation.

In Vitro Antiviral Activity of Germacrone Against Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most serious diseases affecting the swine industry worldwide; however, there is no efficient control strategies against PRRSV at present. Therefore, development of new antiviral treatment strategies is urgently needed. As reported, germacrone can efficiently impair influenza virus replication. In this study, we exploited whether germacrone has the potential to inhibit PRRSV infection. Our results showed that the germacrone significantly inhibited replication of PRRSV in vitro and repressed the synthesis of viral RNA and protein. However, it did not block PRRSV binding and entry. Further studies confirmed that germacrone impaired PRRSV replication at an early stage, and inhibited infection of both classic and highly pathogenic type II PRRSV strains. Collectively, our findings imply that the germacrone has the potential to be used as an anti-PRRSV drug.

Epidemiological and evolutionary characteristics of the PRRSV in Southern China from 2010 to 2013.

In 2006, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV) emerged in China and caused lasting damage to the swine industry. To analyze the genetic variation of PRRSV in Southern China, 126 tissue samples were collected; 41 ORF5 and partial Nsp2 genes were sequenced and analyzed. The results showed that the PRRSV positive rate was 32.54% over the last four years, that there are two main subgenotypes in Southern China, and that the dominant strain is HP-PRRSV. An amino acid analysis of Nsp2 showed that 40 strains contained a 30-amino acid deletion in the hypervariable region. However, the 13YJ6-8 mutant exhibited a unique amino acid deletion at positions 508-514 of Nsp2. A phylogenetic analysis of ORF5 revealed that this mutant and five other strains, belong to an intermediate subgenotype (inter-subgenotype), which is characterized by extensive mutations, especially in the signal peptide and N-glycosylation sites. The results of this study demonstrate the genetic diversity of PRRSV in Southern China and provide basic knowledge of the PRRSV epidemic in this region.

Critical role of cellular cholesterol in bovine rotavirus infection.

BACKGROUND: Bovine rotavirus (BRV) is a non-enveloped dsRNA virus that cause neonatal calf diarrhea. Lipid rafts are cholesterol-enrich membrane mircodomains that play a vital role in many cellular processes. In this study, the effect of cellular cholesterol depletion on infection of MA-104 cells with bovine rotavirus was investigated. RESULTS: We demonstrated that cholesterol depletion of the plasma membrane by MßCD had no effect on BRV binding to cells but significantly impaired BRV entry in a dose-dependent manner and the effect was partially reversed by addition of exogenous cholesterol, suggesting the reduction of BRV infection by MßCD was specifically due to cholesterol depletion. Cholesterol depletion after virus entry did not reduce BRV replication, whereas affected virus assembly. CONCLUSIONS: Taken together, our results demonstrate that cell membrane cholesterol is essential to BRV infectivity.

Seroepidemiological evidence of avian influenza A virus transmission to pigs in southern China.

Recently, three novel avian-origin swine influenza viruses (SIVs) were first isolated from pigs in Guangdong Province, southern China, yet little is known about the seroprevalence of avian influenza viruses among pigs in southern China. Here, we report for the first time the seroprevalence of avian H3, H4, and H6 influenza viruses in swine populations and the lack of seroepidemiological evidence of avian H5 influenza virus transmission to pigs in China.

Complete genome sequence of duck Tembusu virus, isolated from Muscovy ducks in southern China.

We report here the complete genomic sequence of the duck Tembusu virus (DTMUV) WJ-1 strain, isolated from Muscovy ducks. This is the first complete genome sequence of DTMUV reported in southern China. Compared with the other strains (TA, GH-2, YY5, and ZJ-407) that were previously found in eastern China, WJ-1 bears a few differences in the nucleotide and amino acid sequences. We found that there are 47 mutations of amino acids encoded by the whole open reading frame (ORF) among these five strains. The whole-genome sequence of DTMUV will help in understanding the epidemiology and molecular characteristics of duck Tembusu virus in southern China.

Complete genome sequence of a novel field strain of rearranged porcine circovirus type 2 in southern China.

We report here the complete genomic sequence of a novel porcine circovirus type 2 (PCV2) strain, which is supposed to be the result of natural genetic recombination between the ORF1 gene of genotype PCV2b-1B and the ORF2 gene of PCV2b-1C. Further analyses revealed that this novel PCV2 strain arose from recombination between PCV2a and PCV2b strains within the ORF2 gene. To our knowledge, this is the first report of both inter- and intragenotype PCV2 gene rearrangement in the field, and it will help in understanding the epidemiology and molecular characteristics of porcine circovirus type 2(PCV2) in southern China.

Complete genome sequence of a novel duck hepatitis A virus discovered in southern China.

We report here the complete genomic sequence of a novel duck hepatitis A virus (DHAV) isolated from mixed infections with DHAV type 1 (DHAV-1) and DHAV-3 in ducklings in Southern China. The whole nucleotide sequence had the highest homology with the sequence of DHAV-3 (GenBank accession number DQ812093) (96.2%). To our knowledge, this is the first report of gene rearrangement between DHAV-1 and DHAV-3, and it will help to understand the epidemiology and molecular characteristics of duck hepatitis A virus in Southern China.

Complete Genome Sequence of a Novel Avian-Like H3N2 Swine Influenza Virus Discovered in Southern China.

We report here the complete genomic sequence of a novel avian-like H3N2 swine influenza virus containing an H5N1 highly pathogenic avian influenza virus segment that was obtained from swine in southern China. Phylogenetic analysis indicated that this virus might originate from domestic aquatic birds. The sequence information provided herein suggests that continuing study is required to determine if this virus can be established in the swine population and pose potential threats to public health.

Phylogenetic analysis and molecular characteristics of 17 porcine reproductive and respiratory syndrome virus isolates in Southern China from 2010 to 2011.

We analyzed the complete genomic sequences of 17 porcine reproductive and respiratory syndrome virus (PRRSV) isolates from Southern China obtained between 2010 and 2011 and found that four of seven isolates from 2011 were closely related to the JXA1-R strain (vaccine virus of JXA1). This close relationship between field isolates and China domestic vaccine viruses has not been reported to date. The occurrence of vaccine-like viruses potentially creates a threat for the pig breeding industry and brings difficulties for control of this disease.

Molecular epidemiology of PRRSV in South China from 2007 to 2011 based on the genetic analysis of ORF5.

Porcine reproductive and respiratory syndrome virus (PRRSV) has proven to be highly genetically variable; however, comprehensive information regarding the virus's genetic diversity in South China is limited. In this study, a total of 3199 clinical samples were collected from 267 pig farms suspected of PRRSV infection between 2007 and 2011. The ORF5 genes of 51 PRRSV-positive samples were sequenced and analyzed. The 51 study strains were divided into three primary subgenotypes. Fourty-five of the strains belonged to subgenotype I and were closely related to the highly pathogenic PRRSV (HP-PRRSV) strains. The subgenotype I strains were generally clustered into genetically similar groups by year. Only one of the strains belonged to subgenotype II, clustering with the classical North American type, VR2332. Five of the strains were grouped into subgenotype III, which occupied a separate branch and was closely related to the recently isolated novel field strains, QYYZ and GM2. The 5 subgenotype III strains shared an amino acid identity with the remaining 46 study strains ranging from 79.6%-83.6%. Amino acid analysis showed extensive mutations in subgenotype III; the diverse genetic mutations of these novel strains are of great concern.

Serological surveillance of H5 and H9 avian influenza A viral infections among pigs in Southern China.

Pigs are susceptible to both human and avian influenza viruses (AIV). Moreover, they are suspected of being the intermediate hosts or mixing vessels of pandemic influenza viruses. Researchers suspect that the influenza viruses are able to undergo reassortment or to adapt to various mammalian hosts while they incubate in pigs. For the present report, we conducted a serological surveillance of pigs in southern China from 2008 to 2012 to establish the prevalence of antibodies against H5N1 and H9N2 AIV. A total of one hundred pig farms from the Guangdong, Zhejiang, Fujian, and Yunnan Provinces were sampled, yielding a total of 3960 serum specimens. The haemagglutination inhibition (HI) tests revealed no evidence of H5 infection when the Clade 2.3.2 virus was used as the antigen, but a 4.6% positive rate of H9 infection was observed when using the Beijing/1/94-like virus as the antigen. The positive sera for H9 infection were further verified with neutralization tests, which confirmed a 3.7% rate of positive sera of H9 infection. In summary, the results imply that the swine populations in southern China had not been affected greatly by the H5N1 avian influenza virus. Nevertheless, these swine H9N2 influenza viruses might pose a threat to human health, and so researchers should continue to carry out swine influenza virus surveillance in China.

Mutagenesis analysis of porcine reproductive and respiratory syndrome virus nonstructural protein 7.

Nonstructural protein 7 (nsp7), which is flanked by nsp6 and nsp8, is one of the most conserved nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV). Nonstructural protein (nsp)-specific antibodies are produced in high titers in response to virus replication, especially against nsp1a, nsp1b, nsp2, and nsp7. However, many regional aspects of nsp7 are still veiled, such as its impact on viral replication and virulence or the immunological mechanism between virus and host. Based on the structure of the predicted nsp7 domain, we have constructed a series of large mutations and deletions. We ultimately demonstrated all mutations (nsp7, nsp7α/nspß) and the majority of substitutions of nsp7 affected the PRRSV replicative cycle in some ways and were fatal for viral recovery, which indicates that these are significant to structure or function of the nsp7. What's more, the mutant vOKXH-nsp7 (F40A) indeed caused some of the variation compared with the parental virus vOKXH-GD, which shortens the amount of time needed to reach its highest viral titer, and decreases the concentration of the highest viral titer, obstructing viral mRNA and protein synthesis. Consequently, these valuable results possibly provide the first direct evidence that the nsp7 is really a critical protein domain for the RNA synthesis and the translation of viral protein of PRRSV.