Inflammation plays a critical role in damage to the bronchiolar epithelium induced by Trueperella pyogenes in vitro and in vivo.

Trueperella pyogenes can cause severe pulmonary disease in swine, but the mechanism of pathogenesis is not well defined. T. pyogenes-induced damage to porcine bronchial epithelial cells (PBECs), porcine precision-cut lung slices (PCLS), and respiratory epithelium of mice remains unknown. In this study, we used T. pyogenes 20121 to infect PBECs in air-liquid interface conditions and porcine PCLS. T. pyogenes could adhere to, colonize, and induce cytotoxic effect on PBECs and the luminal surface of bronchi in PCLS, which damaged the bronchiolar epithelium. Moreover, bronchiolar epithelial cells showed extensive degeneration in the lungs of infected mice. Furthermore, western blot showed that the NOD-like receptor (NLR)/C-terminal caspase recruitment domain (ASC)/caspase-1 axis and nuclear factor-kappa B pathway were involved in inflammation in PCLS and lungs of mice, which also confirms that porcine PCLS provide a platform to analyze the pulmonary immune response. Meanwhile, the levels of p-c-Jun N-terminal kinase, p-extracellular signal-regulated kinase, and p-protein kinase B (AKT) were increased significantly, which indicated the mitogen-activated protein kinase and Akt pathways were also involved in inflammation in T. pyogenes-infected mice. In addition, we used T. pyogenes 20121 to infect tumor necrosis factor-alpha (tnf-α-/-) mice, and the results indicated that apoptosis and injury in respiratory epithelium of infected tnf-α-/- mice were alleviated. Thus, the pro-inflammatory cytokine TNF-α played a role in apoptosis and the respiratory epithelium injury in mouse lungs. Collectively, our study provides insight into the inflammatory injury induced by T. pyogenes and suggests that blocking NLR may be a potential therapeutic strategy against T. pyogenes infection.

Neuropilin-1 Facilitates Pseudorabies Virus Replication and Viral Glycoprotein B Promotes Its Degradation in a Furin-Dependent Manner.

Pseudorabies virus (PRV), which is extremely infectious and can infect numerous mammals, has a risk of spillover into humans. Virus-host interactions determine viral entry and spreading. Here, we showed that neuropilin-1 (NRP1) significantly potentiates PRV infection. Mechanistically, NRP1 promoted PRV attachment and entry, and enhanced cell-to-cell fusion mediated by viral glycoprotein B (gB), gD, gH, and gL. Furthermore, through in vitro coimmunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays, NRP1 was found to physically interact with gB, gD, and gH, and these interactions were C-end Rule (CendR) motif independent, in contrast to currently known viruses. Remarkably, we illustrated that the viral protein gB promotes NRP1 degradation via a lysosome-dependent pathway. We further demonstrate that gB promotes NRP1 degradation in a furin-cleavage-dependent manner. Interestingly, in this study, we generated gB furin cleavage site (FCS)-knockout PRV (Δfurin PRV) and evaluated its pathogenesis; in vivo, we found that Δfurin PRV virulence was significantly attenuated in mice. Together, our findings demonstrated that NRP1 is an important host factor for PRV and that NRP1 may be a potential target for antiviral intervention. IMPORTANCE Recent studies have shown accelerated PRV cross-species spillover and that PRV poses a potential threat to humans. PRV infection in humans always manifests as a high fever, tonic-clonic seizures, and encephalitis. Therefore, understanding the interaction between PRV and host factors may contribute to the development of new antiviral strategies against PRV. NRP1 has been demonstrated to be a receptor for several viruses that harbor CendR, including SARS-CoV-2. However, the relationships between NRP1 and PRV are poorly understood. Here, we found that NRP1 significantly potentiated PRV infection by promoting PRV attachment and enhanced cell-to-cell fusion. For the first time, we demonstrated that gB promotes NRP1 degradation via a lysosome-dependent pathway. Last, in vivo, Δfurin PRV virulence was significantly attenuated in mice. Therefore, NRP1 is an important host factor for PRV, and NRP1 may be a potential target for antiviral drug development.

Aminopeptidase N Is an Entry Co-factor Triggering Porcine Deltacoronavirus Entry via an Endocytotic Pathway.

Porcine deltacoronavirus (PDCoV) is a recently discovered coronavirus that poses a potential threat to the global swine industry. Although we know that aminopeptidase N (APN) is important for PDCoV replication, it is unclear whether it is the primary functional receptor, and the mechanism by which it promotes viral replication is not fully understood. Here, we systematically investigated the roles of porcine APN (pAPN) during PDCoV infection of nonsusceptible cells, including in viral attachment and internalization. Using a viral entry assay, we found that PDCoV can enter nonsusceptible cells but then fails to initiate efficient replication. pAPN and PDCoV virions clearly colocalized with the endocytotic markers RAB5, RAB7, and LAMP1, suggesting that pAPN mediates PDCoV entry by an endocytotic pathway. Most importantly, our study shows that regardless of which receptor PDCoV engages, only entry by an endocytotic route ultimately leads to efficient viral replication. This knowledge should contribute to the development of efficient antiviral treatments, which are especially useful in preventing cross-species transmission. IMPORTANCE PDCoV is a pathogen with the potential for transmission across diverse species, although the mechanism of such host-switching events (from swine to other species) is poorly understood. Here, we show that PDCoV enters nonsusceptible cells but without efficient replication. We also investigated the key role played by aminopeptidase N in mediating PDCoV entry via an endocytotic pathway. Our results demonstrate that viral entry via endocytosis is a major determinant of efficient PDCoV replication. This knowledge provides a basis for future studies of the cross-species transmissibility of PDCoV and the development of appropriate antiviral drugs.

Streptococcus suis Serotype 2 Infection Causes Host Immunomodulation through Induction of Thymic Atrophy.

Streptococcus suis serotype 2 is an important bacterial pathogen of swine and is also an emerging zoonotic agent that may be harmful to human health. Although the virulence genes of S. suis have been extensively studied, the mechanisms by which they damage the central immune organs have rarely been studied. In the current work, we wanted to uncover more details about the impact and mechanisms of S. suis on specific populations of thymic and immune cells in infected mice. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assays revealed that S. suis infection induced apoptosis in CD3+, CD14+, and epithelial cells from the thymus. S. suis infection resulted in a rapid depletion of mitochondrial permeability and release of cytochrome c (CytC) and apoptosis-inducing factor (AIF) through upregulation of Bax expression and downregulation of Bcl-xl and Bcl2 expression in thymocytes. Moreover, S. suis infection increased cleavage of caspase-3, caspase-8, and caspase-9. Thus, S. suis induced thymocyte apoptosis through a p53- and caspase-dependent pathway, which led to a decrease of CD3+ cells in the thymus, subsequently decreasing the numbers of CD4+ and CD8+ cells in the peripheral blood. Finally, expression dysregulation of proinflammatory cytokines in the serum, including interleukin 2 (IL-2), IL-6, IL-12 (p70), tumor necrosis factor (TNF), and IL-10, was observed in mice after S. suis type 2 infection. Taken together, these results suggest that S. suis infection can cause atrophy of the thymus and induce apoptosis of thymocytes in mice, thus likely suppressing host immunity.

Infection Studies in Pigs and Porcine Airway Epithelial Cells Reveal an Evolution of A(H1N1)pdm09 Influenza A Viruses Toward Lower Virulence.

We analyzed the virulence of pandemic H1N1 2009 influenza A viruses in vivo and in vitro. Selected viruses isolated in 2009, 2010, 2014, and 2015 were assessed using an aerosol-mediated high-dose infection model for pigs as well as air-liquid interface cultures of differentiated airway epithelial cells. Using a dyspnea score, rectal temperature, lung lesions, and viral load in the lung as parameters, the strains from 2014-2015 were significantly less virulent than the strains isolated in 2009-2010. In vitro, the viruses from 2009-2010 also differed from the 2014-2015 viruses by increased release of infectious virus, a more pronounced loss of ciliated cells, and a reduced thickness of the epithelial cell layer. Our in vivo and in vitro results reveal an evolution of A(H1N1)pdm09 viruses toward lower virulence. Our in vitro culture system can be used to predict the virulence of influenza viruses.

Viral Coinfection Replaces Effects of Suilysin on Streptococcus suis Adherence to and Invasion of Respiratory Epithelial Cells Grown under Air-Liquid Interface Conditions.

Streptococcus suis is an important zoonotic pathogen which can infect humans and pigs worldwide, posing a potential risk to global public health. Suilysin, a pore-forming cholesterol-dependent cytolysin, is considered to play an important role in the pathogenesis of S. suis infections. It is known that infection with influenza A viruses may favor susceptibility to secondary bacterial infection, resulting in more severe disease and increased mortality. However, the molecular mechanisms underlying these coinfections are incompletely understood. Applying highly differentiated primary porcine respiratory epithelial cells grown under air-liquid interface (ALI) conditions, we analyzed the contribution of swine influenza viruses (SIV) to the virulence of S. suis, with a special focus on its cytolytic toxin, suilysin. We found that during secondary bacterial infection, suilysin of S. suis contributed to the damage of well-differentiated respiratory epithelial cells in the early stage of infection, whereas the cytotoxic effects induced by SIV became prominent at later stages of infection. Prior infection by SIV enhanced the adherence to and colonization of porcine airway epithelial cells by a wild-type (wt) S. suis strain and a suilysin-negative S. suis mutant in a sialic acid-dependent manner. A striking difference was observed with respect to bacterial invasion. After bacterial monoinfection, only the wt S. suis strain showed an invasive phenotype, whereas the mutant remained adherent. When the epithelial cells were preinfected with SIV, the suilysin-negative mutant also showed an invasion capacity. Therefore, we propose that coinfection with SIV may compensate for the lack of suilysin in the adherence and invasion process of suilysin-negative S. suis.

Sialic acid-dependent interaction of group B streptococci with influenza virus-infected cells reveals a novel adherence and invasion mechanism.

Group B streptococci (GBS) contain a capsular polysaccharide with side chains terminating in α2,3-linked sialic acids. Because of this linkage type, the sialic acids of GBS are recognised by lectins of immune cells. This interaction results in a dampening of the host immune response and thus promotes immune evasion. As several influenza A viruses (IAV) use α2,3-linked sialic acid as a receptor determinant for binding to host cells, we analysed whether GBS and influenza viruses can interact with each other and how this interaction affects viral replication and bacterial adherence to and invasion of host cells. A co-sedimentation assay revealed that viruses with a preference for α2,3-linked sialic acids bind to GBS in a sialic acid-dependent manner. There is, however, a large variation in the efficiency of binding among avian influenza viruses of different subtypes as shown by a hemagglutination-inhibition assay. A delay in the growth curve of IAV indicated that GBS has an inhibitory effect on virus replication. On the other hand, both the adherence and invasion efficiency of GBS were enhanced when the cells were pre-infected by IAV with appropriate receptor specificity. Our results suggest that GBS infection may result in a more severe disease when patients are co-infected by influenza viruses. This co-infection mechanism may have relevance also to other human diseases, as there are more bacterial pathogens with α2,3-linked sialic acids and human viruses binding to this linkage type.

Ciliostasis of airway epithelial cells facilitates influenza A virus infection.

Porcine precision-cut lung slices (PCLS) were used to analyze the effect of the ciliary activity on infection of airway epithelial cells by influenza viruses. Treatment of slices with 2% NaCl for 30 min resulted in reversible ciliostasis. When PCLS were infected by a swine influenza virus of the H3N2 subtype under ciliostatic conditions, the viral yield was about twofold or threefold higher at 24 or 48 h post-infection, respectively, as compared to slices with ciliary activity. Therefore, the cilia beating not only transports the mucus out of the airways, it also impedes virus infection.

Sialic acid-dependent interactions between influenza viruses and Streptococcus suis affect the infection of porcine tracheal cells.

Bacterial co-infections are a major complication in influenza-virus-induced disease in both humans and animals. Either of the pathogens may induce a host response that affects the infection by the other pathogen. A unique feature in the co-infection by swine influenza viruses (SIV) and Streptococcus suis serotype 2 is the direct interaction between the two pathogens. It is mediated by the haemagglutinin of SIV that recognizes the α2,6-linked sialic acid present in the capsular polysaccharide of Streptococcus suis. In the present study, this interaction was demonstrated for SIV of both H1N1 and H3N2 subtypes as well as for human influenza viruses that recognize α2,6-linked sialic acid. Binding of SIV to Streptococcus suis resulted in co-sedimentation of virus with bacteria during low-speed centrifugation. Viruses bound to bacteria retained infectivity but induced only tiny plaques compared with control virus. Infection of porcine tracheal cells by SIV facilitated adherence of Streptococcus suis, which was evident by co-staining of bacterial and viral antigen. Sialic-acid-dependent binding of Streptococcus suis was already detectable after incubation for 30 min. By contrast, bacterial co-infection had a negative effect on the replication of SIV as indicated by lower virus titres in the supernatant and a delay in the kinetics of virus release.

Identification of a linear B-cell epitope on the "puff" loop of the Senecavirus A VP2 protein involved in receptor binding.

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

MARCH8 inhibits pseudorabies virus replication by trapping the viral cell-to-cell fusion complex in the trans-Golgi network.

The membrane-associated RING-CH 8 protein (MARCH8), a member of the E3 ubiquitin ligase family, has broad-spectrum antiviral activity. However, some viruses hijack MARCH8 to promote virus replication, highlighting its dual role in the viral lifecycle. Most studies on MARCH8 have focused on RNA viruses, leaving its role in DNA viruses largely unexplored. Pseudorabies virus (PRV) is a large DNA virus that poses a potential threat to humans. In this study, we found that MARCH8 inhibited PRV replication at the cell-to-cell fusion stage. Interestingly, our findings proved that MARCH8 blocks gB cleavage by recruiting furin but this activity does not inhibit viral infection in vitro. Furthermore, we confirmed that MARCH8 inhibits cell-to-cell fusion independent of its E3 ubiquitin ligase activity but dependent on the interaction with the cell-to-cell fusion complex (gB, gD, gH, and gL). Finally, we discovered that the distribution of the cell-to-cell fusion complex is significantly altered and trapped within the trans-Golgi network. Overall, our results indicate that human MARCH8 acts as a potent antiviral host factor against PRV via trapping the cell-to-cell fusion complex in the trans-Golgi network.

Replication characteristics of swine influenza viruses in precision-cut lung slices reflect the virulence properties of the viruses.

Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes (A/sw/Potsdam/15/1981 H1N1, A/sw/Bad Griesbach/IDT5604/2006 H1N1, A/sw/Bakum/1832/2000 H1N2, A/sw/Damme/IDT5673/2006 H3N2, A/sw/Herford/IDT5932/2007 H3N2). The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses.

A novel viral vaccine platform based on engineered transfer RNA.

In recent years, an increasing number of emerging and remerging virus outbreaks have occurred and the rapid development of vaccines against these viruses has been crucial. Controlling the replication of premature termination codon (PTC)-containing viruses is a promising approach to generate live but replication-defective viruses that can be used for potent vaccines. Here, we used anticodon-engineered transfer RNAs (ACE-tRNAs) as powerful precision switches to control the replication of PTC-containing viruses. We showed that ACE-tRNAs display higher potency of reading through PTCs than genetic code expansion (GCE) technology. Interestingly, ACE-tRNA has a site preference that may influence its read-through efficacy. We further attempted to use ACE-tRNAs as a novel viral vaccine platform. Using a human immunodeficiency virus type 1 (HIV-1) pseudotyped virus as an RNA virus model, we found that ACE-tRNAs display high potency for read-through viral PTCs and precisely control their production. Pseudorabies virus (PRV), a herpesvirus, was used as a DNA virus model. We found that ACE-tRNAs display high potency for reading through viral PTCs and precisely controlling PTC-containing virus replication. In addition, PTC-engineered PRV completely attenuated and lost virulence in mice in vivo, and immunization with PRV containing a PTC elicited a robust immune response and provided complete protection against wild-type PRV challenge. Overall, replication-controllable PTC-containing viruses based on ACE-tRNAs provide a new strategy to rapidly attenuate virus infection and prime robust immune responses. This technology can be used as a platform for rapidly developing viral vaccines in the future.

Senecavirus A Entry Into Host Cells Is Dependent on the Cholesterol-Mediated Endocytic Pathway.

Senecavirus A (SVA), an important member of the Picornaviridae family, causes vesicular disease in pigs. Here, we generated an EGFP-expressing recombinant SVA re-SVA-EGFP, which exhibited similar growth kinetics to its parental virus. The reporter SVA was used to study the role of pig ANTXR1 (pANTXR1) in SVA infection in a porcine alveolar macrophage cell line (PAM-Tang cells). Knockdown of the pANTXR1 significantly reduced SVA infection and replication in PAM-Tang cells, while re-expression of the pANTXR1 promoted the cell susceptibility to SVA infection. The results indicated that pANTXR1 is a crucial receptor mediating SVA infection. Subsequently, the viral endocytosis pathways for SVA entry into pig cells were investigated and the results showed that cholesterol played an essential role in receptor-mediated SVA entry. Together, these results demonstrated that SVA entered into host cells through the pANTXR1-mediated cholesterol pathway. Our findings provide potential targets to develop antiviral drugs for the prevention of SVA infection in the pig population.

The relationship between autophagy and apoptosis during pseudorabies virus infection.

Both autophagy and apoptosis are mechanisms that maintain homeostasis in cells and that play essential roles in viral infections. Previous studies have demonstrated that autophagy and apoptosis pathways occurred with complex relationships in virus-infected cells. However, the regulation between these two processes in Pseudorabies virus (PRV) infection remains unclear. In the present study, we demonstrated that activated autophagy was induced at the early stage of PRV infection and that apoptosis was induced at the late stage of infection. Autophagy induction inhibited apoptosis and decreased viral replication, and autophagy inhibition promoted apoptosis and increased viral replication. We also found that viral infection resulted in an increase in the production of reactive oxygen species (ROS) and activation of apoptosis in autophagy-impaired cells, suggesting that ROS may participate in the cross-talk between autophagy and apoptosis in PRV-infected cells. Our studies provide possible molecular mechanisms for the cross-talk between apoptosis and autophagy induced by PRV infection in porcine cells. This suggests that these two cell death processes should be considered as the same continuum rather than as completely separate processes.

CRISPR-Cas13d Exhibits Robust Antiviral Activity Against Seneca Valley Virus.

In recent years, Seneca Valley virus (SVV) as a newly identified pathogen of porcine vesicular disease spread quickly and has posed a potential threat to the swine industry in several countries resulting in economic losses. Considering the evolution of SVV, attention should be given to controlling SVV epidemics. So far there are no commercial vaccines or drugs available to combat SVV. Therefore, development of strategies for preventing and controlling SVV infection should be taken into account. In the current study, we evaluated whether the CRISPR-Cas13d system could be used as a powerful tool against SVV infection. Besides, selected crRNAs showed different capacity against SVV infection. Our study suggests the CRISPR-Cas13d system significantly inhibited SVV replication and exhibited potent anti-SVV activity. This knowledge may provide a novel alternative strategy to control epidemics of SVV in the future.

Novel characteristics of Chinese NADC34-like PRRSV during 2020-2021.

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) strains were first detected in China in 2017, with epidemic potential. In this study, the phylogenetic, epidemic, and recombinant properties of NADC34-like PRRSV in China were evaluated comprehensively. From 2020 to October 2021, 82 NADC34-like PRRSV isolates were obtained from 433 PRRSV-positive clinical samples. These strains accounted for 11.5% and 28.6% of positives in 2020 and 2021, respectively, and have spread to eight provinces. We selected 15 samples for whole-genome sequencing, revealing genome lengths of 15,009-15,113 nt. Phylogenetic analysis revealed that Chinese NADC34-like strains cluster with American sublineage 1.5 strains and do not form an independent branch. Recombination analysis revealed that six of fifteen complete genome sequences were derived from recombination between NADC34-like and NADC30-like or HP-PRRSV; all of the strains recombined with local strains in China, exhibiting a complex recombination pattern. Partial Nsp2 sequence alignment showed that nine of fifteen isolates had a 100 aa continuous deletion (similar to that in IA/2014/NADC34); other isolates had a 131 aa discontinuous deletion (similar to that in NADC30). Five of them also had additional amino acid deletions, all of which are reported for the first time here. In the last 2 years, NADC34-like PRRSV has become one of the main epidemic strains in some areas of China; it has changed significantly, its homology has decreased significantly, and it has undergone complex recombination with local Chinese strains. These results are of great significance for understanding the current epidemic situation of PRRSV in China.

Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection.

The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell receptors and therefore specific cell types. It is also an important immune target for the host in neutralizing the virus. Four antigenic sites A, B, C, and D that reside near the N-terminal domain have been defined in the S protein. Of these, the region encoding antigenic sites A and to a lesser extent D, herein defined as S-AD, are most critical in eliciting host neutralizing antibodies. Herein, we enzymatically amplified, cloned, and expressed the S-AD fragment from TGEV in the prokaryotic expression vector, pET-30a. Maximum protein expression was achieved at 30°C over a 5-h period post-induction. Rabbit polyclonal antiserum was generated using recombinant S-AD (rS-AD) protein. In contrast to prior studies showing no activity with bacterially produced S protein, results indicated that polyclonal serum recognized TGEV-infected cells and reduced infection by 100%. Furthermore, the truncated rS-AD peptide was able to bind to the surface of cells from swine testes in a competitive manner and completely inhibit viral infection.

Infection of polarized bovine respiratory epithelial cells by bovine viral diarrhea virus (BVDV).

Bovine viral diarrhea virus (BVDV) is affecting cattle populations all over the world causing acute disease, immunosuppressive effects, respiratory diseases, gastrointestinal, and reproductive failure in cattle. The virus is taken up via the oronasal route and infection of epithelial and immune cells contributes to the dissemination of the virus throughout the body. However, it is not known how the virus gets across the barrier of epithelial cells encountered in the airways. Here, we analyzed the infection of polarized primary bovine airway epithelial cells (BAEC). Infection of BAEC by a non-cytopathogenic BVDV was possible via both the apical and the basolateral plasma membrane, but the infection was most efficient when the virus was applied to the basolateral plasma membrane. Irrespective of the site of infection, BVDV was efficiently released to the apical site, while only minor amounts of virus were detected in the basal medium. This indicates that the respiratory epithelium can release large amounts of BVDV to the environment and susceptible animals via respiratory fluids and aerosols, but BVDV cannot cross the airway epithelial cells to infect subepithelial cells and establish systemic infection. Further experiments showed that the receptor, bovine CD46, for BVDV is expressed predominantly on the apical membrane domain of the polarized epithelial cells. In a CD46 blocking experiment, the addition of an antibody directed against CD46 almost completely inhibited apical infection, whereas basolateral infection was not affected. While CD46 serves as a receptor for apical infection of BAEC by BVDV, the receptor for basolateral infection remains to be elucidated.