Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.

Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.

Quantitative proteomic analysis shows involvement of the p38 MAPK pathway in bovine parainfluenza virus type 3 replication.

BACKGROUND: Bovine parainfluenza virus type 3 (BPIV3) infection often causes respiratory tissue damage and immunosuppression and further results in bovine respiratory disease complex (BRDC), one of the major diseases in dairy cattle, caused huge economical losses every year. However, the pathogenetic and immunoregulatory mechanisms involved in the process of BPIV3 infection remain unknown. However, the pathogenetic and immunoregulatory mechanisms involved in the process of BPIV3 infection remain unknown. Proteomics is a powerful tool for high-throughput identification of proteins, which has been widely used to understand how viruses interact with host cells. METHODS: In the present study, we report a proteomic analysis to investigate the whole cellular protein alterations of MDBK cells infected with BPIV3. To investigate the infection process of BPIV3 and the immune response mechanism of MDBK cells, isobaric tags for relative and absolute quantitation analysis (iTRAQ) and Q-Exactive mass spectrometry-based proteomics were performed. The differentially expressed proteins (DEPs) involved in the BPIV3 invasion process in MDBK cells were identified, annotated, and quantitated. RESULTS: A total of 116 proteins, which included 74 upregulated proteins and 42 downregulated proteins, were identified as DEPs between the BPIV3-infected and the mock-infected groups. These DEPs included corresponding proteins related to inflammatory response, immune response, and lipid metabolism. These results might provide some insights for understanding the pathogenesis of BPIV3. Fluorescent quantitative PCR and western blotting analysis showed results consistent with those of iTRAQ identification. Interestingly, the upregulated protein MKK3 was associated with the p38 MAPK signaling pathway. CONCLUSIONS: The results of proteomics analysis indicated BPIV3 infection could activate the p38 MAPK pathway to promote virus replication.

Epidermal growth factor receptor (EGFR) promotes uptake of bovine parainfluenza virus type 3 into MDBK cells.

Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. The pathways of cell entry are highly related to viral transmission and pathogenicity. In previous studies, we demonstrated that macropinocytosis and clathrin-dependent endocytosis play critical roles in the entry of BPIV3 into MDBK cells. Macropinocytosis is special endocytic process which need to activate signaling pathways that remodle the actin cytoskeleton. Parainfluenza viruses (PIVs) initiate infection by binding to sialic acid receptors on cell surfaces. Nevertheless, sialic acids are not able to transmit signals across the plasma membrane, indicating the necessity for additional signaling receptors. Here, we have demonstrated that specific inhibitors and siRNAs targeting EGFR inhibit the entry of BPIV3 into MDBK cells. BPIV3 productive infection in MDBK cells led to activation of EGFR. Inactivation of EGFR suppressed BPIV3-induced rearrangement of the F-actin cytoskeleton. In addition, PI3K-Akt and ERK1/2 were activated in an EGFR-dependent manner during BPIV3 infection. Specific inhibitors targeting these canonical downstream effectors of EGFR could significantly reduce viral entry efficacy. Moreover, we also demonstrated that the important regulators of macropinocytosis, Rac1 and Pak1, are downstream mediators of EGFR during BPIV3 internalization. These results indicated that EGFR is a host-entry cofactor used by BPIV3 to enter MDBK cells.

Bovine Parainfluenza Virus Type 3 (BPIV3) Enters HeLa Cells via Clathrin-Mediated Endocytosis in a Cholesterol- and Dynamin-Dependent Manner.

Bovine parainfluenza virus 3 (BPIV3) is a crucial causative agent of respiratory disease in young and adult cattle. No specific therapies are available for BPIV3 infection. Understanding the internalization pathway of the virus will provide a new strategy for the development of antiviral therapy. Here, the mechanism of BPIV3 entry into HeLa cells was analyzed using RNA silencing and pharmacological inhibitors. Treatment of HeLa cells with hypertonic medium prevented BPIV3 internalization. These results indicated that BPIV3 entered HeLa cells via receptor-mediated endocytosis. Moreover, removing cell membrane cholesterol through MßCD treatment hampered viral penetration but not viral replication. In addition, BPIV3 infection was inhibited by pretreatment with dynasore or chlorpromazine (CPZ) or knockdown of dynamin II or clathrin heavy chain. However, virus entry was unaffected by nystatin, EIPA, wortmannin, or cytochalasin D treatment or caveolin-1 knockdown. These data demonstrated that the entry of BPIV3 into HeLa cells was dependent on clathrin-mediated endocytosis but not on caveolae-mediated endocytosis or the macropinocytosis pathway. Many viruses are transported to endosomes, which provide an acidic environment and release their genome upon separation from primary endocytic vesicles. However, we found that BPIV3 infection required endosomal cathepsins, but not a low pH. In summary, we show, for the first time, that BPIV3 enters HeLa cells through the clathrin-mediated endocytosis pathway, presenting novel insights into the invasion mechanism of Paramyxoviridae.

Amino- and carboxyl-terminal ends of the bovine parainfluenza virus type 3 matrix protein are important for virion and virus-like particle release.

Paramyxovirus matrix (M) proteins are key drivers of virus particle assembly and budding at the plasma membrane. To identify regions important for the M protein function, we generated a series of deletion mutants of the bovine parainfluenza virus type 3 (BPIV3) M protein. We found that M proteins lacking 10 amino acids in the amino-terminal end (ΔN10) or 4 amino acids in the carboxyl-terminal end (ΔC4) did not support M-deficient BPIV3 virion release and M protein-induced virus-like particle (VLP) release. Both ΔN10 and ΔC4 retained M protein-M protein and M protein-nucleocapsid (N) protein interactions. However, neither was transported to the plasma membrane. Our results indicate that both amino- and carboxyl-terminal ends of the BPIV3 M protein are essential for M protein transport to the plasma membrane, where it facilitates virion and VLP release.

Entry of bovine parainfluenza virus type 3 into MDBK cells occurs via clathrin-mediated endocytosis and macropinocytosis in a acid-dependent manner.

Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. No specific therapies are available for BPIV3. Understanding the viral internalization pathway of BPIV3 will provide new strategies for the development of antiviral treatments. Here, the entry mechanism of BPIV3 into MDBK cells was analyzed using chemical inhibitors and RNA silencing. Our data demonstrated that treatment with an inhibitor targeting the clathrin-mediated pathway or clathrin heavy chain (CHC) knockdown suppressed the entry of BPIV3 into MDBK cells. In contrast, sequestration of cellular cholesterol by nystatin or silencing of caveolin-1 had no effect on viral entry. Moreover, inhibition of critical modulators of macropinocytosis significantly reduced BPIV3 uptake. In addition, fluid-phase uptake was significantly increased in cells infected with BPIV3, which is indicative of virus-induced facilitation of macropinocytosis. These results suggest that BPIV3 enters MDBK cells via macropinocytosis and clathrin- but not caveolar-dependent endocytosis. Furthermore, inhibition of endosomal acidification and activation of cathepsin blocked BPIV3 entry, demonstrating that BPIV3 entered MDBK cells in a acid-dependent manner and required cathepsin L. Finally, we demonstrated that macropinocytosis but not clathrin-mediated endocytosis is dependent on actin dynamics during BPIV3 infection.

Cholesterol 25-Hydroxylase inhibits bovine parainfluenza virus type 3 replication through enzyme activity-dependent and -independent ways.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important pathogens associated with bovine respiratory diseases in both young and adult cattle widespreadly around the world. The host factors which participate in the infection of BPIV3 are poorly understood. Here, we found the bovine protein Cholesterol 25-hydroxylase (CH25 H) plays an important role in the infection of BPIV3. CH25H is a multi-transmembrane and endoplasmic reticulum-related enzyme that catalyzes oxidation reaction of cholesterol to production of 25-hydroxycholesterol (25HC) and significantly inhibits the replication of several viruses. In this study, we found that CH25H is an interferon-stimulated gene (ISG), which taken part in the antiviral innate immunity. In addition, the overexpression of CH25H could inhibit the replication of BPIV3, and 25HC significantly inhibited BPIV3 infection by preventing the synthesis of both virus antigenomic RNA (cRNA) and genomic RNA (gRNA) in MDBK cells. Interestingly, CH25H-M, a mutant lacking hydroxylase activity, still had an antiviral effect against BPIV3. Taken together, our findings highlight the antiviral function of CH25H during BPIV3 infection, and suggest that CH25H inhibits viral infection through both enzyme activity-dependent and -independent ways.

Cholesterol-rich lipid rafts play a critical role in bovine parainfluenza virus type 3 (BPIV3) infection.

Lipid rafts are specialized lipid domains enriched in cholesterol and sphingolipid, which can be utilized in the lifecycle of numerous enveloped viruses. Bovine parainfluenza virustype3 (BPIV3) entry to cell is mediated by receptor binding and membrane fusion, but how lipid rafts in host cell membrane and BPIV3 envelope affect virus infection remains unclear. In this study, we investigated the role of lipid rafts in the different stages of BPIV3 infection. The MDBK cells were treated by methyl-ß-cyclodextrin (MßCD) to disrupt cellular lipid raft, and the virus infection was determined. The results showed that MßCD significantly inhibited BPIV3 infection in a dose-dependent manner, but didn't block the binding of virus to the cell membrane. Whereas, the MDBK cells treated by MßCD after virus-entry had no effects on the virus infection, to suggest that BPIV3 infection was associated with lipid rafts in cell membrane during viral entry stage. To further confirm lipid rafts in viral envelope also affected BPIV3 infection, we treated BPIV3 with MßCD to determine the virus titer. We found that disruption of the viral lipid raft caused a significant reduction of viral yield. Cholesterol reconstitution experiment showed that BPIV3 infection was successfully restored by cholesterol supplementation both in cellular membrane and viral envelope, which demonstrated that cholesterol-rich lipid rafts played a critical role in BPIV3 infection. These findings provide insights on our understanding of the mechanism of BPIV3 infection and imply that lipid raft might be a good potential therapeutic target to prevent virus infection.

A single L288I substitution in the fusion protein of bovine parainfluenza virus type 3 enhances virus growth in semi-suitable cell lines.

The bovine parainfluenza virus type 3 BN-CE vaccine strain was obtained by serial passage of the BN-1 strain in chicken embryonic fibroblasts (CEF). We previously identified a substitution (L288I) in the fusion (F) protein between the two strains. To examine the effect of the substitution on CEF adaptation and attenuation, we generated a recombinant BN-1 strain with the L288I substitution in the F protein (FL288I-EGFP). FL288I-EGFP replicated more efficiently than a recombinant BN-1 strain (wt-EGFP) in semi-suitable cell lines, suggesting that the L288I substitution was established in the BN-1 strain during the process of adaptation in CEF.

3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses.

Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation.

UNLABELLED: Parainfluenza viruses are known to inhibit type I interferon (IFN) production; however, there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, IFN-λR1/interleukin-10R2 (IL-10R2), which is primarily expressed by epithelial cells. Parainfluenza virus 3 (PIV-3) infection is highly restricted to the airway epithelium. We therefore sought to examine type III IFN signaling pathways during PIV-3 infection of epithelial cells. We used three strains of PIV-3: human PIV-3 (HPIV-3), bovine PIV-3 (BPIV-3), and dolphin PIV-1 (Tursiops truncatus PIV-1, or TtPIV-1). Here, we show that message levels of IL-29 are significantly increased during PIV-3 infection, yet downstream antiviral signaling molecules are not upregulated to levels similar to those of the positive control. Furthermore, in Vero cells infected with PIV-3, stimulation with recombinant IL-29/-28A/-28B does not cause upregulation of downstream antiviral molecules, suggesting that PIV-3 interferes with the JAK/STAT pathway downstream of the IFN-λR1/IL-10R2 receptor. We used Western blotting to examine the phosphorylation of Stat1 and Stat2 in Vero cells and the bronchial epithelial cell line BEAS-2B. In Vero cells, we observed reduced phosphorylation of the serine 727 (S727) site on Stat1, while in BEAS-2B cells Stat1 phosphorylation was decreased at the tyrosine 701 (Y701) site during PIV-3 infection. PIV-3 therefore interferes with the phosphorylation of Stat1 downstream of the type III IFN receptor. These data provide new evidence regarding strategies employed by parainfluenza viruses to effectively circumvent respiratory epithelial cell-specific antiviral immunity. IMPORTANCE: Parainfluenza virus (PIV) in humans is associated with bronchiolitis and pneumonia and can be especially problematic in infants and the elderly. Also seen in cattle, bovine PIV-3 causes respiratory infections in young calves. In addition, PIV-3 is one of a number of pathogens that contribute to the bovine respiratory disease complex (BRDC). As their name suggests, interferons (IFNs) are produced by cells to interfere with viral replication. Paramyxoviruses have previously been shown to block production and downstream signaling of type I IFNs. For the first time, it is shown here that PIV-3 can induce protective type III IFNs in epithelial cells, the primary site of PIV-3 infection. However, we found that PIV-3 modulates signaling pathways downstream of the type III IFN receptor to block production of several specific molecules that aid in a productive antiviral response. Importantly, this work expands our understanding of how PIV-3 effectively evades host innate immunity.

Infection of the upper respiratory tract of hamsters by the bovine parainfluenza virus type 3 BN-1 strain expressing enhanced green fluorescent protein.

Bovine parainfluenza virus type 3 (BPIV3) is an important pathogen associated with bovine respiratory disease complex (BRDC). We have generated a recombinant BPIV3 expressing enhanced green fluorescent protein (rBPIV3-EGFP) based on the BN-1 strain isolated in Japan. After intranasal infection of hamsters with rBPIV3-EGFP, EGFP fluorescence was detected in the upper respiratory tract including the nasal turbinates, pharynx, larynx, and trachea. In the nasal turbinates, rBPIV3-EGFP attained high titers (>10(6) TCID50/g of tissue) 2-4 days after infection. Ciliated epithelial cells in the nasal turbinates and trachea were infected with rBPIV3-EGFP. Histopathological analysis indicated that mucosal epithelial cells in bronchi were shed by 6 days after infection, leaving non-ciliated cells, which may have increased susceptibility to bacterial infection leading to the development of BRDC. These data indicate that rBPIV3-EGFP infection of hamsters is a useful small animal model for studying the development of BPIV3-associated BRDC.

Infection of differentiated airway epithelial cells from caprine lungs by viruses of the bovine respiratory disease complex.

Bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3) and bovine herpesvirus type 1 (BHV-1) are important pathogens associated with the bovine respiratory disease complex (BRDC). Non-bovine ruminants such as goats may also be infected and serve as a virus reservoir to be considered in the development of control strategies. To evaluate the susceptibility of caprine airway epithelial cells to infection by viruses of BRDC, we established a culture system for differentiated caprine epithelial cells. For this purpose, we generated precision-cut lung slices (PCLS), in which cells are retained in their original structural configuration and remain viable for more than a week. The three bovine viruses were found to preferentially infect different cell types. Ciliated epithelial cells were the major target cells of BPIV3, whereas BHV-1 preferred basal cells. Cells infected by BRSV were detected in submucosal cell layers. This spectrum of susceptible cells is the same as that reported recently for infected bovine PCLS. While infection of caprine cells by BRSV and BPIV3 was as efficient as that reported for bovine cells, infection of caprine cells by BHV-1 required a tenfold higher dose of infectious virus as compared to infection of bovine airway cells. These results support the notion that non-bovine ruminants may serve as a reservoir for viruses of BRDC and introduce a culture system to analyze virus infection of differentiated airway epithelial cells from the caprine lung.

Three viruses of the bovine respiratory disease complex apply different strategies to initiate infection.

Bovine respiratory disease complex (BRDC) is the major cause of serious respiratory tract infections in calves. The disease is multifactorial, with either stress or reduced immunity allowing several pathogens to emerge. We investigated the susceptibility of bovine airway epithelial cells (BAEC) to infection by the three major viruses associated with the BRDC: bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1 (BHV-1) and bovine parainfluenza virus type 3 (BPIV3). For this purpose, two culture systems for well-differentiated BAEC were used: the air-liquid interface (ALI) system, where filter-grown BAEC differentiate into a pseudostratified respiratory epithelium and precision-cut lung slices (PCLS) where BAEC are maintained in the original tissue organisation. Comparative infection studies demonstrated that entry and release of BPIV3 occurred specifically via the apical membrane with ciliated cells being the major target cells. By contrast, airway epithelial cells were largely resistant to infection by BHV-1. When the epithelial barrier was abolished by opening tight junctions or by injuring the cell monolayer, BHV-1 infected mainly basal cells. Respiratory epithelial cells were also refractory to infection by BRSV. However, this virus infected neither differentiated epithelial cells nor basal cells when the integrity of the epithelial barrier was destroyed. In contrast to cells of the airway epithelium, subepithelial cells were susceptible to infection by BRSV. Altogether, these results indicate that the three viruses of the same disease complex follow different strategies to interact with the airway epithelium. Possible entry mechanisms are discussed.

Chimeric bovine/human parainfluenza virus type 3 expressing respiratory syncytial virus (RSV) F glycoprotein: effect of insert position on expression, replication, immunogenicity, stability, and protection against RSV infection.

UNLABELLED: A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109-114, 2012; C.-F. Yang et al., Vaccine 31:2822-2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity. IMPORTANCE: The research findings presented here will be instrumental for improving the design of a bivalent pediatric vaccine for respiratory syncytial virus and parainfluenza virus type 3, two major causes of severe respiratory tract infection in infants and young children. Moreover, this knowledge has general application to the development and clinical evaluation of other mononegavirus vectors and vaccines.

Subcutaneous, but not intratracheal administration of the TLR9 agonist, CpG DNA transiently reduces parainfluenza-3 virus shedding in newborn lambs.

Synthetic oligodeoxynucleotides (ODN) containing CpG motifs signal through TLR9 and activate innate immunity resulting in protection against a variety of parasitic, bacterial and viral pathogens in mouse models. However, few studies have demonstrated protection in humans and large animals. In the present investigations, we evaluated protection by CpG ODN in a parainfluenza-3 (PI-3) virus infection in neonatal lambs. Subcutaneous (SC) injection of CpG ODN induced high levels of 2'5'-A synthetase and significantly reduced PI-3 virus shedding in newborn lambs. Furthermore, pre-treatment of newborn lambs with SC CpG ODN 2 days, but not 6 days prior to the virus challenge was protective. In contrast, intratracheal (IT) administration of CpG ODN induced 2'5'-A synthetase but had no significant impact on PI-3 virus shedding in nasal secretions. We conclude that a systemic administration of CpG ODN and the timing of the treatment are critical for the protection of neonatal lambs against a respiratory viral infection.

Bighorn sheep fetal lung cell line for detection of respiratory viruses.

Pneumonia is an important disease of bighorn sheep (BHS) that is primarily responsible for the drastic decline in numbers of these animals in North America. Members of the genus Mannheimia and Pasteurella have frequently been isolated from the pneumonic lungs of BHS. Antibodies to several respiratory viruses, including bovine parainfluenza virus 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine viral diarrhea virus (BVDV), and bovine herpesvirus 1 (BoHV-1), have been detected in herds of BHS. The availability of BHS fetal lung cell lines is likely to enhance the chances of isolation of these viruses. Here we report the development of such a cell line. This line is permissive for BPIV-3, BRSV, BVDV, and BoHV-1 infection, as revealed by an enzyme immunoassay of virus-infected cells with antibodies specific for each of these viruses. This cell line should be valuable for detecting these 4, and possibly other, respiratory viruses in BHS.

Lentiviruses inefficiently incorporate human parainfluenza type 3 envelope proteins.

We have previously shown that the envelope glycoproteins of human parainfluenza type 3 (HPIV3), F and HN, are able to pseudotype lentiviruses, but the titers of these viruses are too low for use in clinical gene transfer. In this study we investigated the cause of these low titers. We compared the mRNA and protein expression levels of HN and F in transfected cells and in cells infected with wild-type HPIV3. Transfected cells contained similar levels of HN and F cytosolic mRNA, but fewer cell-surface HN and F proteins (3.8- and 1.3-fold less, respectively), than cells infected with wild-type HPIV3. To increase expression of HN in transfected cells, we codon-optimized HN and used it to transfect lentivirus producer cells. Cell surface expression of HN, as well as the amount of HN incorporated into virus particles, increased two- to threefold. Virus titers increased 1.2- to 6.4-fold, and the transduction efficiency of polarized MDCK cells via their apical surfaces increased 1.4-fold. Interestingly, even though codon optimization improved the expression levels of HN and virus titers, we found that HPIV3 pseudotyped viruses contained about 14-fold fewer envelope proteins than lentiviruses pseudotyped with the amphotropic envelope protein. Taken together, our findings suggest that titers are low, not because virus producer cells express levels of HPIV3 envelope proteins that are too low, but because too few of these proteins are incorporated by the lentiviruses for them to be able to efficiently transduce cells.

Determinants of the host range restriction of replication of bovine parainfluenza virus type 3 in rhesus monkeys are polygenic.

The Kansas strain of bovine parainfluenza virus type 3 (BPIV3) is 100- to 1,000-fold restricted in replication in the respiratory tracts of nonhuman primates compared to human PIV3 (HPIV3), an important pathogen of infants and young children. BPIV3 is also restricted in replication in human infants and children, yet it is immunogenic and is currently being evaluated in clinical trials as a vaccine candidate to protect against illness caused by HPIV3. We have examined the genetic basis for the host range attenuation phenotype of BPIV3 by exchanging each open reading frame (ORF) of a recombinant wild-type HPIV3 with the analogous ORF from BPIV3, with the caveats that the multiple ORFs of the P gene were exchanged as a single unit and that the HN and F genes were exchanged as a single unit. Recombinant chimeric bovine-human PIV3s were recovered from cDNA, and the levels of viral replication in vitro and in the respiratory tract of rhesus monkeys were determined. Recombinant chimeric HPIV3s bearing the BPIV3 N or P ORF were highly attenuated in the upper and lower respiratory tracts of monkeys, whereas those bearing the BPIV3 M or L ORF or the F and HN genes were only moderately attenuated. This indicates that the genetic determinants of the host range restriction of replication of BPIV3 for primates are polygenic, with the major determinants being the N and P ORFs. Monkeys immunized with these bovine-human chimeric viruses, including the more highly attenuated ones, developed higher levels of HPIV3 hemagglutination-inhibiting serum antibodies than did monkeys immunized with BPIV3 and were protected from challenge with wild-type HPIV3. Furthermore, host range determinants could be combined with attenuating point mutations to achieve an increased level of attenuation. Thus, chimeric recombinant bovine-human PIV3 viruses that manifest different levels of attenuation in rhesus monkeys are available for evaluation as vaccine candidates to protect infants from the severe lower respiratory tract disease caused by HPIV3.

Mucosal immunization of rhesus monkeys against respiratory syncytial virus subgroups A and B and human parainfluenza virus type 3 by using a live cDNA-derived vaccine based on a host range-attenuated bovine parainfluenza virus type 3 vector backbone.

Reverse genetics was used to develop a two-component, trivalent live attenuated vaccine against human parainfluenza virus type 3 (HPIV3) and respiratory syncytial virus (RSV) subgroups A and B. The backbone for each of the two components of this vaccine was the attenuated recombinant bovine/human PIV3 (rB/HPIV3), a recombinant BPIV3 in which the bovine HN and F protective antigens are replaced by their HPIV3 counterparts (48). This chimera retains the well-characterized host range attenuation phenotype of BPIV3, which appears to be appropriate for immunization of young infants. The open reading frames (ORFs) for the G and F major protective antigens of RSV subgroup A and B were each placed under the control of PIV3 transcription signals and inserted individually or in homologous pairs as supernumerary genes in the promoter proximal position of rB/HPIV3. The level of replication of rB/HPIV3-RSV chimeric viruses in the respiratory tract of rhesus monkeys was similar to that of their parent virus rB/HPIV3, and each of the chimeras induced a robust immune response to both RSV and HPIV3. RSV-neutralizing antibody titers induced by rB/HPIV3-RSV chimeric viruses were equivalent to those induced by infection with wild-type RSV, and HPIV3-specific antibody responses were similar to, or slightly less than, after infection with the rB/HPIV3 vector itself. This study describes a novel vaccine strategy against RSV in which vaccine viruses with a common attenuated backbone, specifically rB/HPIV3 derivatives expressing the G and/or F major protective antigens of RSV subgroup A and of RSV subgroup B, are used to immunize by the intranasal route against RSV and HPIV3, which are the first and second most important viral agents of pediatric respiratory tract disease worldwide.