Comparison of mutations in human parainfluenza viruses during passage in primary human bronchial/tracheal epithelial air-liquid interface cultures and cell lines.

Human parainfluenza virus (HPIV) causes respiratory infections, which are exacerbated in children and older people. Correct evaluation of viral characteristics is essential for the study of countermeasures. However, adaptation of viruses to cultured cells during isolation or propagation might select laboratory passage-associated mutations that modify the characteristics of the virus. It was previously reported that adaptation of HPIV3, but not other HPIVs, was avoided in human airway epithelia. To examine the influence of laboratory passage on the genomes of HPIV1-HPIV4, we evaluated the occurrence of mutations after passage in primary human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) culture and conventional cultured cells (Vero cells expressing the transmembrane protease, serine 2, and normal Vero cells). The occurrence of mutations was significantly lower in HBTEC-ALI than in conventional culture. In HBTEC-ALI culture, most of the mutations were silent or remained at low variant frequency, resulting in less impact on the viral consensus sequence. In contrast, passage in conventional culture induced or selected genetic mutations at high frequency with passage-associated unique substitutions. High mutagenesis of hemagglutinin-neuraminidase was commonly observed in all four HPIVs, and mutations even occurred in a single passage. In addition, in HPIV1 and HPIV2, mutations in the large protein were more frequent. These results indicate that passage in HBTEC-ALI culture is more suitable than conventional culture for maintaining the original characteristics of clinical isolates in all four HPIVs, which can help with the understanding of viral pathogenesis. IMPORTANCE: Adaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies.

A Point Mutation in the Human Parainfluenza Virus Type 2 Nucleoprotein Leads to Two Separate Effects on Virus Replication.

Paramyxovirus genomes, like that of human parainfluenza virus type 2 (hPIV2), have lengths of precisely multiples-of-six nucleotides ("rule of six"), where each nucleoprotein subunit (NP) binds exactly six nucleotides. Ten residues of its RNA binding groove contact the genome RNA; but only one, Q202, directly contacts a nucleotide base. The mutation of NPQ202 leads to two phenotypes: the ability of the viral polymerase to replicate minigenomes with defective bipartite promoters where NPwt is inactive, and the inability to rescue rPIV2 carrying this point mutation by standard means. The absence of an rPIV2 NPQ202A prevented further study of the latter phenotype. By extensive and repeated cocultivation of transfected cells, an rPIV2 carrying this mutation was finally recovered, and this virus was apparently viable due to the presence of an additional NP mutation (I35L). Our results suggest that these two phenotypes are due to separate effects of the Q202 mutation, and that the problematic rescue phenotype may be due to the inability of the transfected cell to incorporate viral nucleocapsids during virus budding. IMPORTANCE Paramyxovirus genomes are contained within a noncovalent homopolymer of its nucleoprotein (NP) and form helical nucleocapsids (NC) whose 3' ends contain the promoters for the initiation of viral RNA synthesis. This work suggests that these NC 3' ends may play another role in the virus life cycle via their specific interaction with virus-modified cell membranes needed for the incorporation of viral NCs into budding virions.

Human Parainfluenza Virus Type 2 V Protein Modulates Iron Homeostasis.

Intracellular iron concentration is tightly controlled for cell viability. It is known to affect the growth of several viruses, but the molecular mechanisms are not well understood. We found that iron chelators inhibit growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, infection with hPIV-2 alters ferritin localization from granules to a homogenous distribution within cytoplasm of iron-stimulated cells. The V protein of hPIV-2 interacts with ferritin heavy chain 1 (FTH1), a ferritin subunit. It also binds to nuclear receptor coactivator 4 (NCOA4), which mediates autophagic degradation of ferritin, so-called ferritinophagy. V protein consequently interferes with interaction between FTH1 and NCOA4. hPIV-2 growth is inhibited in FTH1 knockdown cell line where severe hPIV-2-induced apoptosis is shown. In contrast, NCOA4 knockdown results in the promotion of hPIV-2 growth and limited apoptosis. Our data collectively suggest that hPIV-2 V protein inhibits FTH1-NCOA4 interaction and subsequent ferritinophagy. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.IMPORTANCE hPIV-2 V protein interferes with interaction between FTH1 and NCOA4 and inhibits NCOA4-mediated ferritin degradation, leading to the inhibition of iron release to the cytoplasm. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.

Common and unique mechanisms of filamentous actin formation by viruses of the genus Orthorubulavirus.

We previously found that infection with human parainfluenza virus type 2 (hPIV-2), a member of the genus Orthorubulavirus, family Paramyxoviridae, causes filamentous actin (F-actin) formation to promote viral growth. In the present study, we investigated whether similar regulation of F-actin formation is observed in infections with other rubulaviruses, such as parainfluenza virus type 5 (PIV-5) and simian virus 41 (SV41). Infection with these viruses caused F-actin formation and RhoA activation, which promoted viral growth. These results indicate that RhoA-induced F-actin formation is important for efficient growth of these rubulaviruses. Only SV41 and hPIV-2 V and P proteins bound to Graf1, while the V and P proteins of PIV-5, mumps virus, and hPIV-4 did not bind to Graf1. In contrast, the V proteins of these rubulaviruses bound to both inactive RhoA and profilin 2. These results suggest that there are common and unique mechanisms involved in regulation of F-actin formation by members of the genus Orthorubulavirus.

The V protein of human parainfluenza virus type 2 promotes RhoA-induced filamentous actin formation.

We previously demonstrated that human parainfluenza virus type 2 (hPIV-2) induces RhoA activation, which promotes its growth. RhoA controls the equilibrium between globular and filamentous actin (F-actin). We found that F-actin formation is induced by wild type (wt) hPIV-2 infection, and that inhibition of F-actin formation by cytochalasin D decreases hPIV-2 growth. In wt RhoA-expressing cells, F-actin formation occurs and hPIV-2 growth is promoted. Overexpression of T19N RhoA, a dominant negative (DN) form of RhoA, inhibits hPIV-2-induced F-actin formation, and suppresses hPIV-2 growth. Immunoprecipitation assays reveal that hPIV-2 V protein binds only to DN RhoA, and this interaction requires its C-terminal Trp residues. F-actin formation is not observed during infection of recombinant hPIV-2 expressing Trp-mutated V protein (VW178H/W182E/W192A). Overexpression of V protein, but not that of VW178H/W182E/W192A, causes F-actin formation. Our results suggest that hPIV-2 V protein enhances hPIV2 growth through RhoA-induced F-actin formation, by selectively binding to inactive RhoA.

Evidence that Receptor Destruction by the Sendai Virus Hemagglutinin-Neuraminidase Protein Is Responsible for Homologous Interference.

UNLABELLED: Receptor destruction has been considered one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach to or penetrate the infected cells and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that the α2,3-linked sialic acids were specifically reduced in the SeV-infected cells, but the level of α2,6-linked sialic acids had not changed. As infection with IAV removed both α2,3- and α2,6-linked sialic acids, especially α2,3-linked sialic acids, IAV-infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, α2,3-linked sialic acids, is relevant to homologous interference by SeV. IMPORTANCE: Viral interference is a classically observed phenomenon, but the precise mechanism is not clear. Using SeV interference, we provide concrete evidence that reduction of the α2,3-linked sialic acid receptor by the HN of SeV is closely related with viral interference. Since SeV infection resulted in decrease of only α2,3-linked sialic acids, IAV, which also utilized α2,6-linked sialic acids to initiate infection, superinfected the SeV-infected cells. In contrast, SeV could not superinfect the IAV-infected cells because both α2,3- and α2,6-linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

Parainfluenza virus chimeric mini-replicons indicate a novel regulatory element in the leader promoter.

Gene expression of paramyxoviruses is regulated by genome-encoded cis-acting elements; however, whether all the required elements for viral growth have been identified is not clear. Using a mini-replicon system, it has been shown that human parainfluenza virus type 2 (hPIV2) polymerase can recognize the promoter elements of parainfluenza virus type 5 (PIV5), but reporter activity is lower in this case. We constructed a series of luciferase-encoding chimeric PIV2/5 mini-genomes that are basically hPIV2, but whose leader (le), mRNA start signal and trailer sequence are partially replaced with those of PIV5. Studies of the chimeric PIV2/5 mini-replicons demonstrated that replacement of hPIV2 le with PIV5 le results in remarkably weak luciferase expression. Further mutagenesis identified the responsible region as positions 25-30 of the PIV5 le. Using recombinant hPIV2, the impact of this region on viral life cycles was assessed. Insertion of the mutation at this region facilitated viral growth, genomic replication and mRNA transcription at the early stage of infection, which elicited severe cell damage. In contrast, at the late infection stage it caused a reduction in viral transcription. Here, we identify a novel cis-acting element in the internal region of an le sequence that is involved in the regulation of polymerase, and which contributes to maintaining a balance between viral growth and cytotoxicity.

Enhanced growth of influenza A virus by coinfection with human parainfluenza virus type 2.

It has been reported that dual or multiple viruses can coinfect epithelial cells of the respiratory tract. However, little has been reported on in vitro interactions of coinfected viruses. To explore how coinfection of different viruses affects their biological property, we examined growth of influenza A virus (IAV) and human parainfluenza virus type 2 (hPIV2) during coinfection of Vero cells. We found that IAV growth was enhanced by coinfection with hPIV2. The enhanced growth of IAV was not reproduced by coinfection with an hPIV2 mutant with reduced cell fusion activity, or by ectopic expression of the V protein of hPIV2. In contrast, induction of cell fusion by ectopic expression of the hPIV2 HN and F proteins augments IAV growth. hPIV2 coinfection supported IAV growth in cells originated from the respiratory epithelium. The enhancement correlated closely with cell fusion ability of hPIV2 in those cells. These results indicate that cell fusion induced by hPIV2 infection is beneficial to IAV replication and that enhanced viral replication by coinfection with different viruses can modify their pathological consequences.

Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys.

Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32 degrees C and exhibited little productive replication at 37 degrees C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates.

The conserved carboxyl terminus of human parainfluenza virus type 2 V protein plays an important role in virus growth.

Our previous results have shown that some residues of V protein-specific domain in human parainfluenza virus type 2 (hPIV2) are essential not only for STAT protein degradation but also for promoting virus growth. Here, we demonstrated that the virus growth of these recombinant hPIV2s (rPIV2) expressing mutated V proteins were improved in HeLa cell transiently expressing the wild-type V protein, but not in the cells constitutively expressing it. Consequently, we identified the region of the V protein that is essential for its oligomerization and for complex formation with NP protein. We also identified a host protein, AlP1/Alix, involved in apoptosis and efficient budding of several enveloped viruses as an interacting partner of the V and NP proteins. Depletion of AIP1/Alix by small interfering RNA suppressed virus growth. These data suggest that the conserved carboxyl terminus of the V protein plays an important role in virus growth.

Evaluation of R-Mix shell vials for the diagnosis of viral respiratory tract infections.

Respiratory viruses cause significant morbidity and mortality. The management of these infections can be improved by a rapid diagnosis and administration of available virus-specific therapy. The goal of this study was to compare R-Mix, an engineered tissue monolayer for rapid shell vial (SV) diagnosis of viral respiratory infections, with conventional tissue culture (TC) and conventional respiratory SV (primary rhesus monkey kidney (RhMK) and Hep2 monolayers). The primary outcome measure was sensitivity for detection of influenza A and B, respiratory syncytial virus, parainfluenza 1-3, and adenovirus. The study was performed in two phases: (1) the three methods were compared using 250 nasal washes from children with lower respiratory tract infections; (2) a modified R-Mix SV harvesting schedule (SV were harvested at 24 and 120 h) was compared with TC and conventional RhMK/Hep2 SV using 311 respiratory specimens. A total of 110 viruses were identified in the first and 55 in the second phase. Diagnostic accuracies of R-Mix harvested at 24, 48, and 120 h were 98%, whereas for TC varied between 99 and 100%, and for RhMK/Hep2 SV between 98 and 99%. Sensitivities of R-Mix harvested at 24, 48, and 120 h were 26, 75, and 47%, respectively, whereas for TC varied between 60 and 94%, and for RhMK/Hep2 SV between 62 and 85%. R-Mix harvested at 48 h represent a valuable substitute for RhMK/Hep2 SV because they have comparable sensitivities and diagnostic accuracies, but R-Mix offers several technical advantages. In contrast, R-Mix harvested at 24h did not seem a very useful diagnostic tool. The utility of R-Mix harvested at 120 h, which accelerated the diagnosis of 16% of positive specimens in study phase 2, needs further investigation.

Antiviral potency of mistletoe (Viscum album ssp. album) extracts against human parainfluenza virus type 2 in Vero cells.

Various extracts from the leaves of mistletoe (Viscum album L. ssp. album) were investigated for their antiviral activity on human parainfluenza virus type 2 (HPIV-2) growth in Vero cells. Plant extracts were prepared using distilled water, 50% ethanol, petroleum ether, chloroform and acetone. The 50% effective dose (ED(50)) of aqueous extract for HPIV-2 replication was 0.53 +/- 0.12 micro g/mL, and the antiviral index (AI), which was based on the ratio of the 50% inhibitory concentration (CD(50)) for host cell viability to the ED(50) for parainfluenza virus replication, was 10.05. The aqueous extract was found to be the most selective inhibitor. Furthermore, the aqueous extract at a concentration of 1 micro g/mL was found to inhibit HPIV-2 replication and the virus production was suppressed to more than 99% without any toxic effect on host cells. The chloroform extract was also found to be moderately active. In an effort to further analyse the mechanism of antiviral activity, the effectiveness of the aqueous extract on different steps of virus replication was examined. The antiviral activity could neither be attributed to the direct inactivation of the HPIV-2 nor to the inhibition of adsorption to Vero cells. The active aqueous extract has shown a dose-dependent antiviral activity on virus replication.

The genome length of human parainfluenza virus type 2 follows the rule of six, and recombinant viruses recovered from non-polyhexameric-length antigenomic cDNAs contain a biased distribution of correcting mutations.

Members of the Paramyxovirinae subfamily of the Paramyxoviridae family of viruses have the unusual requirement that the nucleotide length of the viral genome must be an even multiple of six in order for efficient RNA replication, and hence virus replication, to occur. Human parainfluenza virus type 2 (HPIV2) is the only member of the genus that has been reported to have a genome length that is not an even multiple of six, and it has also been recovered from a full-length antigenomic-sense cDNA that did not conform to the "rule of six." To reexamine the issue of nucleotide length in natural isolates of HPIV2, a complete consensus genomic sequence was determined for three HPIV2 strains: Greer, Vanderbilt/1994 (V94), and Vanderbilt/1998. Each of these strains was found to have a genome length of 15,654 nucleotides (nt), thus conforming in each case to the rule of six. To directly examine the requirement that the genomic length of HPIV2 be an even multiple of six, we constructed six full-length antigenomic HPIV2/V94 cDNAs that deviated from a polyhexameric length by 0 to 5 nt. Recombinant HPIV2s were readily recovered from all of the cDNAs, including those that did not conform to the rule of six. One recombinant HPIV2 isolate was completely sequenced for each of the nonpolyhexameric antigenomic cDNAs. These were found to contain small nucleotide insertions or deletions that conferred polyhexameric length to the recovered genome. Interestingly, almost all of the length corrections occurred within the hemagglutinin-neuraminidase and large polymerase genes or the intervening intergenic region and thus were proximal to the insert that caused the deviation from the rule of six. These results demonstrate, in the context of complete infectious virus, that HPIV2 has a strong and seemingly absolute requirement for a polyhexameric genome.

R-Mix cells are faster, at least as sensitive and marginally more costly than conventional cell lines for the detection of respiratory viruses.

OBJECTIVE: To evaluate shell vials of R-Mix, a combination of mink lung cells and human adenocarcinoma cells (strains Mv1Lu and A549, respectively, Diagnostic Hybrids, Athens, OH) to detect respiratory viruses from prospective clinical respiratory specimens and frozen stocks. STUDY DESIGN: We compared the performance of R-Mix to conventional culture (CC) using tubes of PMK, Hep-2, and MRC5 to detect respiratory viruses from fresh clinical specimens. For each respiratory specimen submitted to virology, two shell vials of R-Mix were inoculated and examined twice (generally after 24 and 48 h) by an indirect test with a pool of immunofluorescent antisera to influenza A and B, adenovirus, parainfluenza 1-3 and RSV (DAKO, Carpinteria, CA). If positive, testing with monoclonal antisera was done. CCs were incubated for 10 days, examined daily for cytopathological effect, hemadsorbed twice and stained if positive. Cost comparison was done. Lastly, respiratory viruses frozen from previous years were inoculated onto R-Mix. RESULTS: R-Mix was positive for all 29 frozen virus stocks. In the clinical trial, 396 prospective specimens were inoculated into R-Mix and CC. R-Mix identified 21 specimens as respiratory virus positive; CC identified 19. Turn-around time of R-Mix for positive specimens was 1.4 days; for CC it was 5.2 days. Turn-around time of R-Mix for all specimens (positive and negative) was 2.0 days; for CC it was 9.8 days. The overall cost of R-Mix was approximately 11% more than that of CC. CONCLUSION: R-Mix enabled rapid identification of all the frozen virus stocks representing the seven major respiratory viral groups. When compared to CC, R-Mix was slightly more sensitive than three cell lines (four tubes) used in CC but it was several days faster.

Enhancement of human parainfluenza virus-induced cell fusion by pradimicin, a low molecular weight mannose-binding antibiotic.

Oligosaccharides, especially mannose residues, expressed on the cell surface, are thought to be important for virus-induced membrane fusion. We examined the effect of mannose-binding compounds, pradimicin derivative BMY-28,864 (PRM) and concanavalin A (Con A), on cell fusion of human parainfluenza type 2 virus (hPIV2)-infected HeLa cells. Syncytium formation of hPIV2-infected HeLa cells was suppressed in the presence of Con A. On the other hand, PRM enhanced cell fusion of hPIV2-infected HeLa cells. These effects were blocked by addition of mannose-rich mannan. However, PRM shows little effect on virus growth and the expression of viral glycoproteins on the cell surface in hPIV2-infected HeLa cells. Fluorescein-isothiocyanate-labeled pradimicin and Con A bound to both uninfected and hPIV2-infected mononuclear cells, indicating that these compounds have an affinity to several cellular component(s). In contrast to Con A, PRM had little affinity to the viral glycoproteins. It is inferred from these results that the enhancement of hPIV2-induced cell fusion is probably due to the interaction between PRM and cellular component(s).

Growth properties and F protein cleavage site sequences of naturally occurring human parainfluenza type 2 viruses.

The growth properties of 24 clinical isolates of PIV-2 obtained from six independent areas in Japan were examined using Vero and primary monkey kidney cells. These viruses could be subdivided into three groups on the basis of the ability of syncytium formation on the two primate cell systems. The distinct correlation between the F protein cleavability and the fusogenic effect was observed in Vero cells, and the importance of consecutive basic residues in the F protein cleavage site for efficient cleavage was suggested by the sequence analyses of their F genes. On the other hand, in PMK cells, their fusogenic activities could not be directly attributed to the F cleavability, fusion peptide sequence, and replication efficiency, indicating that unidentified structural features play an important role in cytopathic activities of naturally occurring PIV-2s.

Incomplete replication of human parainfluenza virus type 2 in mouse L929 cells.

Human parainfluenza virus type 2 (HPIV-2) was tested for its ability to replicate in murine L929 cells. L929 cells were non-permissive for replication of HPIV-2. Interferon produced endogenously played no role in its incomplete replication. The mechanism by which growth of HPIV-2 was suppressed in L929 cells was studied. Synthesis of virus-specific polypeptides, particularly glycoprotein(s), was suppressed in HPIV-2-infected L929 cells. The HN mRNA could scarcely be detected in virus-infected L929 cells.

Comparative inhibition of influenza and parainfluenza virus replication by ribavirin in MDCK cells.

Actinomycin D inhibited the yield of influenza virus hemagglutinin from MDCK cells infected at high multiplicity, but had little effect on the yield of parainfluenza virus hemagglutinin. In similar hemagglutinin yield experiments, ribavirin was only slightly more active (threefold) against influenza virus than against parainfluenza virus replication. In plaque inhibition experiments, ribavirin depressed influenza virus plaque formation by 50% at a concentration of approximately 3 micrograms/ml, whereas the corresponding figure for parainfluenza viruses was threefold higher. The concentration of ribavirin demonstrating anti-influenza activity was indistinguishable for that inhibiting host cell growth. It is concluded that, unlike actinomycin D, ribavirin is unlikely to have a major effect on the provision of host cell 5-germinal methylated cap structures and the subsequent priming of influenza messenger ribonucleic acid synthesis.

Plaque formation by human-origin parainfluenza type 2 virus in established cell lines.

Two strains of parainfluenza type 2 virus formed well-defined plaques in cultures of Vero cells, an established line of African green monkey kidney cells. In the absence of trypsin, satisfactory plaques were formed by the Toshiba strain of virus. When trypsin was added in the overlay medium of Vero cell monolayers, another strain (62-M786) of virus produced plaques. The Toshiba strain was also able to make plaques in HeLa, BHK, and LLC-MK2 (an established line of monkey kidney) cells without trypsin, but not in mouse L cells. The sensitivity of plaque assay was about equal to that of the hemadsorption method.

Interferon production and virus replication in lymphoblastoid cells infected with different viruses.

A semicontinuous infection system was used to test viral replication and interferon induction in lymphoblastoid cells: measles virus, Newcastle disease virus (NDV), Sendai virus, human parainfluenza virus (type II and III), Semliki forest virus (SFV) and Vesicular stomatitis virus (VSV). With the exception of Sendai virus, all viruses replicated in the Namalva cell line. Only measles virus was able to induce high levels of interferon. Three other cell lines, NC37, Raji (TK+-variant) and Raji (TK--variant) were tested using measles virus as inducer. The interferon yields from these cells were inferior to those obtained from Namalva cells.