Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells.

IFNs, produced during viral infections, induce the expression of hundreds of IFN-stimulated genes (ISGs). Some ISGs have specific antiviral activity, whereas others regulate the cellular response. Besides functioning as an antiviral effector, ISG15 is a negative regulator of IFN signaling, and inherited ISG15 deficiency leads to autoinflammatory IFNopathies, in which individuals exhibit elevated ISG expression in the absence of pathogenic infection. We have recapitulated these effects in cultured human A549-ISG15-/- cells and (using A549-UBA7-/- cells) confirmed that posttranslational modification by ISG15 (ISGylation) is not required for regulation of the type I IFN response. ISG15-deficient cells pretreated with IFN-α were resistant to paramyxovirus infection. We also showed that IFN-α treatment of ISG15-deficient cells led to significant inhibition of global protein synthesis, leading us to ask whether resistance was due to the direct antiviral activity of ISGs or whether cells were nonpermissive because of translation defects. We took advantage of the knowledge that IFN-induced protein with tetratricopeptide repeats 1 (IFIT1) is the principal antiviral ISG for parainfluenza virus 5. Knockdown of IFIT1 restored parainfluenza virus 5 infection in IFN-α-pretreated, ISG15-deficient cells, confirming that resistance was due to the direct antiviral activity of the IFN response. However, resistance could be induced if cells were pretreated with IFN-α for longer times, presumably because of inhibition of protein synthesis. These data show that the cause of virus resistance is 2-fold; ISG15 deficiency leads to the early overexpression of specific antiviral ISGs, but the later response is dominated by an unanticipated, ISG15-dependent loss of translational control.

Structure-based design of a quadrivalent fusion glycoprotein vaccine for human parainfluenza virus types 1-4.

Parainfluenza virus types 1-4 (PIV1-4) are highly infectious human pathogens, of which PIV3 is most commonly responsible for severe respiratory illness in newborns, elderly, and immunocompromised individuals. To obtain a vaccine effective against all four PIV types, we engineered mutations in each of the four PIV fusion (F) glycoproteins to stabilize their metastable prefusion states, as such stabilization had previously enabled the elicitation of high-titer neutralizing antibodies against the related respiratory syncytial virus. A cryoelectron microscopy structure of an engineered PIV3 F prefusion-stabilized trimer, bound to the prefusion-specific antibody PIA174, revealed atomic-level details for how introduced mutations improved stability as well as how a single PIA174 antibody recognized the trimeric apex of prefusion PIV3 F. Nine combinations of six newly identified disulfides and two cavity-filling mutations stabilized the prefusion PIV3 F immunogens and induced 200- to 500-fold higher neutralizing titers in mice than were elicited by PIV3 F in the postfusion conformation. For PIV1, PIV2, and PIV4, we also obtained stabilized prefusion Fs, for which prefusion versus postfusion titers were 2- to 20-fold higher. Elicited murine responses were PIV type-specific, with little cross-neutralization of other PIVs. In nonhuman primates (NHPs), quadrivalent immunization with prefusion-stabilized Fs from PIV1-4 consistently induced potent neutralizing responses against all four PIVs. For PIV3, the average elicited NHP titer from the quadrivalent immunization was more than fivefold higher than any titer observed in a cohort of over 100 human adults, highlighting the ability of a prefusion-stabilized immunogen to elicit especially potent neutralization.

Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice.

We previously reported that Ag85B-expressing human parainfluenza type 2 virus (Ag85B-rHPIV2) was effective as a nasal vaccine against tuberculosis in mice; however, the mechanism by which it induces an immune response remains to be investigated. In the present study, we found that organogenesis of inducible bronchus-associated lymphoid tissue (iBALT) played a role in the induction of antigen-specific T cells and IgA antibody responses in the lung of mice intra-nasally administered Ag85B-rHPIV2. We found that expression of Ag85B was dispensable for the development of iBALT, suggesting that HPIV2 acted as an iBALT-inducing vector. When iBALT organogenesis was disrupted in Ag85B-rHPIV2-immunized mice, either by neutralization of the lymphotoxin pathway or depletion of CD11b+ cells, Ag85B-specific immune responses (i.e. IFN γ-producing T cells and IgA antibody) were diminished in the lung. Furthermore, we found that immunization with Ag85B-rHPIV2 induced neutrophil and eosinophil infiltration temporally after the immunization in the lung. Thus, our results show that iBALT organogenesis contributes to the induction of antigen-specific immune responses by Ag85B-rHPIV2 and that Ag85B-rHPIV2 provokes its immune responses without inducing long-lasting inflammation.

Genotype replacement of the human parainfluenza virus type 2 in Croatia between 2011 and 2017 - the role of neutralising antibodies.

Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.

Human parainfluenza virus type 2 V protein inhibits induction of tetherin.

Tetherin is an anti-viral factor that restricts viral budding through tethering virions to the cell surface. The human parainfluenza virus type 2 (hPIV-2) V protein decreases cell surface tetherin in HeLa cells, which constitutively express endogenous tetherin. However, the role of the hPIV-2 V protein in tetherin induction remains unclear. Here, we examined whether hPIV-2 infection itself induces tetherin in HEK293 cells that have no basal expression of tetherin. Unlike influenza A virus (IAV) infection, hPIV-2 infection induced neither tetherin mRNA nor protein expression. In contrast, robust tetherin induction was observed by infection of rPIV-2s carrying V mutants, in which either three Trp residues (W178H/W182E/W192A) or Cys residues (C209/211/214A) that are important for decreasing cell surface tetherin are mutated. hPIV-2 infection also inhibited the induction of tetherin expression by IFN-α and IAV infection. Furthermore, hPIV-2 V protein but not P and VW178H/W182E/W192A suppressed tetherin induction. Our data collectively suggest that the hPIV-2 V protein inhibits tetherin expression induced by several external stimuli.

Synthesis of human parainfluenza virus 4 nucleocapsid-like particles in yeast and their use for detection of virus-specific antibodies in human serum.

The aim of this study was to produce human parainfluenza virus type 4 (HPIV4) nucleocapsid (N) protein in yeast Saccharomyces cerevisiae expression system, to explore its structural and antigenic properties and to evaluate its applicability in serology. The use of an optimized gene encoding HPIV4 N protein amino acid (aa) sequence GenBank AGU90031.1 allowed high yield of recombinant N protein forming nucleocapsid-like particles (NLPs) in yeast. A substitution L332D disrupted self-assembly of NLPs, confirming the role of this position in the N proteins of Paramyxovirinae. Three monoclonal antibodies (MAbs) were generated against the NLP-forming HPIV4 N protein. They recognised HPIV4-infected cells, demonstrating the antigenic similarity between the recombinant and virus-derived N proteins. HPIV4 N protein was used as a coating antigen in an indirect IgG ELISA with serum specimens of 154 patients with respiratory tract infection. The same serum specimens were tested with previously generated N protein of a closely related HPIV2, another representative of genus Rubulavirus. Competitive ELISA was developed using related yeast-produced viral antigens to deplete the cross-reactive serum antibodies. In the ELISA either without or with competition using heterologous HPIV (2 or 4) N or mumps virus N proteins, the seroprevalence of HPIV4 N-specific IgG was, respectively, 46.8, 39.6 and 40.3% and the seroprevalence of HPIV2 N-specific IgG-47.4, 39.0 and 37.7%. In conclusion, yeast-produced HPIV4 N protein shares structural and antigenic properties of the native virus nucleocapsids. Yeast-produced HPIV4 and HPIV2 NLPs are prospective tools in serology.

Current status of vaccines for parainfluenza virus infections.

Parainfluenza viruses (PIV) have been generally disregarded as pathogens in spite of their importance in pediatric lower respiratory illness. Because PIVs account for 17% of hospitalized illness associated virus isolation, the development of PIV vaccine would be a major advance in preventing lower respiratory tract infection in infants and young children. We will review in detail several PIV vaccine candidates and recent newer approaches to PIV vaccine development. Intranasally administered bovine PIV3 (bPIV3) vaccine and cold-adapted PIV3 vaccine have been evaluated throughout the pediatric age spectrum. BPIV3 does not give a robust response to the heterotypic human strain although seroconversion rate to bPIV3 is 57-65%. However, bPIV3 vaccine is being used as an attenuated backbone for insertion of human PIV3 hemagglutinin-neuraminidase and fusion (F) proteins and a surface protein, F, of respiratory syncytial virus. The effectiveness of this vaccine against both PIV3 and RSV challenge has been demonstrated in African green monkeys. The cold-adapted PIV3 vaccine has been extensively evaluated and is safe and immunogenic in seronegative children with a seroconversion rate of 79%. These promising candidates deserve to enter into efficacy trials both for their ability to prevent PIV3 disease and as a model of protection against respiratory illness by mucosal vaccination.

Demonstration of 1-year duration of immunity for attenuated Bordetella bronchiseptica vaccines in dogs.

Three groups of healthy dogs with low antibody titers to Bordetella bronchiseptica (Bb), canine parainfluenza virus (CPI), and canine adenovirus type 2 (CAV-2) were used in this study. One group was vaccinated with a single dose of monovalent attenuated Bb vaccine and one group with a trivalent vaccine containing attenuated Bb, CPI, and CAV-2; dogs were vaccinated intranasally with a single dose of the respective vaccines. The third group served as unvaccinated controls. All vaccinated dogs subsequently developed serum antibody titers to Bb that persisted for at least 1 year. Following Bb challenge 1 year after vaccination, all vaccinated dogs, regardless of group, showed significantly fewer clinical signs and shed significantly fewer challenge organisms than unvaccinated controls. These results demonstrate that intranasal administration of a single dose of monovalent attenuated Bb vaccine or trivalent vaccine containing attenuated Bb, CPI, and CAV-2 provides 1 year of protection against Bb.

An improved method for the recovery of recombinant paramyxovirus vaccine candidates suitable for use in human clinical trials.

We describe a method for the generation of clinical grade, live-attenuated vaccines in Vero cells entirely from cDNA plasmids. The entire electroporation procedure can be completed in less than 15 minutes and this is a significant improvement over previous lipid or electroporation based transfection techniques that also involve a heat-shock step. Importantly, the virus preparations can be generated with a minimal use of animal product derived materials, an important consideration for a vaccine candidate that is to be tested in humans. Since it is likely that all live-attenuated parainfluenza virus and pneumovirus vaccines in the future will be generated using reverse genetics, this simplified method provides guidance on how this can be achieved.

Exposure to human respiratory viruses among urban performing monkeys in Indonesia.

Performing monkeys, a common phenomena in Asia, occupy a unique urban niche that comprises a number of factors influencing the likelihood of cross-species transmission of pathogens. Here we present the first documented evidence of exposure to measles, rubella, and parainfluenza in a population of performing monkeys. Evidence of exposure to these endemic human respiratory viruses in the performing monkeys confirms human-to-primate transmission and suggests the possibility of primate-to-human transmission. Urban animal markets, the likely source of these performing monkeys, may represent an environment conducive to the mixing of animals and pathogens, making these monkeys a potential conduit for infectious agents passing from a variety of animals found in animal markets to humans. The potential significance of these results to human public health and the unique contexts of disease transmission associated with the urban ecology of the performance monkeys are discussed. Given the level of overseas travel, this potential threat is not confined solely to Asia.

Distinct hemagglutinin and neuraminidase epitopes involved in antigenic variation of recent human parainfluenza virus type 2 isolates.

A panel of fourteen neutralizing anti-HN monoclonal antibodies (mAbs) to the prototype Greer strain of human parainfluenza virus type 2 (PI2) was used to determine the extent of antigenic variation in recent virus isolates. Competitive binding analysis with the mAbs indicated the presence of at least five distinct antigenic sites (I to V) on the HN glycoprotein molecule. MAbs recognizing different antigenic sites were found to be associated with the hemagglutinin (sites I, IV and V), hemagglutinin and neuraminidase (site II), or neuraminidase (site III) activities. The location of two distinct epitopes identifying the neuraminidase sites (II and III) was further verified from the generation of escape mutants. Antibodies directed to sites I and III failed to show any detectable binding or neutralizing activity against a number of natural PI2 virus isolates collected in Texas between 1986 and 1987. Interestingly, these natural variants, unlike the prototype virus, did not show any detectable neuraminidase activity with fetuin as a substrate and the enzyme activity was only detected with N-acetylneuramin-lactose as an alternative substrate. Despite the observed variation in the antigenic sites, primary infection with the prototype virus or the natural variants generated a protective immune response against challenge infection with the other virus strains.

The value of virus serology in epidemiological studies of acute otitis media in children.

BACKGROUND: Acute otitis media (AOM) is a major health problem in young children. There is a general conception that AOM is a bacterial disease but with the availability of sensitive diagnostic methods, it has gradually become evident that viruses play an important role in the pathogenesis of AOM. Paired blood samples are seldom taken from infants although valuable information could be obtained by serological methods. During the recent Finnish Otitis Media (FinOM) Cohort Study, in addition to nasopharyngeal aspirates (NPA) and middle ear fluids (MEF), paired acute and convalescent serum samples were collected from children with AOM. OBJECTIVES: To establish the diagnostic value of serological methods in etiological and epidemiological studies of AOM. STUDY DESIGN: A complete set of NPA, MEF, and paired sera was collected during 447 events of AOM experienced by 179 children between 2 months and 2 years of age. Antigens of respiratory syncytial virus (RSV), adenoviruses, influenza A and B, and parainfluenza types 1-3 in NPAs and MEFs were detected by time-resolved fluoroimmunoassay (TR-FIA), and antibody titers were determined by complement fixation test (CFT) or by enzyme immunoassay. RESULTS: A total of 163 virus-positive events were identified. Of those, only 34 were positive by TR-FIA and by serology. From 48 events a positive result was obtained only by TR-FIA and from 81 only by serology. CONCLUSION: Although serological methods are usually of little use in clinical practice, epidemiological studies clearly gain value if serology is included. The number of virus-positive findings dramatically increased by including serological tests in the diagnostic work-up of these AOM events.

Respiratory virus antibodies in adults of a Norwegian community: prevalences and risk factors.

BACKGROUND: The aims were to examine prevalences as well as demographic and environmental predictors of respiratory virus antibodies in serum. METHODS: In a cross-sectional study of 18-73 year old Norwegian adults a random stratified sample (n = 1512) was invited to attend an examination at an outpatient clinic. Seven respiratory virus antibodies were assessed by the complement fixation test. RESULTS: The attendance rate was 84%. The most frequent virus antibodies with titre of > or = 1:8 were influenza virus type A with a population standardized prevalence of 44%, adenovirus 25% and influenza virus type B 22%. The prevalences of antibodies against parainfluenza virus type 1, 2 and 3 increased with age. Smokers compared to non-smokers had an adjusted odds ratio (OR) of 1.7 (95% confidence interval [CI]: 1.3-2.4) for having one or more of the seven examined virus antibodies. The presence of one or more of the virus antibodies increased from summer to winter months (adjusted OR = 1.3 per month; 95% CI: 1.2-1.4) and it was higher in occupational dust or gas exposed smokers (adjusted OR = 2.0; 95% CI: 1.1-3.7) compared with unexposed smokers. CONCLUSIONS: Ageing, smoking, occupational dust or gas exposure as well as season of the year may thus be predictors for levels of respiratory virus antibodies in adults. These observations should be taken into account when comparing prevalences of virus antibodies in various communities as well as when examining the relationship between presence of virus antibodies and airway disease.

Rapid diagnosis of respiratory virus infections in patients with acute respiratory disease.

Viral respiratory infections represent a significant segment of the total respiratory disease spectrum; however, until recently the laboratory diagnosis of viral respiratory infections was relatively inefficient. Development of new and improved immunologic assay systems has paved the way for accurate and reliable rapid diagnostic tests that detect viral antigens in clinical specimens. We conducted a careful and elaborate study in which radioimmunoassay for antigen detection was compared with a battery of tissue culture systems for viral isolation and identification. Using a fine plastic catheter, a specimen of mucus was aspirated from the nasopharynx of patients with clinical signs and symptoms of acute viral upper respiratory tract infections. Each specimen was divided into two portions; one was used to inoculate a variety of tissue culture cell lines and the other was used for radioimmunoassay tests for influenza A and B, adenovirus, parainfluenza 1, 2, and 3, and respiratory syncytial virus. Radioimmunoassay results compared very favorably with the tissue culture data with only one exception--adenovirus. Essentially this degree of accuracy and reproducibility was obtained with an enzyme-linked immunosorbent assay test, which has replaced radioimmunoassay. Tissue cultures are still used for backup, but with a rapid antigen detection system in place, coupled with a modern computer program to facilitate the laboratory data to the clinician, considerable strides have been made, and will continue to be made, in the diagnosis and therapy of viral respiratory tract infections.

Enzyme immunoassays for detection of IgG and IgM antibodies to parainfluenza types 1, 2 and 3.

Quantitative enzyme immunoassays for parainfluenza type 1, 2 and 3 IgG antibodies were developed. Serum specimens were tested at a single dilution of 1:1000 and results expressed in units by the use of a standard curve. The unit values correlated well with titres obtained by testing the same specimens in serial dilutions. All serum pairs with significant titre rises also showed significant rises in unit values. Parainfluenza IgG and IgM serology was evaluated in 66 patients with a proven parainfluenza infection. Diagnostic IgG antibody increases were detected in 70, 69 and 87% of parainfluenza type 1, 2 and 3 infections, respectively. Heterologous titre rises between parainfluenza types 1 and 3 were common. IgM antibodies were detected in 42% of the patients, most commonly in those below two years of age and rarely in adults.

Avidity of IgG antibodies against mumps, parainfluenza 2 and Newcastle disease viruses after mumps infection.

Paired serum samples from 39 patients with recent mumps infection were assayed for IgG antibodies against mumps, parainfluenza 2 and Newcastle disease virus (NDV). A modified enzyme immunoassay was used, giving separate estimates of high avidity antibodies (EHAA) and total specific antibodies (ETSA). A marked cross-reaction was seen between mumps and parainfluenza 2 virus, with changes of ETSA between paired samples of about the same magnitude against both these viruses. The mean change of EHAA against mumps was, however, significantly greater than that against parainfluenza 2. There were 16 patients who had a change of ETSA greater against parainfluenza 2 than against mumps. When the EHAA responses were compared, there were only 8 such patients. The responses against NDV were negligible. Estimation of antibody avidity, even by the arbitrary method used, can distinguish between homotypic and cross-reactive heterotypic antibodies after mumps infection. The implications for expressing the results of enzyme immunoassay are discussed.

Elevated parainfluenza virus type 1 antibody in patients with subacute sclerosing panencephalitis.

Parainfluenza virus hemagglutination inhibition (HI) antibodies were determined 3 times in the sera of 9 patients with subacute sclerosiing panencephalitis (SSPE) and 20 healthy controls matched for age and place of residence. Serum antibody against parainfluenza virus type 1 was significantly elevated in SSPE patients as compared with controls, whereas antibodies against type 2 and 3 were found to be in normal ranges. Higher titres of parainfluenza virus type 1 antibody might depend on: (1) dual viral infection, (2) cross-reaction between antigens of SSPE virus and parainfluenza virus type 1, and (3) non-specific activation of latent virus type 1 genome. The latter explanation seems to be particularly interesting since the parainfluenza type 1 antibody titres remained constant despite the clinical progression. This finding is comparable to the elevated titres against Epstein-Barr virus of adenovirus which have been found occasionally in this disease.

[Detection of antibodies against infectious viral laryngotracheitis and parainfluenza 2 in dogs bred in Czechoslovakia]. / Dôkaz protilátok proti vírusom infekcnej laryngotracheitídy a parainfluenzy 2 u psov v chovov na území CSSR.

A total of 398 blood serums of dogs of various breeds and age categories, coming from 72 places in Bohemia and Slovakia, were examined for the content of haemagglutination-inhibiting (HI) antibodies to the infectious laryngotracheitis virus (CADV-2) and parainfluenza 2 virus (CPIV-2). Out of this total number, 203 serums (51.1%) reacted against CADV-2 in titres from 1:16 to 1:2048 and 115 serums (28.9%) against CPIV-2 in titres from 1:2 to 1:256. The results indicate that the dog population is considerably infected with viruses affecting the respiratory organs.

Development of hypertrophic osteodystrophy and antibody response in a litter of vaccinated Weimaraner puppies.

Two different vaccination protocols were compared with regard to the development of hypertrophic osteodystrophy (HOD) (also termed metaphyseal osteopathy) and effectiveness of immunisation in a litter of 10 Weimaraner puppies. Five puppies (group 1) were vaccinated with a modified live canine parvovirus vaccine (CPV) and then two weeks later with a trivalent vaccine containing modified live canine distemper virus and adenovirus type 2 combined with a Leptospira bacterin (DHL). The CPV and DHL vaccine protocols were administered a further two times, at two-week intervals. Group 2 was vaccinated with three consecutive multivalent vaccines containing modified live canine distemper virus, canine parvovirus, parainfluenza and adenovirus type 2 combined with a Leptospira bacterin, at four-week intervals. All puppies were first vaccinated at the age of eight weeks. Three dogs in group 1 developed HOD, while all five dogs in group 2 developed HOD during the study period. Dogs in group 2 had more episodes of HOD than those in group 1. Dogs in group 1 developed higher antibody titres to canine distemper virus and parvovirus compared with those in group 2. Only two out of the 10 dogs developed protective antibody titres to parvovirus. The results of this study suggest that the two different vaccination protocols affected the pattern of appearance of HOD and immunisation in this litter of Weimaraner puppies. The results obtained and the previously reported data suggest that a larger controlled study is needed to further elucidate the effect of different vaccination protocols on HOD and immunisation in Weimaraner puppies.