Viral and Atypical Bacterial Etiology of Acute Respiratory Infections in Children under 5 Years Old Living in a Rural Tropical Area of Madagascar

Background

In Madagascar, very little is known about the etiology and prevalence of acute respiratory infections (ARIs) in a rural tropical area. Recent data are needed to determine the viral and atypical bacterial etiologies in children with defined clinical manifestations of ARIs.

Methods

During one year, we conducted a prospective study on ARIs in children between 2 to 59 months in the community hospital of Ampasimanjeva, located in the south-east of Madagascar. Respiratory samples were analyzed by multiplex real-time RT-PCR, including 18 viruses and 2 atypical bacteria. The various episodes of ARI were grouped into four clinical manifestations with well-documented diagnosis: “Community Acquired Pneumonia”(CAP, group I), “Other acute lower respiratory infections (Other ALRIs, group II)”, “Upper respiratory tract infections with cough (URTIs with cough, group III)”and “Upper respiratory tract infections without cough (URTIs without cough, group IV)”.

Results

295 children were included in the study between February 2010 and February 2011. Viruses and/or atypical bacteria respiratory pathogens were detected in 74.6% of samples, the rate of co-infection was 27.3%. Human rhinovirus (HRV; 20.5%), metapneumovirus (HMPV A/B, 13.8%), coronaviruses (HCoV, 12.5%), parainfluenza virus (HPIV, 11.8%) and respiratory syncytial virus A and B (RSV A/B, 11.8%) were the most detected. HRV was predominantly single detected (23.8%) in all the clinical groups while HMPV A/B (23.9%) was mainly related to CAP (group I), HPIV (17.3%) to the “Other ALRIs” (group II), RSV A/B (19.5%) predominated in the group “URTIs with cough” (group III) and Adenovirus (HAdV, 17.8%) was mainly detected in the “without cough” (group IV).

Interpretation

This study describes for the first time the etiology of respiratory infections in febrile children under 5 years in a malaria rural area of Madagascar and highlights the role of respiratory viruses in a well clinically defined population of ARIs.     

Effects of human respiratory syncytial virus, metapneumovirus, parainfluenza virus 3 and influenza virus on CD4+ T cell activation by dendritic cells

BACKGROUND: Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells. METHODOLOGY, PRINCIPAL FINDINGS: We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro. IAV was included because symptomatic re-infection without antigenic change is less frequent, suggesting that immune protection is more complete and durable. We examined virus-specific memory responses and superantigen-induced responses by multiparameter flow cytometry. Live virus was more stimulatory than inactivated virus in inducing DC-mediated proliferation of virus-specific memory CD4+ T cells, suggesting a lack of strong suppression by live virus. There were trends of increasing proliferation in the order: HMPV相似文献   

A single intranasal inoculation with a paramyxovirus-vectored vaccine protects guinea pigs against a lethal-dose Ebola virus challenge

To determine whether intranasal inoculation with a paramyxovirus-vectored vaccine can induce protective immunity against Ebola virus (EV), recombinant human parainfluenza virus type 3 (HPIV3) was modified to express either the EV structural glycoprotein (GP) by itself (HPIV3/EboGP) or together with the EV nucleoprotein (NP) (HPIV3/EboGP-NP). Expression of EV GP by these recombinant viruses resulted in its efficient incorporation into virus particles and increased cytopathic effect in Vero cells. HPIV3/EboGP was 100-fold more efficiently neutralized by antibodies to EV than by antibodies to HPIV3. Guinea pigs infected with a single intranasal inoculation of 10(5.3) PFU of HPIV3/EboGP or HPIV3/EboGP-NP showed no apparent signs of disease yet developed a strong humoral response specific to the EV proteins. When these animals were challenged with an intraperitoneal injection of 10(3) PFU of EV, there were no outward signs of disease, no viremia or detectable EV antigen in the blood, and no evidence of infection in the spleen, liver, and lungs. In contrast, all of the control animals died or developed severe EV disease following challenge. The highly effective immunity achieved with a single vaccine dose suggests that intranasal immunization with live vectored vaccines based on recombinant respiratory viruses may be an advantageous approach to inducing protective responses against severe systemic infections, such as those caused by hemorrhagic fever agents.     

Inhibition of parainfluenza virus type 3 and Newcastle disease virus hemagglutinin-neuraminidase receptor binding: effect of receptor avidity and steric hindrance at the inhibitor binding sites

Zanamivir (4-guanidino-Neu5Ac2en [4-GU-DANA]) inhibits not only the neuraminidase activity but also the receptor interaction of the human parainfluenza virus type 3 (HPIV3) hemagglutinin-neuraminidase (HN), blocking receptor binding and subsequent fusion promotion. All activities of the HPIV3 variant ZM1 HN (T193I/I567V) are less sensitive to 4-GU-DANA's effects. The T193I mutation in HN confers both increased receptor binding and increased neuraminidase activity, as well as reduced sensitivities of both activities to 4-GU-DANA inhibition, consistent with a single site on the HN molecule carrying out both catalysis and binding. We now provide evidence that the HPIV3 variant's resistance to receptor-binding inhibition by 4-GU-DANA is related to a reduced affinity of the HN receptor-binding site for this compound as well as to an increase in the avidity of HN for the receptor. Newcastle disease virus (NDV) HN and HPIV3 HN respond differently to inhibition in ways that suggest a fundamental distinction between them. NDV HN-receptor binding is less sensitive than HPIV3 HN-receptor binding to 4-GU-DANA, while its neuraminidase activity is highly sensitive. Both HPIV3 and NDV HNs are sensitive to receptor-binding inhibition by the smaller molecule DANA. However, for NDV HN, some receptor binding cannot be inhibited. These data are consistent with the presence in NDV HN of a second receptor-binding site that is devoid of enzyme activity and has a negligible, if any, affinity for 4-GU-DANA. Avidity for the receptor contributes to resistance by allowing the receptor to compete effectively with inhibitors for interaction with HN, while the further determinant of resistance is the reduced binding of the inhibitor molecule to the binding pocket on HN. Based upon our data and recent three-dimensional structural information on the HPIV3 and NDV HNs, we propose mechanisms for the observed sensitivity and resistance of HN to receptor-binding inhibition and discuss the implications of these mechanisms for the distribution of HN functions.     

Human parainfluenza virus type 1 C proteins are nonessential proteins that inhibit the host interferon and apoptotic responses and are required for efficient replication in nonhuman primates

Recombinant human parainfluenza virus type 1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C', C, Y1, and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. Early during infection in vitro, rHPIV1-P(C-) replicated as efficiently as wild-type (wt) HPIV1, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect not observed with wt rHPIV1. rHPIV1-P(C-) infection, but not wt rHPIV1 infection, induced caspase 3 activation and nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to wt rHPIV1, rHPIV1-P(C-) and rHPIV1-C(F170S), a mutant encoding an F170S substitution in C, induced interferon (IFN) and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C-) induced apoptosis. Thus, the anti-IFN and antiapoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C-), whereas only the anti-IFN activity is disabled in rHPIV1-C(F170S). In African green monkeys (AGMs), rHPIV1-P(C-) was considerably more attenuated than rHPIV1-C(F170S), suggesting that disabling the anti-IFN and antiapoptotic activities of HPIV1 had additive effects on attenuation in vivo. Although rHPIV1-P(C-) protected against challenge with wt HPIV1, its highly restricted replication in AGMs and in primary human airway epithelial cell cultures suggests that it might be overattenuated for use as a vaccine. Thus, the C proteins of HPIV1 are nonessential but have anti-IFN and antiapoptosis activities required for virulence in primates.     

A chimeric human-bovine parainfluenza virus type 3 expressing measles virus hemagglutinin is attenuated for replication but is still immunogenic in rhesus monkeys

The chimeric recombinant virus rHPIV3-N(B), a version of human parainfluenza virus type 3 (HPIV3) that is attenuated due to the presence of the bovine PIV3 nucleocapsid (N) protein open reading frame (ORF) in place of the HPIV3 ORF, was modified to encode the measles virus hemagglutinin (HA) inserted as an additional, supernumerary gene between the HPIV3 P and M genes. This recombinant, designated rHPIV3-N(B)HA, replicated like its attenuated rHPIV3-N(B) parent virus in vitro and in the upper and lower respiratory tracts of rhesus monkeys, indicating that the insertion of the measles virus HA did not further attenuate rHPIV3-N(B) in vitro or in vivo. Monkeys immunized with rHPIV3-N(B)HA developed a vigorous immune response to both measles virus and HPIV3, with serum antibody titers to both measles virus (neutralizing antibody) and HPIV3 (hemagglutination inhibiting antibody) of over 1:500. An attenuated HPIV3 expressing a major protective antigen of measles virus provides a method for immunization against measles by the intranasal route, a route that has been shown with HPIV3 and respiratory syncytial virus vaccines to be relatively refractory to the neutralizing and immunosuppressive effects of maternally derived virus-specific serum antibodies. It should now be possible to induce a protective immune response against measles virus in 6-month-old infants, an age group that in developing areas of the world is not responsive to the current measles virus vaccine.     

Generation of recombinant human parainfluenza virus type 1 vaccine candidates by importation of temperature-sensitive and attenuating mutations from heterologous paramyxoviruses

Human parainfluenza virus type 1 (HPIV1) is a significant cause of respiratory tract disease in infants and young children for which a vaccine is needed. In the present study, we sought to attenuate HPIV1 by the importation of one or more known attenuating point mutations from heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations; (ii) the respiratory syncytial virus cpts530 with an attenuating mutation in the L polymerase protein; and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Recombinant HPIV1 (rHPIV1) mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature-sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild-type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates.     

Peripheral blood T cells response in human parainfluenza virus-associated lower respiratory tract infection in children

Human Parainfluenza virus (HPIV) causes lower respiratory tract infections (LRTI) mostly in young children. Respiratory viral infections may decline T cells in circulation and display enhanced pathogenicity. This study is aimed to analyze T cells alterations due to HPIV in children with LRTIs. Children (N = 152) with bronchitis or pneumonia, admitted in tertiary care hospitals were included in the study. Respiratory samples (throat or nasopharyngeal swabs) were taken and HPIV genotypes (1–4) were analyzed through RT-PCR. Peripheral blood T cells, CD3+, CD4+, CD8+, and CD19+, were analyzed in confirmed HPIV positive and healthy control group children through flow cytometry. The positivity rate of HPIV was 24.34% and the most prevalent genotype was HPIV-3 (20.40%). HPIV-1 and HPIV-2 were detected in 0.66% and 02% children respectively. The T lymphocyte counts were observed significantly reduced in children infected with HPIV-3. CD4+ cell (1580 ± 97.87) counts did not change significantly but the lowest CD8+ T cell counts (518.5 ± 74.00) were recorded. Similarly, CD3+ and CD19 cell ratios were also reduced. The CD4/CD8 ratio was significantly higher (3.12 ± 0.59) in the study population as compared to the control group (2.18 ± 0.654). Changes in the count of CD8+ T cells were more pronounced in patients with bronchiolitis and pneumonia. It is concluded that CD8+ T cells show a reduced response to HPIV-3 in children with severe LRTIs suggesting a strong association of these cells with disease severity.     

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.     

The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome

Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.     

Efficient isolation of human parainfluenza viruses 1 and 3 using MNT‐1, a human malignant melanoma cell line system that exhibits an apparent cytopathic effect

Isolation of human parainfluenza virus (HPIV) serotypes 1 and 3 from clinical specimens is not very efficient because of the lack of a cell culture system capable of inducing CPE. In this study, the utility of a melanoma cell line, MNT‐1, that allows HPIV growth and displays CPE was demonstrated. In particularly, the efficiency of isolating HPIV1 and HPIV3 using MNT‐1 was greater than for cell lines conventionally used for HPIV isolation. Our demonstrated efficacy of HPIV1 and HPIV3 isolation with apparent CPE using the MNT‐1 cell culture system has the potential to improve virus isolation from clinical specimens.     

Natural killer cells regulate T-cell proliferation during human parainfluenza virus type 3 infection

Human parainfluenza virus type 3 (HPIV3) is a major respiratory pathogen in humans. Failure to induce immunological memory associated with HPIV3 infection has been attributed to inhibition of lymphocyte proliferation. We demonstrate that the inability of mixed lymphocytes to respond to virally infected antigen-presenting cells is due to an interleukin-2-dependent, nonapoptotic mechanism involving natural killer (NK) cells and their influence is exerted in a contact-dependent manner. These results suggest a novel regulatory mechanism for NK cells during HPIV3 infection, offering an explanation for viral persistence and poor memory responses.     

Lentiviral vectors pseudotyped with envelope glycoproteins derived from human parainfluenza virus type 3

We describe the generation of lentiviruses pseudotyped with human parainfluenza type 3 envelope (HPIV3) glycoproteins. Lentivirus particles, expressed in 293T/17 cells, incorporate HPIV3 hemagglutinin-neuraminidase (HN) and fusion (F) proteins into their lipid bilayers and are able to transduce human kidney epithelial cells and polarized MDCK cells. Neuraminidase, AZT, and anti-HPIV3 antisera block transduction, which is consistent with lentiviral-mediated transduction via sialated receptors for HPIV3. Our findings show that HPIV3 pseudotyped lentiviruses can be formed and may have a number of useful properties for human gene transfer.     

A recombinant human parainfluenza virus type 3 (PIV3) in which the nucleocapsid N protein has been replaced by that of bovine PIV3 is attenuated in primates

The shipping fever (SF) and Kansas (Ka) strains of bovine parainfluenza virus type 3 (BPIV3) are restricted in their replication in rhesus monkeys 100- to 1,000-fold compared to human parainfluenza virus type 3 (HPIV3), and the Ka strain also was shown to be attenuated in humans. To initiate an investigation of the genetic basis of the attenuation of BPIV3 in primates, we produced viable chimeric HPIV3 recombinants containing the nucleoprotein (N) open reading frame (ORF) from either BPIV3 Ka or SF in place of the HPIV3 N ORF. These chimeric recombinants were designated cKa-N and cSF-N, respectively. Remarkably, cKa-N and cSF-N grew to titers comparable to those of their HPIV3 and BPIV3 parents in LLC-MK2 monkey kidney and Madin-Darby bovine kidney cells. Thus, the heterologous nature of the N protein did not impede replication in vitro. However, cKa-N and cSF-N were each restricted in replication in rhesus monkeys to a similar extent as Ka and SF, respectively. This identified the BPIV3 N protein as a determinant of the host range restriction of BPIV3 in primates. These chimeras thus combine the antigenic determinants of HPIV3 with the host range restriction and attenuation phenotype of BPIV3. Despite their restricted replication in rhesus monkeys, the chimeric viruses induced a level of resistance to HPIV3 challenge in these animals which was indistinguishable from that conferred by immunization with HPIV3. The infectivity, attenuation, and immunogenicity of these BPIV3/HPIV3 chimeras suggest that the modified Jennerian approach described in the present report represents a novel method to design vaccines to protect against HPIV3-induced disease in humans.     

Nucleotide sequence of the bovine parainfluenza 3 virus genome: the genes of the F and HN glycoproteins.

By analysing complementary DNA clones constructed from genomic RNA of bovine parainfluenza 3 virus (BPIV3), we determined the nucleotide sequence of the region containing the entire F and HN genes. Their deduced amino acid sequences showed about 80% homologies with those of human parainfluenza 3 virus (HPIV3), about 45% with those of Sendai virus, and about 20% with those of SV5 and Newcastle disease virus (NDV), indicating, together with the results described in the preceding paper on the NP, P, C and M proteins of BPIV3, that BPIV3, HPIV3 and Sendai virus constitute a paramyxovirus subgroup, and that BPIV3 and HPIV3 are very closely related. The F and HN proteins of all these viruses, including SV5 and NDV, however, were shown to have protein-specific structures as well as short but well-conserved amino acid sequences, suggesting that these structures and sequences are related to the activities of these glycoproteins.     

Bovine parainfluenza virus type 3 (BPIV3) fusion and hemagglutinin-neuraminidase glycoproteins make an important contribution to the restricted replication of BPIV3 in primates

This study examines the contribution of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoprotein genes of bovine parainfluenza virus type 3 (BPIV3) to its restricted replication in the respiratory tract of nonhuman primates. A chimeric recombinant human parainfluenza type 3 virus (HPIV3) containing BPIV3 F and HN glycoprotein genes in place of its own and the reciprocal recombinant consisting of BPIV3 bearing the HPIV3 F and HN genes (rBPIV3-F(H)HN(H)) were generated to assess the effect of glycoprotein substitution on replication of HPIV3 and BPIV3 in the upper and lower respiratory tract of rhesus monkeys. The chimeric viruses were readily recovered and replicated in simian LLC-MK2 cells to a level comparable to that of their parental viruses, suggesting that the heterologous glycoproteins were compatible with the PIV3 internal proteins. HPIV3 bearing the BPIV3 F and HN genes was restricted in replication in rhesus monkeys to a level similar to that of its BPIV3 parent virus, indicating that the glycoprotein genes of BPIV3 are major determinants of its host range restriction of replication in rhesus monkeys. rBPIV3-F(H)HN(H) replicated in rhesus monkeys to a level intermediate between that of HPIV3 and BPIV3. This observation indicates that the F and HN genes make a significant contribution to the overall attenuation of BPIV3 for rhesus monkeys. Furthermore, it shows that BPIV3 sequences outside the F and HN region also contribute to the attenuation phenotype in primates, a finding consistent with the previous demonstration that the nucleoprotein coding sequence of BPIV3 is a determinant of its attenuation for primates. Despite its restricted replication in the respiratory tract of rhesus monkeys, rBPIV3-F(H)HN(H) conferred a level of protection against challenge with HPIV3 that was indistinguishable from that induced by previous infection with wild-type HPIV3. The usefulness of rBPIV3-F(H)HN(H) as a vaccine candidate against HPIV3 and as a vector for other viral antigens is discussed.