Development of A recombinant nucleocapsid based indirect ELISA for the detection of antibodies to avian metapneumovirus subtypes, A, B, and C.

Nucleocapsid (N) protein is the most highly expressed of all avian metapneumovirus (aMPV) viral proteins and stimulates a substantial immune response in infected animals. Codon optimized recombinant N (rec-N) protein from aMPV subtypes A, B, and C were expressed using the baculoviral expression system in Trichoplusia ni (Tni) insect cells. A mixture of purified rec-N antigens from each subtype was used as a coating antigen and was evaluated in indirect ELISA (iELISA) to assess antibody response in serum samples collected from experimentally infected chickens and turkeys with different aMPV subtypes. Also, archived field serum samples that were collected from different poultry submissions were used. Receiver operating characteristic (ROC) analysis was performed using chicken and turkey serum samples that were confirmed by indirect fluorescent antibody (IFA) test for serostatus (positive n = 270, negative n = 610). The ROC analysis showed sensitivity and specificity of 97 % at a cut-off value of 0.25. The rec-N iELISA was compared with a commercial whole virus-based APV kit. The rec-N iELISA showed comparable results in detecting antibody response in aMPV infected chicken sera but was more sensitive in detecting early antibody response in aMPV infected turkey serum samples. Our results further confirm the presence of aMPV antibodies in Canadian domestic poultry populations. The developed aMPV-rec N iELISA offers a safe and valuable alternative to whole virus-based iELISA for serodiagnosis and seroepidemiological surveillance of the disease in domestic poultry.

Seroreactive Profiling of Filoviruses in Chinese Bats Reveals Extensive Infection of Diverse Viruses.

Southern China is a hot spot of emerging infectious diseases, in which diverse species of bats dwell, a large group of flying mammals considered natural reservoirs for zoonotic viruses. Recently, divergent filoviruses (FiVs) have been identified in bats within this region, which pose a potential risk to public health, but the true infection situation in bats remains largely unclear. Here, 689 archived bat serum samples were analyzed by enzyme-linked immunosorbent assay (ELISA), Western blotting, and neutralization assay to investigate the seroprevalence and cross-reactivity of four divergent FiVs and two other viruses (rabies virus and Tuhoko pararubulavirus 1) of different families within the order Mononegavirales Results showed no cross-antigenicity between FiVs and other mononegaviruses but different cross-reactivity among the FiVs themselves. The total FiV seroreactive rate was 36.3% (250/689), with infection by the indigenous Chinese FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Further viral metagenomic analysis of fruit bat tissues also identified the gene sequence of a novel FiV. These results indicate the likely prevalence of other so far unidentified FiVs within the Chinese bat population, with frugivorous Rousettus leschenaultii and Eonycteris spelaea bats and insectivorous Myotis horsfieldii and Miniopterus schreibersii bats being their major reservoirs.IMPORTANCE Bats are natural hosts of many FiVs, from which diverse FiVs were serologically or virologically detected in Africa, Europe, and East Asia. Recently, very divergent FiVs were identified in the Chinese bat population, but their antigenic relationship with other known FiVs remains unknown. Here, we conducted serological characterization and investigation of Chinese indigenous FiVs and prototypes of other viruses in bats. Results indicated that Chinese indigenous FiVs are antigenically distant to other FiVs, and infection of novel or multiple FiVs occurred in Chinese bats, with FiV DH04 or an antigenically related one being the most widely and the most highly prevalent. Additionally, besides Rousettus leschenaultii and Eonycteris spelaea bats, the insectivorous Myotis horsfieldii and M. schreibersii bats are highly preferential hosts of FiVs. Seroreactive and viral metagenomic results indicated that more as yet unknown bat-borne FiVs circulate in Southern China, and to uncover them further, investigation and timely surveillance is needed.

Risk Factors for Parainfluenza Virus Lower Respiratory Tract Disease after Hematopoietic Cell Transplantation.

Parainfluenza virus (PIV) infection can progress from upper respiratory tract infection (URTI) to lower respiratory tract disease (LRTD) in immunocompromised hosts. Risk factors for progression to LRTD and presentation with LRTD without prior URTI are poorly defined. Hematopoietic cell transplant (HCT) recipients with PIV infection were retrospectively analyzed using standardized definitions of LRTD. PIV was detected in 540 HCT recipients; 343 had URTI alone and 197 (36%) had LRTD (possible, 76; probable, 19; proven, 102). Among 476 patients with positive nasopharyngeal samples, the cumulative incidence of progression to probable/proven LRTD by day 40 was 12%, with a median time to progression of 7 days (range, 2 to 40). In multivariable analysis monocytopenia (hazard ratio, 2.22; P = .011), steroid use ≥1mg/kg prior to diagnosis (hazard ratio, 1.89; P = .018), co-pathogen detection in blood (hazard ratio, 3.21; P = .027), and PIV type 3 (hazard ratio, 3.57; P = .032) were associated with increased progression risk. In the absence of all 4 risk factors no patients progressed to LRTD, whereas progression risk increased to >30% if 3 or more risk factors were present. Viral load or ribavirin use appeared to have no effect on progression. Among 121 patients with probable/proven LRTD, 64 (53%) presented LRTD without prior URTI, and decreased lung function before infection and lower respiratory co-pathogens were risk factors for this presentation. Mortality was unaffected by the absence of prior URTI. We conclude that the risk of progression to probable/proven LRTD exceeded 30% with ≥3 risk factors. To detect all cases of LRTD, virologic testing of lower respiratory samples is required regardless of URTI symptoms.

Animal infection studies of two recently discovered African bat paramyxoviruses, Achimota 1 and Achimota 2.

Bats are implicated as the natural reservoirs for several highly pathogenic viruses that can infect other animal species, including man. Here, we investigate the potential for two recently discovered bat rubulaviruses, Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2), isolated from urine collected under urban bat (Eidolon helvum) roosts in Ghana, West Africa, to infect small laboratory animals. AchPV1 and AchPV2 are classified in the family Paramyxoviridae and cluster with other bat derived zoonotic rubulaviruses (i.e. Sosuga, Menangle and Tioman viruses). To assess the susceptibility of AchPV1 and AchPV2 in animals, infection studies were conducted in ferrets, guinea pigs and mice. Seroconversion, immunohistological evidence of infection, and viral shedding were identified in ferrets and guinea pigs, but not in mice. Infection was associated with respiratory disease in ferrets. Viral genome was detected in a range of tissues from ferrets and guinea pigs, however virus isolation was only achieved from ferret tissues. The results from this study indicate Achimota viruses (AchPVs) are able to cross the species barrier. Consequently, vigilance for infection with and disease caused by these viruses in people and domesticated animals is warranted in sub-Saharan Africa and the Arabian Peninsula where the reservoir hosts are present.

Human Metapneumovirus Infection: Pneumonia Risk Factors in Patients With Solid Organ Transplantation and Computed Tomography Findings.

BACKGROUND: Human metapneumovirus (HMPV) is a newly detected pathogen that can cause lower respiratory tract disease. Clinical characteristics, computed tomography (CT) findings, and outcomes of HMPV pneumonia in patients with solid organ transplantation (SOT) have not been well demonstrated. METHODS: Between January 2010 and February 2016, clinical and imaging findings of 59 patients with SOT (types of organ: kidney, 37; liver, 16; heart, 4; and pancreas and kidney, 2) who had HMPV infection detected in nasopharyngeal or bronchoalveolar lavage by reverse transcription polymerase chain reaction were retrospectively evaluated. RESULTS: Most (90%) of the patients were detected between March and June. In the 59 patients with SOT with upper respiratory tract infection (URI), 29 (49%) progressed to lower respiratory tract disease after a median of 7 days (range, 2-31 days). Coinfection was noted in 39% of the patients. In Cox proportional hazards analysis, low lymphocyte count (≤0.7 × 10/µL; hazard ratio, 2.24; 95% confidence interval, 1.04-4.85; P = 0.04) and high C-reactive protein (>10 mg/dL; hazard ratio, 2.93; 95% confidence interval, 1.19-7.21; P = 0.02) at URI diagnosis were associated with HMPV pneumonia. On CT, HMPV pneumonia presented as bilateral ill-defined centrilobular nodules, consolidation and ground-glass opacities, whereas lymphadenopathy or effusion is not common. There were no significantly different imaging CT findings between patients with HMPV infection alone and those with coinfection. CONCLUSIONS: Human metapneumovirus pneumonias were detected in nearly half of patients with SOT showing URI symptoms with positive HMPV, and low lymphocyte count and high C-reactive protein at URI diagnosis were significant factors associated with HMPV pneumonia.

Low Retinol-Binding Protein and Vitamin D Levels Are Associated with Severe Outcomes in Children Hospitalized with Lower Respiratory Tract Infection and Respiratory Syncytial Virus or Human Metapneumovirus Detection.

Retinol binding protein and vitamin D were measured in children aged <5 years hospitalized with lower respiratory tract infection and respiratory syncytial virus and/or human metapneumovirus detections. Low vitamin levels were observed in 50% of the children and were associated with significantly elevated risk of the need for intensive care unit admission and invasive mechanical ventilation.

Cytokine Profiles in Human Metapneumovirus Infected Children: Identification of Genes Involved in the Antiviral Response and Pathogenesis.

Human metapneumovirus (hMPV) causes severe airway infection in children that may be caused by an unfavorable immune response. The nature of the innate immune response to hMPV in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. We examined nasopharynx aspirates and blood samples from hMPV-infected children without detectable co-infections. The expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mRNA quantification while blood plasma proteins were determined by a multiplex immunoassay. Several genes were significantly up-regulated at mRNA and protein level in the hMPV infected children. Most apparent was the expression of the chemokine IP-10, the pro-inflammatory cytokine IL-18 in addition to the interferon inducible gene ISG54. Interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes IL-1ß and NLRP3. Overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hMPV mediated lung disease and the antiviral response in children with severe infection. Our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hMPV-infected children.

DAS181 for Treatment of Parainfluenza Virus Infections in Hematopoietic Stem Cell Transplant Recipients at a Single Center.

Parainfluenza virus (PIV) causes severe respiratory infections in hematopoietic stem cell transplant (HSCT) recipients. Currently, no effective therapies are available. DAS181 is a novel antiviral agent that inhibits attachment of PIV to respiratory cells, but clinical data on the use of DAS181 for PIV infection are limited to case reports. We report the clinical manifestations and outcomes of 16 HSCT recipients who received DAS181 daily for the treatment of PIV infection through a compassionate-use protocol or a single-arm clinical trial. Of the 16 patients (clinical trial: 9; compassionate use: 7), 13 were allogeneic HSCT recipients and 8 had graft-versus-host disease. PIV types were 3 (n = 7), 4 (n = 5), 1 (n = 3), and type 3 and 4 coinfection (n = 1). Fourteen patients had pneumonia. All patients presented with cough, 14 had dyspnea, 11 had hypoxia, and 8 had a fever. Patients received 5 to 10 days of treatment. Nine patients (56%) had a complete clinical response after DAS181 therapy and 4 (25%) had a partial response. The 3 patients without a clinical response had coinfections with other pathogens. Of the 7 patients with virologic and spirometric data, 5 had >1-log reduction in nasopharyngeal swab PIV viral load and 4 had improved forced expiratory volumes by the end of treatment. Three patients (19%) died within 30 days and 2 of these deaths were related to PIV infection. Our data suggest that DAS181 may be an effective therapy for PIV pneumonia in HSCT recipients. Randomized placebo-controlled trials are needed to better evaluate its efficacy.

Viral etiologies of hospitalized acute lower respiratory infection patients in China, 2009-2013.

BACKGROUND: Acute lower respiratory infections (ALRIs) are an important cause of acute illnesses and mortality worldwide and in China. However, a large-scale study on the prevalence of viral infections across multiple provinces and seasons has not been previously reported from China. Here, we aimed to identify the viral etiologies associated with ALRIs from 22 Chinese provinces. METHODS AND FINDINGS: Active surveillance for hospitalized ALRI patients in 108 sentinel hospitals in 24 provinces of China was conducted from January 2009-September 2013. We enrolled hospitalized all-age patients with ALRI, and collected respiratory specimens, blood or serum collected for diagnostic testing for respiratory syncytial virus (RSV), human influenza virus, adenoviruses (ADV), human parainfluenza virus (PIV), human metapneumovirus (hMPV), human coronavirus (hCoV) and human bocavirus (hBoV). We included 28,369 ALRI patients from 81 (of the 108) sentinel hospitals in 22 (of the 24) provinces, and 10,387 (36.6%) were positive for at least one etiology. The most frequently detected virus was RSV (9.9%), followed by influenza (6.6%), PIV (4.8%), ADV (3.4%), hBoV (1.9), hMPV (1.5%) and hCoV (1.4%). Co-detections were found in 7.2% of patients. RSV was the most common etiology (17.0%) in young children aged <2 years. Influenza viruses were the main cause of the ALRIs in adults and elderly. PIV, hBoV, hMPV and ADV infections were more frequent in children, while hCoV infection was distributed evenly in all-age. There were clear seasonal peaks for RSV, influenza, PIV, hBoV and hMPV infections. CONCLUSIONS: Our findings could serve as robust evidence for public health authorities in drawing up further plans to prevent and control ALRIs associated with viral pathogens. RSV is common in young children and prevention measures could have large public health impact. Influenza was most common in adults and influenza vaccination should be implemented on a wider scale in China.

Acute encephalopathy associated with human metapneumovirus infection.

We encountered a 3.5-year-old girl with acute encephalopathy associated with human metapneumovirus (hMPV) infection. She had pyrexia and status epilepticus, followed by a coma. Cerebrospinal fluid analysis showed no pleocytosis or elevation of protein levels. hMPV RNA was detected in tracheal aspirate. Acute encephalopathy in the patient was probably related to the hMPV infection. Serum levels of interleukin-6 and matrix metalloproteinase-9 were elevated on admission, and these factors were presumed to be related to acute encephalopathy, associated with her viral infection, or due to status epilepticus. She was treated with dexamethasone pulse therapy, intravenous immunoglobulin, and continuous thiopental infusion. She recovered without neurological sequelae.

Serologic evidence of avian metapneumovirus infection among adults occupationally exposed to Turkeys.

Genetically similar, the avian metapneumovirus (aMPV) and the human MPV (hMPV) are the only viruses in the Metapneumovirus genus. Previous research demonstrated the ability of hMPV to cause clinical disease in turkeys. In this controlled, cross-sectional, seroepidemiological study, we examined the hypothesis that aMPV might infect humans. We enrolled 95 adults occupationally exposed to turkeys and 82 nonexposed controls. Sera from study participants were examined for antibodies against aMPV and hMPV. Both in bivariate (OR=3.2; 95% CI: 1.1-9.2) and in multivariate modelling adjusting for antibody to hMPV (OR=4.1; 95% CI: 1.3-13.1), meat-processing workers were found to have an increased odds of previous infection with aMPV compared to controls. While hMPV antibody cross-reactivity is evident, these data suggest that occupational exposure to turkeys is a risk factor for human infection with aMPV. More studies are needed to validate these findings, to identify modes of aMPV transmission, and to determine risk factors associated with infection.

Association between human metapneumovirus seroprevalence and hypertension in elderly subjects in a long-term care facility.

Recently, relations between hypertension and infections caused by several pathogens have been reported. However, few studies have examined the relationship between human metapneumovirus (hMPV) and hypertension in elderly inpatients. To assess the association between anti-hMPV-immunoglobulin G (IgG) titer and the prevalence of hypertension, we conducted a case-control study in a Japanese long-term care facility (LTCF). The participants included 84 hypertensive patients aged 65 years, and 84 age- and sex-matched normotensive controls (38 males and 46 females in each group; cases, 79.9±8.4 (s.d.) years; controls, 80.1±8.3 years). Data on underling chronic clinical conditions were collected. Titers were measured using an immunofluorescence assay kit. The significance of risk factor differences was analyzed using univariate and multivariate comparisons of cases and controls. All serum samples were positive for hMPV, and IgG titers ranged from 40-fold to more than 5120-fold. There were no significant sex- or age-related differences in log(2) (anti-hMPV-IgG titer/10) among the subjects. Compared with normotensive subjects, hypertensive patients presented significantly higher log(2) (anti-hMPV-IgG titer/10) values (P<0.001). After adjustment with multiple logistic analysis, the odds ratio for log(2) (anti-hMPV-IgG titer/10) was 1.42 (95% confidence interval 1.16-1.75, P=0.001) relative to normotensive subjects. In all subjects, stepwise multiple regression analysis revealed that both hypertension and a poor nutritional state independently contributed to increased log(2) (anti-hMPV-IgG titer/10). These observations suggest that an increased anti-hMPV-IgG titer was closely related to hypertension in elderly subjects in a Japanese LTCF.

Pathogenic and immunogenic responses in turkeys following in ovo exposure to avian metapneumovirus subtype C.

Commercial turkey eggs, free of antibodies to avian metapneumovirus subtype C (aMPV/C), were inoculated with aMPV/C at embryonation day (ED) 24. There was no detectable effect of virus inoculation on the hatchability of eggs. At 4 days post inoculation (DPI) (the day of hatch (ED 28)) and 9 DPI (5 days after hatch), virus replication was detected by quantitative RT-PCR in the turbinate, trachea and lung but not in the thymus or spleen. Mild histological lesions characterized by lymphoid cell infiltration were evident in the turbinate mucosa. Virus exposure inhibited the mitogenic response of splenocytes and thymocytes and upregulated gene expression of IFN-γ and IL-10 in the turbinate tissue. Turkeys hatching from virus-exposed eggs had aMPV/C-specific IgG in the serum and the lachrymal fluid. At 3 week of age, in ovo immunized turkeys were protected against a challenge with pathogenic aMPV/C.

[Genetic variability of matrix (M), small hydrophobic (SH) and attachment (G) proteins of human metapneumovirus circulating in children in Beijing from 2006 to 2010].

Human metapneumovirus (hMPV) is associated with acute respiratory tract infections (ARTI) in all age groups. However, there is limited information of genetic analysis of hMPV circulating in Beijing. To learn the characteristics of structural protein genes of human metapneumovirus circulating in children in Beijing, sequence analysis of matrix (M), small hydrophobic (SH) and attachment (G) proteins of hMPV from 2006 to 2010 was performed. Phylogenetic analysis of nucleotide sequences of 42 full length M genes, 49 SH gene and 55 G gene revealed that the hMPVs from pediatric patients were divided into sub-genotypes A2, B1 and B. There were highly conserved identities among M gene, with 7 conserved mutations of amino acids between A and B genotypes which were fairly conserved in the same genotype A or B. The amino acid identities of SH were 60.7% to 64.4% between different genotypes, 93.3% - 100% among same sub-genotype and 84.7% - 88.7% between different sub-genotypes. Use of alternative transcription-termination codon, nucleotide deletion and insertion resulted in variable length of nucleotide and deduced amino acid of G protein. Amino acid identities within same genotype ranged from 81.5% - 100%, whereas sequence identities between two genotypes ranged from 34.0% - 38.6% at the amino acid level. A new cluster of G genes in sub-genotype B2 appeared due to the same mutations and insertion of two amino acids in G protein encoding genes amplified from specimens collected from 2008 to 2010. Prediction of antigen sites of SH and G protein indicated that the variation of antigen sites between different sub-genotypes existed.

High seroprevalence of neutralizing capacity against human metapneumovirus in all age groups studied in Bonn, Germany.

Human metapneumovirus (hMPV) infections occur frequently despite high rates of perpetual seroprevalence for all age groups. Analyses of approximately 2,000 archived, randomly selected serum samples demonstrated that neutralizing capacities remain high, with a minor decrease for individuals over 69 years of age, leading to the hypothesis that reinfections occur because humoral immune responses play minor roles in the clearance of hMPV infections.

Effects of cyclosporin A induced T-lymphocyte depletion on the course of avian Metapneumovirus (aMPV) infection in turkeys.

The avian Metapneumovirus (aMPV) causes an economically important acute respiratory disease in turkeys (turkey rhinotracheitis, TRT). While antibodies were shown to be insufficient for protection against aMPV-infection, the role of T-lymphocytes in the control of aMPV-infection is not clear. In this study we investigated the role of T-lymphocytes in aMPV-pathogenesis in a T-cell-suppression model in turkeys. T-cell-intact turkeys and turkeys partly depleted of functional CD4(+) and CD8(+) T-lymphocytes by Cyclosporin A (CsA) treatment were inoculated with the virulent aMPV subtype A strain BUT 8544. CsA-treatment resulted in a significant reduction of absolute numbers of circulating CD4(+) and CD8alpha(+) T-lymphocytes by up to 82 and 65%, respectively (P<0.05). Proportions of proliferating T-cells within mitogen-stimulated peripheral blood mononuclear cells were reduced by similar levels in CsA-treated birds compared to untreated controls (P<0.05). CsA-treated turkeys showed delayed recovery from aMPV-induced clinical signs and histopathological lesions and a prolonged detection of aMPV in choanal swabs. The results of this study show that T-lymphocytes play an important role in the control of primary aMPV-infection in turkeys.

Diagnostic utility of egg yolk for the detection of avian metapneumovirus antibodies in laying hens.

Surveillance and diagnosis of avian metapneumovirus (AMPV) infection typically involve measurement of serum antibodies. In the current study, eggs instead of serum samples were used for the detection of AMPV antibodies in egg-laying chicken hens by enzyme-linked immunosorbent assay (ELISA). AMPV-free commercial layer hens were experimentally challenged with AMPV strain SC1509 through intravenous or oculonasal administration. Antibody levels were determined by ELISA. AMPV antibodies were detected in egg yolks from challenged hens by 7 days postinoculation (dpi), with the peak titer at 16 dpi. Antibody levels in eggs laid at 28 dpi correlated well (r = 0.93) with sera taken 28 dpi from the same hens. In a field trial of the yolk ELISA, six broiler breeder farms were surveyed, and all tested positive for AMPV antibodies in hen eggs, although positivity varied from farm to farm. Abnormal discolored eggs collected from outbreak farms had significantly higher titers of AMPV yolk antibodies than normal eggs from the same farm, unlike clinically healthy farms, where normal and abnormal eggs had similar antibody titers. These results indicate that diagnosis of AMPV infection by yolk ELISA to detect anti-AMPV antibodies may be a suitable alternative to serologic testing.

Generation of recombinant nucleocapsid protein of human metapneumovirus in baculovirus for detecting antibodies in the Beijing population.

Human metapneumovirus (hMPV) is associated with respiratory infections in both adults and children. Recombinant nucleocapsid (N) proteins of hMPV from two major groups of hMPV in Beijing with a 6x His-tag at the C terminus were constructed in a baculovirus and expressed in transfected Sf21 insect cells. The antigenicity and specificity of the expressed recombinant proteins were examined by immunofluorescence assay and western blotting. Preliminary use of the expressed proteins for antibody detection in 187 serum specimens collected from different age groups in Beijing, China, indicated that the purified recombinant N-His proteins were of good antigenicity and specificity and could be a potential antigen for further seroprevalence study of hMPV, especially in the Chinese population. The positive rate for antibody detection suggested that hMPV from two clusters was co-circulating in Beijing and that most of the people in Beijing have been exposed to the virus by age 60.