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Epidemiological studies report the impact of co-infection with pneumococcus and respiratory viruses upon disease rates and outcomes, but their effect on pneumococcal carriage acquisition and bacterial load is scarcely described. Here, we assess this by combining natural viral infection with controlled human pneumococcal infection in 581 healthy adults screened for upper respiratory tract viral infection before intranasal pneumococcal challenge. Across all adults, respiratory syncytial virus (RSV) and rhinovirus asymptomatic infection confer a substantial increase in secondary infection with pneumococcus. RSV also has a major impact on pneumococcal density up to 9 days post challenge. We also study rates and kinetics of bacterial shedding through the nose and oral route in a subset. High levels of pneumococcal colonization density and nasal inflammation are strongly correlated with increased odds of nasal shedding as opposed to cough shedding. Protection against respiratory viral infections and control of pneumococcal density may contribute to preventing pneumococcal disease and reducing bacterial spread., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Parechovirus-A3 (PeV-A3) and the enteroviruses (EVs) are the most common viral pathogens responsible for sepsis and meningoencephalitis in neonates and young infants; however, differences in the clinical presentations of two infections are not well described., Objectives: To describe the clinical presentations of PeV-A3- and EVs-related diseases and develop a novel scoring system to differentiate two diseases., Study Design: This prospective study used real-time PCR and genetic sequencing to evaluate viral etiologies of febrile neonates and infants <4 months with suspected sepsis or meningoencephalitis in Niigata area, Japan, in 2014-2016. The clinical manifestations of PeV-A3- and EVs-infected patients were compared, and a novel scoring system was developed after identifying the most distinguishable clinical findings, followed by the external cohort validation., Results: In 210 patients evaluated, we identified 56 PeV-A3-infected (27%) and 43 EVs-infected (20%) patients. The following clinical manifestations were significant in PeV-A3-infected patients, as compared with EVs-infected patients; a higher body temperature (38.9°C vs. 38.5°C, P < .01) and heart rate (181/min vs. 168/min, P = .01), cold extremities (72% vs. 34%, P < .01) and skin mottling (65% vs. 23%, P < .01), lower white blood cell count (5,200/μL vs. 8,900/μL, P < .01) and incidence of cerebrospinal fluid (CSF) pleocytosis (2% vs. 63%, P < .01). Using some of these significant findings, the scoring system successfully distinguished the diseases (accuracy: 86% and 83% for the derivative and external validation cohorts, respectively)., Conclusions: We found significant clinical manifestations in PeV-A3-infected patients compared to EVs-infected patients. The scoring system may be helpful to distinguish two infections, especially at onset of outbreak., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Enteric viruses infect the mammalian gastrointestinal tract which is home to a diverse community of intestinal bacteria. Accumulating evidence suggests that certain enteric viruses utilize these bacteria to promote infection. While this is not surprising considering their proximity, multiple viruses from different viral families have been shown to bind directly to bacteria or bacterial components to aid in viral replication, pathogenesis, and transmission. These data suggest that the concept of a single virus infecting a single cell, independent of the environment, needs to be reevaluated. In this review, I will discuss the current knowledge of enteric virus-bacterial interactions and discuss the implications for viral pathogenesis and transmission., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Little is known regarding the nasal microbiome in early childhood and the impact of respiratory infection on the infants' nasal microbial composition. Here we investigated the temporal dynamics and diversity of the bacterial composition in the anterior nares in children attending daycare centers., Results: For our investigation, we considered 76 parental-taken nasal swabs of 26 children (aged 13 to 36 months) collected over a study period of 3 months. Overall, there was no significant age-specific effect or seasonal shift in the nasal bacterial community structure. In a sub-sample of 14 healthy children the relative abundance of individual taxa as well as the overall diversity did not reveal relevant changes, indicating a stable community structure over the entire study period. Moreover, the nasal bacterial profiles clustered subject-specific with Bray-Curtis similarities being elevated in intra-subject calculations compared to between-subject calculations. The remaining subset of 12 children provided samples taken during picornavirus infection (PVI) and either before or after a PVI. We detected an association between the relative abundance of members of the genus Streptococcus and PV when comparing both (i) samples taken during PVI with samples out of 14 healthy children and (ii) samples taken during PVI with samples taken after PVI within the same individual. In addition, the diversity was higher during PVI than after infection., Conclusions: Our findings suggest that a personalized structure of the nasal bacterial community is established already in early childhood and could be detected over a timeframe of 3 months. Studies following infants over a longer time with frequent swab sampling would allow investigating whether certain parameter of the bacterial community, such as the temporal variability, could be related to viral infection.

Background: Patients with chronic obstructive pulmonary disease (COPD) have increased susceptibility to respiratory tract infection, which contributes to disease progression and mortality, but mechanisms of increased susceptibility to infection remain unclear., Objectives: The aim of this study was to determine whether glucose concentrations were increased in airway samples (nasal lavage fluid, sputum, and bronchoalveolar lavage fluid) from patients with stable COPD and to determine the effects of viral infection on sputum glucose concentrations and how airway glucose concentrations relate to bacterial infection., Methods: We measured glucose concentrations in airway samples collected from patients with stable COPD and smokers and nonsmokers with normal lung function. Glucose concentrations were measured in patients with experimentally induced COPD exacerbations, and these results were validated in patients with naturally acquired COPD exacerbations. Relationships between sputum glucose concentrations, inflammatory markers, and bacterial load were examined., Results: Sputum glucose concentrations were significantly higher in patients with stable COPD compared with those in control subjects without COPD. In both experimental virus-induced and naturally acquired COPD exacerbations, sputum and nasal lavage fluid glucose concentrations were increased over baseline values. There were significant correlations between sputum glucose concentrations and sputum inflammatory markers, viral load, and bacterial load. Airway samples with higher glucose concentrations supported more Pseudomonas aeruginosa growth in vitro., Conclusions: Airway glucose concentrations are increased in patients with stable COPD and further increased during COPD exacerbations. Increased airway glucose concentrations might contribute to bacterial infections in both patients with stable and those with exacerbated COPD. This has important implications for the development of nonantibiotic therapeutic strategies for the prevention or treatment of bacterial infection in patients with COPD., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The role of nasal and fecal microbiota in viral respiratory infections has not been established. We collected nasal swabs and washes, and fecal samples in a clinical study assessing the effect of probiotic Bifidobacterium animalis subsp. lactis Bl-04 on experimental rhinovirus infection. The nasal and fecal microbiota were characterized by 16S rRNA gene sequencing. The resulting data were compared with nasal inflammatory marker concentrations, viral load, and clinical symptoms. By using unsupervised clustering, the nasal microbiota divided into six clusters. The clusters predominant of Staphylococcus, Corynebacterium/Alloiococcus, Moraxella, and Pseudomonadaceae/Mixed had characteristic inflammatory marker and viral load profiles in nasal washes. The nasal microbiota clusters of subjects before the infection associated with the severity of clinical cold symptoms during rhinovirus infection. Rhinovirus infection and probiotic intervention did not significantly alter the composition of nasal or fecal microbiota. Our results suggest that nasal microbiota may influence the virus load, host innate immune response, and clinical symptoms during rhinovirus infection, however, further studies are needed.

Rationale: The nasopharyngeal (NP) microbiota of newborns and infants plays a key role in modulating airway inflammation and respiratory symptoms during viral infections. Premature (PM) birth modifies the early NP environment and is a major risk factor for severe viral respiratory infections. However, it is currently unknown if the NP microbiota of PM infants is altered relative to full-term (FT) individuals., Objectives: To characterize the NP microbiota differences in preterm and FT infants during rhinovirus (RV) infection., Methods: We determined the NP microbiota of infants 6 months to ≤2 years of age born FT (n=6) or severely PM<32 weeks gestation (n=7). We compared microbiota composition in healthy NP samples and performed a longitudinal analysis during naturally occurring RV infections to contrast the microbiota dynamics in PM versus FT infants., Results: We observed significant differences in the NP bacterial community of PM versus FT. NP from PM infants had higher within-group dissimilarity (heterogeneity) relative to FT infants. Bacterial composition of NP samples from PM infants showed increased Proteobacteria and decreased in Firmicutes . There were also differences in the major taxonomic groups identified, including Streptococcus , Moraxella , and Haemophilus . Longitudinal data showed that these prematurity-related microbiota features persisted during RV infection., Conclusions: PM is associated with NP microbiota changes beyond the neonatal stage. PM infants have an NP microbiota with high heterogeneity relative to FT infants. These prematurity-related microbiota features persisted during RV infection, suggesting that the NP microbiota of PM may play an important role in modulating airway inflammatory and immune responses in this vulnerable group., Competing Interests: Competing interests: None declared., (Copyright © 2017 American Federation for Medical Research.)

The study of the etiological agents of community-acquired pulmonary infections is important to guide empirical therapy, requires constant updating, and has a substantial impact on the prognosis of patients. The objective of this study is to determine prospectively the etiology of community-acquired pulmonary infections in hospitalized adults living with HIV. Patients were submitted to an extended microbiological investigation that included molecular methods. The microbiological findings were evaluated according to severity of the disease and pneumococcal vaccine status. Two hundred twenty-four patients underwent the extended microbiological investigation of whom 143 (64%) had an etiology determined. Among the 143 patients with a determined etiology, Pneumocystis jirovecii was the main agent, detected in 52 (36%) cases and followed by Mycobacterium tuberculosis accounting for 28 (20%) cases. Streptococcus pneumoniae and Rhinovirus were diagnosed in 22 (15%) cases each and influenza in 15 (10%) cases. Among atypical bacteria, Mycoplasma pneumoniae was responsible for 12 (8%) and Chlamydophila pneumoniae for 7 (5%) cases. Mixed infections occurred in 48 cases (34%). S pneumoniae was associated with higher severity scores and not associated with vaccine status. By using extended diagnostics, a microbiological agent could be determined in the majority of patients living with HIV affected by community-acquired pulmonary infections. Our findings can guide clinicians in the choice of empirical therapy for hospitalized pulmonary disease., Competing Interests: The authors have no conflicts of interest to disclose.

Background: Human Rhinovirus (HRV) is responsible for the majority of common colds and is frequently accompanied by secondary bacterial infections through poorly understood mechanisms. We investigated the effects of experimental human HRV serotype 16 infection on the upper respiratory tract microbiota., Methods: Six healthy volunteers were infected with HRV16. We performed 16S ribosomal RNA-targeted pyrosequencing on throat swabs taken prior, during and after infection. We compared overall community diversity, phylogenetic structure of the ecosystem and relative abundances of the different bacteria between time points., Results: During acute infection strong trends towards increases in the relative abundances of Haemophilus parainfluenzae and Neisseria subflava were observed, as well as a weaker trend towards increases of Staphylococcus aureus. No major differences were observed between day-1 and day 60, whereas differences between subjects were very high., Conclusions: HRV16 infection is associated with the increase of three genera known to be associated with secondary infections following HRV infections. The observed changes of upper respiratory tract microbiota could help explain why HRV infection predisposes to bacterial otitis media, sinusitis and pneumonia.

Background: Detection of either viral or bacterial pathogens is associated with wheezing in children; however, the influence of both bacteria and viruses on illness symptoms has not been described., Objective: We evaluated bacterial detection during the peak rhinovirus season in children with and without asthma to determine whether an association exists between bacterial infection and the severity of rhinovirus-induced illnesses., Methods: Three hundred eight children (166 with asthma and 142 without asthma) aged 4 to 12 years provided 5 consecutive weekly nasal samples during September and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were performed on all nasal samples., Results: Detection rates were 53%, 17%, and 11% for H influenzae, S pneumoniae, and M catarrhalis, respectively, with detection of rhinovirus increasing the risk of detecting bacteria within the same sample (odds ratio [OR], 2.0; 95% CI, 1.4-2.7; P < .0001) or the following week (OR, 1.6; 95% CI, 1.1-2.4; P = .02). In the absence of rhinovirus, S pneumoniae was associated with increased cold symptoms (mean, 2.7 [95% CI, 2.0-3.5] vs 1.8 [95% CI, 1.5-2.2]; P = .006) and moderate asthma exacerbations (18% [95% CI, 12% to 27%] vs 9.2% [95% CI, 6.7% to 12%]; P = .006). In the presence of rhinovirus, S pneumoniae was associated with increased moderate asthma exacerbations (22% [95% CI, 16% to 29%] vs 15% [95% CI, 11% to 20%]; P = .01). Furthermore, M catarrhalis detected alongside rhinovirus increased the likelihood of experiencing cold symptoms, asthma symptoms, or both compared with isolated detection of rhinovirus (OR, 2.0 [95% CI, 1.0-4.1]; P = .04). Regardless of rhinovirus status, H influenzae was not associated with respiratory symptoms., Conclusion: Rhinovirus infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M catarrhalis and S pneumoniae contribute to the severity of respiratory tract illnesses, including asthma exacerbations., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Rationale: Rhinovirus infection is followed by significantly increased frequencies of positive, potentially pathogenic sputum cultures in chronic obstructive pulmonary disease (COPD). However, it remains unclear whether these represent de novo infections or an increased load of organisms from the complex microbial communities (microbiome) in the lower airways., Objectives: To investigate the effect of rhinovirus infection on the airway bacterial microbiome., Methods: Subjects with COPD (n = 14) and healthy control subjects with normal lung function (n = 17) were infected with rhinovirus. Induced sputum was collected at baseline before rhinovirus inoculation and again on Days 5, 15, and 42 after rhinovirus infection and DNA was extracted. The V3-V5 region of the bacterial 16S ribosomal RNA gene was amplified and pyrosequenced, resulting in 370,849 high-quality reads from 112 of the possible 124 time points., Measurements and Main Results: At 15 days after rhinovirus infection, there was a sixfold increase in 16S copy number (P = 0.007) and a 16% rise in numbers of proteobacterial sequences, most notably in potentially pathogenic Haemophilus influenzae (P = 2.7 × 10(-20)), from a preexisting community. These changes occurred only in the sputum microbiome of subjects with COPD and were still evident 42 days after infection. This was in contrast to the temporal stability demonstrated in the microbiome of healthy smokers and nonsmokers., Conclusions: After rhinovirus infection, there is a rise in bacterial burden and a significant outgrowth of Haemophilus influenzae from the existing microbiota of subjects with COPD. This is not observed in healthy individuals. Our findings suggest that rhinovirus infection in COPD alters the respiratory microbiome and may precipitate secondary bacterial infections.

Background: The main aim of this study was to evaluate whether nasopharyngeal Streptococcus pneumoniae colonization in children with alveolar community-acquired pneumonia (CAP) and respiratory syncytial virus (RSV) or rhinovirus (RV) infection indicates a mixed lung infection., Methods: The nasopharyngeal secretions of 530 children with radiographically confirmed CAP were tested using the Luminex x TAG respiratory virus panel fast assay. Real-time polymerase chain reaction for the autolysin-A (LytA) and wzg (cpsA) genes of S. pneumoniae was performed on the RSV- and RV-positive samples., Results: Sixty-five of the 126 RSV-positive children (51.6%) were colonized with S. pneumoniae. Mean bacterial load was significantly higher in the patients with alveolar involvement (4.54±1.47 log10 DNA copies/mL vs. 3.75±1.62 log10 DNA copies/mL; P=0.04). Serotypes 5 and 19A were almost exclusively identified in the children with RSV and alveolar CAP, although the difference was statistically significant only for serotype 19A (P=0.03). Eighty-three of the 134 RV-positive children (61.9%) were colonized with S. pneumoniae and again mean bacterial load was significantly higher in the patients with alveolar involvement (4.21±1.37 log10 DNA copies/mL vs. 3.41±1.47 log10 DNA copies/mL; P=0.03). Serotypes 1, 5 and 19A were more frequently identified in the children with RV and alveolar CAP, although the difference was statistically significant only for serotype 5 (P=0.04)., Conclusions: In children with alveolar CAP and RSV or RV infection, the determination of nasopharyngeal pneumococcal bacterial load and identification of the serotypes can contribute to the diagnosis of mixed lung infection.

The natural history of asthma involves relatively stable periods that are often punctuated by significant exacerbations of symptoms. There are many aetiologies that may lead to an increase in asthma severity including respiratory infection (bacterial/viral), allergens, irritants, and occupational exposures. Each trigger probably acts through different mechanisms, but a final common pathway of multicellular inflammation, enhanced bronchial responsiveness, and airflow obstruction is a likely consequence. This review discusses the most common causes of asthma exacerbations with a focus on their microbiology and immunopathogenesis. Through an understanding of underlying causes of asthma exacerbations, treatments with increased effectiveness may be developed, and it is these future developments that may directly influence the morbidity and mortality of the disease.

Equine rhinitis viruses (ERVs) are the causative agents of mild to severe upper respiratory infections in horses worldwide. Immunologically, four serotypes of ERVs have been identified. Equine rhinitis A virus (ERAV) and Equine rhinitis B virus 1 (ERBV1) are the most frequent serotypes in Europe. Both viruses have a broad host range in cultured cells with ERAV being able to infect humans. Since there is neither information on the seroprevalence of ERAV and ERBV1 in Austria nor on the zoonotic potential of ERBV1, we investigated 200 horse and 137 veterinary sera for the presence of neutralizing antibodies relating to ERAV and ERBV1. One hundred and eighty (90%) and 173 (86%) horse sera neutralized ERAV and ERBV1, respectively. In contrast, only four (2.7%) and five (3.6%) human sera showed weak neutralizing activity to ERAV and ERBV1, respectively. These results indicate that ERAV and ERBV1 are widespread in the Austrian horse population; however, the risk of acquiring zoonotic infection among veterinarians appears low.

Both virus-mediated damage to airway tissues and induction of pro-inflammatory cytokines such as interleukin-8 (IL-8) could contribute to symptom severity during viral respiratory infections in children. To test the hypothesis that IL-8 contributes to the pathogenesis of respiratory symptoms during naturally acquired respiratory viral infections in children, nasal wash samples collected from infants with acute viral infections (n = 198) or from healthy uninfected infants (n = 31) were analysed for IL-8. Nasal wash IL-8 was positively related to age in uninfected children (rs = 0.36, p < 0.05). Respiratory syncytial virus (RSV) infection caused more severe respiratory symptoms compared to infections with influenza A, parainfluenza viruses, or rhinoviruses. In addition, RSV, parainfluenza and rhinovirus infections increased levels of IL-8 in nasal lavage fluid, and there were some differences in the ability of the viruses to induce IL-8 production (RSV>influenza, p < 0.05). Finally, there were significant correlations between nasal wash IL-8 levels and symptom scores during infections with rhinovirus (rs = 0.56, p < 0.001) or influenza A (rs = 0.45, p < 0.05), but not with parainfluenza virus or RSV. These findings provide evidence of a close relationship between the generation of IL-8 and symptoms during acute community-acquired infections with rhinovirus or influenza A. In contrast, for RSV and parainfluenza infections, factors in addition to IL-8 production appear to contribute to the generation of clinical symptoms.

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.

Diagnosis of enterovirus infection in turkey poults is currently done using electron microscopy. We have developed an antigen-capture enzyme-linked immunosorbent assay (ELISA) for use in diagnosis of enterovirus infection in turkey intestinal contents. Intestinal contents from naturally and experimentally infected turkey poults were evaluated for the presence of astrovirus and enterovirus by immune electron microscopy (IEM). The results were compared with the findings obtained using the ELISA for the presence of enterovirus. The ELISA, using the samples from the naturally infected poults, had a sensitivity of 0.963 and a specificity of 0.879. The Kappa value of 0.834 for the naturally infected samples and 0.97 for the experimentally infected samples showed excellent agreement between the ELISA and IEM for detection of enterovirus. Our results indicate that the ELISA is a rapid, sensitive, and specific method for diagnosis of turkey enterovirus infection.

The Marin County strain of type 5 astrovirus was associated with two separate outbreaks of nonbacterial gastroenteritis in California in 1978. A safety-tested, bacterium-free filtrate prepared from a stool specimen of an individual who was ill during the original outbreak was given orally to 19 adult volunteers. One volunteer developed a gastrointestinal illness, and nine had serologic responses. Several diarrheal stool specimens from the ill volunteer contained a large number of 27-nm particles. By using immune electron microscopy with acute- and convalescent-phase sera from the original outbreak, these 27-nm particles were shown to be identical to the viral inoculum. The Marin County virus, purified from the stool of the ill volunteer, was shown by immunoprecipitation and polyacrylamide gel electrophoresis to contain a single structural protein with a molecular weight of 30,000. The buoyant density of the virion was 1.39 g/cm3 in cesium chloride. By electron microscopy, approximately 5% of the particles had the characteristic stellate configuration of astrovirus, and serologic studies by immunofluorescence technique confirmed previous classification of the Marin County virus as a type 5 astrovirus. Radioimmunoassay and biotin-avidin immunoassay were used to detect antibody to the Marin County virus in paired acute- and convalescent-phase sera from 32 outbreaks of nonbacterial gastroenteritis, but none of these outbreaks could be attributed to this virus. Prevalence of antibody to this strain of astrovirus was approximately 13% in children 6 months to 3 years of age and increased to 41% in older children and young adults.

The efficacy of oral WIN 54954 for the prevention of rhinovirus infection and illness was tested in two randomized, double-blinded, placebo-controlled volunteer challenge studies. Volunteers were inoculated with rhinovirus type 39 (MIC of WIN 54954, 0.17 microgram/ml) or rhinovirus type 23 (MIC, 0.016 microgram/ml). The volunteers received two doses of drug (600 mg per dose) or placebo on the first day; this was followed by three doses on each of the subsequent 5 days. All volunteers were challenged with virus after the third dose of study drug. No significant antiviral or clinical effect was detected in either study. Pharmacokinetic studies revealed that on the last day of drug administration, 38 of 39 (97%) volunteers had trough levels of WIN 54954 in plasma greater than the MIC for the respective virus. Nasal wash specimens collected on the same day revealed a detectable level in only 6 of 24 (25%) volunteers at the peak (range, 6 to 24 ng/ml) and in only 2 of 14 (14%) volunteers at the trough (range, 6 and 7 ng/ml). These results suggest that the lack of efficacy of WIN 54954 against rhinovirus may be related to an inability to deliver sufficient drug to the site of viral infection.

Purified and concentrated preparations of virus from liver extracts of infected rabbits contain virus specific components with sedimentation coefficients of about 175, 110 and sometimes 133S and more slow units. Full and empty virus particles with a diameter of about 34 nm were shown electron microscopically in the corresponding 175 and 110S fractions of the sucrose density gradient. The average of buoyant density of the 175, 133, 110S and more slow units are 1.36, 1.32 and 1.31 g/ml respectively. The extinction coefficient E260 nm is 4.3 +/- 0.7 cm2/mg. The RNA content is 17 +/- 4%. SDS-PAGE shows a "65" kD protein as a single or major component. Beside smaller polypeptides with lower intensities, the 67 kD polypeptide reacts positively in the Western blot with polyclonal antibodies of rabbits. The molecular weight of the virus is 15 +/- 4 x 10(6)D. The pH stability of the 175S unit was also tested.

Formalin fixed liver, spleen, kidney, heart, lung, duodenum and appendix tissues from nine rabbits, experimentally infected with rabbit haemorrhagic disease virus (RHDV), were investigated for evidence of RHDV antigen by the direct avidin-biotin peroxidase complex immunohistochemical method. In all the rabbits examined, RHDV antigen was detected in degenerative and necrotic hepatocytes of the liver tissues. The area involved coincided with histopathological lesions on serial liver sections. The RHDV antigen was expressed in the cytoplasm of the hepatocytes, suggesting that RHDV replicated in these cells. RHDV antigen was also detected in the spleen. The results of immunohistochemistry were supported by the demonstration of RHDV protein by Western blot analysis and of RHDV particles by protein A-gold immunoelectron microscopy in the liver homogenate from all the rabbits that were examined.

It is now well known that several viruses are responsible for acute diarrhoea or gastroenteritis in both children and adults. These viruses are difficult to identify since most of them cannot be isolated by stool cultures on cells. The reality of proven reinfection by some of these organisms is not always clearly understood, even though the existence of several serotypes in the same group (notably rotavirus) can be blamed, and this explains why vaccines are difficult to develop.

The virus recovered from cases of European brown hare syndrome in the U.K. contains one major capsid protein of approximately 60 k molecular weight and morphologically resembles known caliciviruses. It has been compared with a European isolate of rabbit haemorrhagic disease calicivirus and, although it shows some antigenic similarity, it is not identical. In transmission and protection studies the virus from U.K. hares failed to produce disease in rabbits and did not effectively protect against subsequent challenge with the rabbit calicivirus.

Causative agent of rabbit haemorrhagic disease (RHD) was purified by CsCl density gradient centrifugation from the liver homogenate of rabbits infected with RHD virus which originated from Korea. The viral particles were 35-40 nm in diameter, and had hollow depressions on their surface. Protein A-gold immunoelectron microscopy clearly showed that the convalescent antisera of diseased rabbits reacted specifically with the virus particles. SDS-PAGE and Western blot analyses demonstrated that the structural protein of the virus was composed of a single major polypeptide of 63 kD. These findings indicate that the causative agent of RHD, tentatively named as picornavirus in Korea, belongs to calicivirus.

Rhinoviruses (HRVs) were isolated from 307 children (7.1%) in the virological surveillance of 4334 children with acute respiratory tract illnesses in Morioka, Japan (September 1973-December 1983). Although HRVs were isolated throughout the year, frequency of HRV infection was significantly higher (p less than 0.001) during the April-November (233/2853; 8.2%) than during the December-March (47/1481; 5.0%). There were two peaks of incidence in May (9.5%) and September (9.1%). During the May-September, the rate of HRV infection was higher in patients under the age of 11 months than the next higher group of 1-2 years old (p less than 0.001). The incidence decreased with increasing age. The illnesses of HRV infection were analysed in 294 patients, except one patient who had symptoms of measles, from whom HRV was isolated singly. Although HRV-associated illnesses were generally mild (57.5%). Upper respiratory tract illnesses (URTIs) with fever were found in 22.1% and lower respiratory tract illnesses (LRTIs) in 20.4% of these. The rate of LRTI was higher during the epidemic period (April-September) than other periods (p less than 0.02). Major symptoms of HRV-associated illnesses observed were sore throat (87.4%), cough (84.0%), and nasal obstruction and/or discharge (72.8%). Wheezing was observed in 21.8% of these. From 19 (21.8%) of 47 patients clinically diagnosed as asthmatic bronchitis in this survey, viruses were isolated. HRV was detected most frequently in 12.8% of these patients, followed by respiratory syncytial virus (RSV, 6.4%) and adenovirus (2.1%). HRV- and RSV-associated asthmatic bronchitis were observed during April-September and November-February, respectively. Viral dual infections were detected in total 20 cases included 12 HRV-associated cases. In no case was the illness of greater severity than might have been caused by either agent acting singly.

Two hundred and twenty-six cats from the Veterinary Medical Teaching Hospital (VMTH), a cat shelter, and a purebred cattery were tested for chronic feline calicivirus (FCV), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) infections. Chronic oral carriage of FCV was present in about one-fifth of the cats in each of the groups. FIV infection was not present in the purebred cattery, was moderately prevalent (8%) in the pet population of cats examined at the VMTH for various complaints and was rampant in the cat shelter (21%). Unexpectedly high FeLV infection rates were found in the hospital cat population (28%) and in the purebred cattery (36%), but not in the cat shelter (1.4%). FCV and FeLV infections tended to occur early in life, whereas FIV infections tended to occur in older animals. From 43 to 100% of the cats in these environments had oral cavity disease ranging from mild gingivitis (23-46%), proliferative gingivitis (18-20%), periodontitis (3-32%) and periodontitis with involvement of extra-gingival tissues (7-27%). Cats infected solely with FCV did not have a greater likelihood of oral lesions, or more severe oral disease, than cats that were totally virus free. This was also true for cats infected solely with FeLV, or for cats dually infected with FeLV and FCV. Cats infected solely with FIV appeared to have a greater prevalence of oral cavity infections and their oral cavity disease tended to be more severe than cats without FIV infection. FIV-infected cats that were coinfected with either FCV, or with FCV and FeLV, had the highest prevalence of oral cavity infections and the most severe oral lesions.

EMC-M virus causes a monophasic paralytic syndrome characterized by encephalitic lesions in the brain and patchy demyelinating lesions in the spinal cord and nerve roots of BALB/c mice. Since the replication of EMC virus in vitro is inhibited by tumor necrosis factor (TNF)-alpha we have studied the effect of in vivo administration of this cytokine on the acute disease. Our studies show that periodic administration of TNF-alpha to animals infected with EMC-M reduces viral titers in the brain, and decreases the degree of clinical paralysis and the severity of the inflammatory lesions in the brain.

Development of methods for the diagnosis of viral haemorrhagic disease and the European brown hare syndrome has proceeded at a steady pace over the last few years. The studies conducted by the authors demonstrate that, like VHDV, EBHSV is a calicivirus. The degree of correlation between the two viruses is a key question both for understanding their biology and interpreting the diagnostic results. A discussion of the similarities and differences between VHD and EBHS is followed by the presentation of the latest antigenic correlation results of the two viruses. Considering the absence of culture procedures to isolate either virus, the diagnostic methods discussed in this review are the haemagglutination (HA) test, immune electron microscopy (IEM) and enzyme-linked immunosorbent assay (ELISA) for virus detection, and the haemagglutination inhibition test (HI) and ELISA for antibody detection. The major obstacles, especially for the diagnosis of EBHS, are described; these are represented by morphological, structural and antigenic modifications due to proteolytic degradation. A differential diagnostic method for the two diseases, based on MAb ELISA, is presented. A final conclusion, drawn from the epidemiological analysis of the virological and serological data, is that EBHS and VHD should be considered as two distinct diseases, each caused by its own aetiological agent.

Liver tissue from rabbits which had died of viral haemorrhagic disease (VHD) was used to identify the causative agent of the disease. After extraction of liver homogenates and density gradient ultracentrifugation, viral particles with buoyant densities between 1.32 to 1.38 g/ml and estimated sedimentation coefficients between 100 S and 175 S were obtained. The isolated virions were determined to have a diameter of 40 nm. The particles morphologically resembled those of the Caliciviridae family, had positive reactions both in ELISA and haemagglutination assays, and were infective for rabbits. By immunoblotting, a major structural protein with a molecular weight of 60 kDa was identified. RNA of high purity and of approximately 8 kb was isolated from virions. Labelled cDNA of virion RNA detected two RNAs of 8 kb and 2 kb in Northern blots of liver RNA from animals infected with the VHD virus (VHDV). Finally, isolated virion RNA injected directly into the liver or rabbits produced a disease with clinical symptoms and pathological findings typical of VHD. A calicivirus originally designated "rabbit haemorrhagic disease virus (RHDV)" was thus identified as the causative agent of VHD. The agent causing the European brown hare syndrome (EBHS) is also a calicivirus (EBHSV). VHDV and EBHSV are different members of the Caliciviridae family; their relationship can be clearly demonstrated by using different approaches of investigation.

Rabbit production is of considerable economic importance in Italy. In the last thirty years, meat production has risen and the number of intensive husbandry establishments has grown. The major region of production (about 60%) lies in the northern part of the country. In addition, approximately one million live animals and more than 14,000 tons of meat are imported yearly. More than 150,000 hares are also imported for restocking and hunting purposes. During the second half of the 1980s a "new" disease (X disease), characterised by haemorrhagic and degenerative lesions and high mortality, was observed on traditional farms in some regions. This form spread throughout the country between 1986 and 1988. In 1988, diagnostic research led to identification of a viral agent. Because of its morphology, the virus was at first considered a picornavirus. More recent research has included the virus in the Caliciviridae family. This agent reproduces the disease experimentally. Between 1988 and 1989, many intensive establishments in several regions were affected by the disease, known as viral haemorrhagic disease (VHD); the losses were always very high. In 1989, after mortality among free-living hares had been observed in the northern part of Italy for some years, the agent of the European brown hare syndrome (EBHS) was identified as being morphologically similar to the VHD virus. The antigenic relationship between VHD and EBHS and the possibility of cross-infection between rabbits and hares are currently being studied. Hygienic measures and vaccination are the most reliable methods of control. International cooperation has greatly helped in the study of the disease and in the adoption of control measures.

Two groups of cats were inoculated oro-nasally with one of two isolates of feline calicivirus (FCV) from clinical cases of chronic stomatitis. All cats developed signs typical of acute FCV infection; namely, ocular and nasal discharge, conjunctivitis, and marked oral ulceration. None of the cats shed virus beyond 28 days. Seronegative control cats were then infected with a lower dose of one isolate, but again only acute signs were seen and no carriers produced. The original cats were then re-infected with the heterologous isolate. As before, only signs of acute disease were seen, but the range of clinical signs and severity was reduced. Virus shedding patterns in one group were similar to those seen originally, but in the other the duration was reduced. No chronic stomatitis developed over the 10 months of the study. Serum virus neutralising and serum and salivary class specific immunoglobulin responses were investigated. Although long-term carriers were not induced, no relationship between cessation of virus shedding in an individual animal and systemic and local antibody responses was seen.

To facilitate understanding of human rhinovirus (HRV) pathogenesis, methods were developed for detection of HRV infection in vitro using in situ hybridization (ISH). HRV-14 RNA probes and oligonucleotide probes representing conserved sequences in the 5'-non-translated region were labeled with 35S and used to detect infected HeLa or WI-38 strain human embryonic lung cells in cytological preparations. ISH was shown to be specific for detection of HRV on a single-cell basis. Subsequently, in human nasal polyps infected in vitro, both oligonucleotide- and ribo-probes produced a strong signal in association with ciliated epithelial cells. In human adenoids infected in vitro, a signal was observed in non-ciliated epithelial cells. This study shows that HRV replicates in ciliated cells in the epithelium of human nasal polyps infected in vitro, and the presence of viral RNA in non-ciliated cells of the human adenoid infected in vitro suggests that other cell types may also support rhinovirus replication.

The G-4260 strain of avian nephritis virus (ANV) was passaged using five different methods as follows: method 1, passage three times in chorioallantoic membrane (CAM) of 11-day-old embryonated eggs (CAM3); method 2, passage twice in CAM and further passage once in yolk sac (YS) of 6-day-old embryonated eggs (CAM2-YS1); method 3, passage 11 times in CAM (CAM11); method 4, passage 10 times in CAM and further passage twice in YS (CAM10-YS2); method 5, passage as in Method 4 and then passage three times in chicken kidney cell culture (CAM10-YS2-CK3). CAM11 and CAM10-YS2 were each inoculated orally into 25 one-day-old specific-pathogen-free (SPF) chicks. Seven chicks in the CAM11-inoculated group and six chicks in the CAM10-YS2-inoculated group died or were killed because they were moribund; all had either nephrosis or urate deposition. CAM3, CAM2-YS1, CAM10-YS2, and CAM10-YS2-CK3 were each inoculated intraperitoneally into 15 one-day-old SPF chicks. No chicks inoculated with CAM3 or CAM2-YS1 died, but wo chicks inoculated with CAM10-YS2 and three inoculated with CAM10-YS2-CK3 died with urate deposition. At 14 postinoculation, plasma urate values of the CAM10-YS2- and CAM10-YS2-CK3-inoculated chicks were significantly higher than those of CAM3- and CAM2-YS1-inoculated chicks and control chicks (P less than 0.01). However, interstitial nephritis was observed in most of the ANV-inoculated birds.

Between January and March 1988, an outbreak of gastroenteritis occurred among children and staff at a day-care center in Sydney, New South Wales, Australia. Over an 11-week period, 53 persons had 101 episodes of gastroenteritis; some patients had 5 separate episodes. The principal etiologic agent in the outbreak, human calicivirus (HCV), was detected by electron microscopy in 32% of fecal specimens from children and staff members with symptoms but in only 8% of asymptomatic individuals (P less than 0.01). HCV was confirmed by both an enzyme immunoassay and solid-phase immune electron microscopy. HCV infection was a particular problem in infants, who had the highest age-specific attack rates, had the greatest symptomatic/asymptomatic infection ratio, and were most likely to have a second symptomatic episode. The mode of transmission of this virus was not identified, and extensive efforts to control the 11-week outbreak had little effect. Prolonged excretion of HCV by some symptomatic patients and high rates of asymptomatic infection may have contributed to the extended duration of the outbreak. HCV may be a common cause of gastroenteritis in children that is under-recognized because of insensitive methods of detection.

An inactivated vaccine against rabbit haemorrhagic disease (RHD), developed and tested in our laboratory, is produced commercially by Bioveta, Ivanovice, Czechoslovakia. Rabbits developed full protection against infection 3 weeks after the administration of a single dose. Antibodies were detectable from day 5 after vaccination. Naturally acquired antibodies were demonstrated in some rabbits kept on commercial farms. The virus survived at least 225 days in an organ suspension kept at 4 degrees C, at least 105 days in the dried state on cloth at room temperature (around 20 degrees C), and at least 2 days at 60 degrees C, both in organ suspension and in the dry state. Experimental infection of rabbits younger than 2 months was successful in some animals. Hares, guinea pigs, white mice, golden and Chinese hamsters, chinchillas and hysterectomy-derived, colostrum-deprived piglets were resistant to infection.

Turkey viral hepatitis (TVH) was experimentally reproduced in two experiments in 1-day-old poults. In the first experiment, an infectious inoculum was prepared from filtered yolk materials harvested from dead embryonating chicken eggs (ECE) previously inoculated with suspensions of liver and pancreas tissues collected from TVH-affected birds in commercial turkey flocks. One-day-old poults given a yolk-sac inoculation or oral gavage with this preparation developed lesions in the liver and pancreas characteristic of TVH at 20 days postinoculation (PI) in 60% and 14% of the experimentally infected birds, respectively. With the identical inoculum, embryo mortality occurred at 8 and 10 days PI in embryonating turkey eggs (ETE) inoculated into the yolk sac. In the second experiment, an infectious inoculum was prepared from filtered yolk materials from dead ETE harvested in the first experiment. One-day-old poults given a yolk-sac inoculation with this filtered yolk material developed lesions in the liver and pancreas within 5 days PI. At 20 days PI, 67% of the experimentally infected birds had similar lesions. With the inoculum given to these poults, embryo mortality occurred at 6, 8, and 10 days PI in ETE inoculated into the yolk sac. Virus particles 26-28 nm in diameter with icosahedral morphology typical of picornaviruses were identified by EM in the yolk sacs of ETE that died in both experiments, and inoculated ETE that died following passage of filtered suspensions of pancreatic tissues collected from affected birds in the first experiment.

The G-4260, IR-N, M-6, and M-8 strains of avian nephritis virus (ANV) were inoculated orally into 1-day-old specific-pathogen-free chicks of the line PDL-1 for pathological and serological study. Five of 15 chicks inoculated with the G-4260 strain died with visceral urate depositions. One of 15 chicks each inoculated with the M-6 and M-8 strains died with nephrosis and visceral urate deposition, respectively. No chicks inoculated with the IR-N strain died. Mean body weights of ANV-inoculated chicks, except for the IR-N-inoculated chicks, at 14 days postinoculation (PI) were significantly lower than those of control chicks (P less than 0.01). However, interstitial nephritis was observed in all ANV-inoculated birds that were histopathologically examined at 14 days PI. In the serological study, the G-4260 and IR-N strains were classified as the same serotype, and the M-6 and M-8 strains were classified as a different serotype from the G-4260 and IR-N strains. These results indicate that there at least two serotypes of ANV and its strains differ in pathogenicity.

Epidemiological studies on SRSVs, human calicivirus and astroviruses have been limited by the problems of establishing them in cell culture and the inability to transmit them to animals or to use strains from animals as a source of antigen for diagnostic tests. The use of EM and the subsequent development of RIAs and EIAs in a few research centres has shown that they are a cause of outbreaks and sporadic cases of diarrhoea and vomiting. SRSVs have increasingly been recognized as a major cause of outbreaks of gastroenteritis in the community and in hospital wards. The symptoms of illness are generally mild and of short duration and patients seldom require medical attention. However, because of the high attack rates and large numbers of persons of all age groups involved, there is often considerable economic loss and disruption of services. Evidence is accumulating that polluted water, molluscan shellfish, and contaminated cold foods are major sources of infection. Recently a SRSV has been shown to be the cause of epidemics and sporadic cases of waterborne enterically transmitted non-A, non-B hepatitis (hepatitis E virus) which have occurred in the USSR, India, Mexico and Africa. Astroviruses and human caliciviruses are occasional causes of outbreaks of vomiting and diarrhoea in infants and the elderly which can necessitate the closure of hospital wards and cause considerable disruption. Symptoms are generally mild and of short duration and therefore the majority of cases are unlikely to be investigated by laboratories. Diagnosis of infections is at present limited to the few laboratories that have developed their own assays or have access to electronmicroscopy facilities.

The neutralisation patterns of 103 recent isolates of feline calicivirus from cats with chronic stomatitis or acute feline calicivirus disease, and from cats with neither oral nor respiratory disease were compared. There were no statistically significant differences between the proportions of isolates from each clinical source neutralised by individual feline calicivirus cat antisera. Different antisera showed widely differing degrees of cross reactivity; antisera to the most widely used vaccine strain F9 being the most cross reactive, neutralising 54 per cent of all the field isolates, and antisera to a field isolate LS015 the next most cross reactive, neutralising 29 per cent of the field isolates. However, the cross reactivity of antisera to early British isolates (A4, 68/40 and 69/1112) was much reduced (overall less than 10 per cent) whereas in the early 1970s 65 per cent of 117 field isolates from clinically normal cats were neutralised by A4 antiserum, and 40 per cent by each of 68/40 and 69/1112 antisera. This suggests a change in the spectrum of antigenicity among feline calicivirus isolates over the past 15 years. However, the cross reactivity of F9 antisera appeared to be similar to that in earlier studies. The relevance of these findings to vaccination is discussed.

A new serotype of calicivirus was isolated from California sea lions (Zalophus californianus) with severe vesicular disease. Neutralizing antibodies were found in 27 of 82 (32.9%) serum samples from California sea lions and in 15 of 146 (10.3%) serum samples from Steller sea lions (Eumetopias jubatus) tested. The seropositive animals were widely dispersed along the margins of the eastern Pacific basin, from the Bering Sea to the Santa Barbara Channel. Seropositive samples were found from as early as 1976 through the present time. This new calicivirus serotype, San Miguel sea lion virus type 13, was inoculated into weaned pigs, resulting in induction of severe vesicular disease, which spread to all pigs, including uninoculated pen contacts. Virus was continually shed by most of the pigs throughout the 2-week duration of the experiment.

Avian nephritis virus (ANV), G-4260 strain, was inoculated orally into 1-day-old specific-pathogen-free chicks of the line 15I for virological, pathological, and serological study of the ANV infection of chicks. The virus titer and incidence of appearance of fluorescent antigens in the kidneys were highest at 4 days postinoculation (PI) and decreased gradually. The degeneration of proximal tubules was the principal pathological change in kidneys of chicks at 4 days PI, and interstitial nephritis was the principal pathological change in kidneys of chicks from 6 to 14 days PI. Moreover, gouty nodules were apparent in the kidneys of more than half of the chicks at 10 days PI. The plasma urate value of infected chicks was clearly higher than that of control chicks, reaching a maximum at 10 days PI, then gradually decreasing. The serum antibody was detected from 4 days PI and increased gradually. The chicks affected with visceral urate deposits or urate deposits in the digitated joints showed high plasma urate values (more than 100 mg/dl).

Viruses change the mRNA repertoire of the tissues they infect. They add viral mRNAs and they specifically alter the expression of some host genes. These events can play important parts in pathogenesis. In principle, it should be possible to isolate viral mRNAs and to identify changes in host gene expression using subtractive hybridization. We tested this approach in the persistent infection of mouse central nervous system by Theiler's virus. A cDNA library was constructed with poly A+ RNA from infected mouse spinal cords. The library was screened with a subtracted probe. We identified one mitochondrial gene, coding for subunit 1 of cytochrome oxidase, which is overexpressed in infected tissues whereas another mitochondrial gene, URF 2, is not. Subtractive hybridization should prove to be invaluable in studying the pathogenesis of chronic human central nervous system diseases of unknown etiology.