Proteolytic processing of the astrovirus capsid

To further characterize the nature of proteolytic processing of the astrovirus capsid, we infected Caco-2 cells with a high multiplicity of astrovirus without trypsin in the presence of 5 to 10% fetal calf serum. These infections were characterized by pulse-chase labeling with [35S]Smethionine, electron microscopy, gel electrophoresis of purified viral particles, and analysis of infectivity of such particles with and without added trypsin. Pulse-chase experiments showed that the astrovirus capsid protein was initially translated as an approximately 87-kDa protein. The 87-kDa capsid protein was rapidly converted intracellularly to a 79-kDa form which was found in smaller amounts in the cell supernatant. Purification by differential centrifugation yielded particles that appeared quite similar to trypsin-grown astrovirus particles by negatively stained electron microscopy. These particles were antigenically distinct from trypsin-treated virions as demonstrated by their various reactions with monoclonal antibodies in a solid-phase immunoassay. The purified trypsin-free particles were mainly composed of the 79-kDa capsid protein which was found to have an amino terminus at residue 71 of the entire open reading frame 2 (ORF2) product. The cleavage site was identified in a highly conserved region of the astrovirus ORF2 product. These trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious (10(5)-fold increase) after trypsin but not chymotrypsin treatment. This trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 34, 29, and 26 kDa.     

Sequence analysis of the human virome in febrile and afebrile children

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF. We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.     

Natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease

We describe the natural history, viral dynamics, and immunobiology of feline infectious peritonitis (FIP), a highly lethal coronavirus infection. A severe recurrent infection developed, typified by viral persistence and acute lymphopenia, with waves of enhanced viral replication coinciding with fever, weight loss, and depletion of CD4+ and CD8+ T cells. Our combined observations suggest a model for FIP pathogenesis in which virus-induced T-cell depletion and the antiviral T-cell response are opposing forces and in which the efficacy of early T-cell responses critically determines the outcome of the infection. Rising amounts of viral RNA in the blood, consistently seen in animals with end-stage FIP, indicate that progression to fatal disease is the direct consequence of a loss of immune control, resulting in unchecked viral replication. The pathogenic phenomena described here likely bear relevance to other severe coronavirus infections, in particular severe acute respiratory syndrome, for which multiphasic disease progression and acute T-cell lymphopenia have also been reported. Experimental FIP presents a relevant, safe, and well-defined model to study coronavirus-mediated immunosuppression and should provide an attractive and convenient system for in vivo testing of anticoronaviral drugs.     

Pathogens in children with severe combined immune deficiency disease or AIDS.

We evaluated the frequency and severity of illnesses caused by various microbial pathogens in 15 children with severe combined immune deficiency disease (SCID) and 8 with acquired immune deficiency syndrome (AIDS). There were 35 viral, 23 bacterial, 19 mycotic and 13 parasitic infections. Nineteen of the 23 patients died of infection; Pneumocystis carinii pneumonia, giant-cell pneumonia due to paramyxoviruses and various disseminated viral infections were responsible for most deaths in both groups. The emerging role of paramyxoviruses was illustrated by the fact that they were responsible for giant-cell pneumonia in seven patients. Viral enteric infections were frequent in both groups. The variety of infectious microorganisms and the severity of resulting illnesses in the patients with AIDS were similar to those in the patients with SCID.     

Small spherical viruses in faeces from gastroenteritis patients

Faecal samples from 238 patients with gastroenteritis were examined by direct electron microscopy using grids with thin carbon film. Of these samples 18 were found to contain Norwalk agent-like particles, calicivirus, astrovirus and parvovirus-like particles. Immune electron microscopy was performed on a serum pair and faeces from one of the patients with astrovirus. An antibody response was demonstrated, suggesting that the virus was the etiological agent of the infection.     

Temporal synthesis of proteins and RNAs during human astrovirus infection of cultured cells

Astroviruses are nonenveloped particles with a distinctive star-shaped surface structure that have been detected by electron microscopy in stool samples from humans and animals with gastroenteritis. We examined the patterns of macromolecular synthesis in astrovirus-infected cells with a goal of establishing a molecular basis for taxonomic classification. Trypsin is required for continuous replication of astrovirus in cultured cells; however, during a single cycle of infection, astrovirus antigen was synthesized earlier and at higher levels when serum, rather than trypsin, was included in the growth medium. This enhanced production of antigen, as measured by enzyme immunoassay, was accompanied by the appearance of aggregates of virus particles in the cytoplasm of infected cells. During astrovirus replication in cells cultured in the presence of serum, we detected a single infection-specific protein (90 kDa) beginning at 12 h postinfection. This protein was recognized by antiastrovirus rabbit serum and was sensitive to trypsin digestion in vitro, with the concomitant appearance of three smaller immunoreactive proteins (31, 29, and 20 kDa). We also detected two dactinomycin-resistant RNAs (7.2 and 2.8 kb), both of which were polyadenylated, in the cytoplasm of astrovirus-infected cells. The larger of these two RNAs is presumably the viral genome, whereas the smaller species may be a subgenomic messenger. Comparison of the proteins and RNAs synthesized in astrovirus-infected cells with those of the recognized families of nonenveloped single-stranded RNA animal viruses suggests that astroviruses should not be classified as members of either Caliciviridae or Picornaviridae.     

Serial observations of chronic rotavirus infection in an immunodeficient child.

Chronic rotavirus infection of an infant with severe combined immunodeficiency (SCID) was studied by virological examinations in association with long-term observation of his symptoms and immune status. During eleven months of hospitalization, the patient was suffering from incurable severe diarrhea with persisting excretion of rotaviruses detected by electron microscopy and the reversed-passive hemagglutination (R-PHA) test and had transient hepatitis symptom despite multiple administrations of human gammaglobulin and high calorie fluids. The detected viruses were morphologically recognized as rotavirus with double capsid structure. Polyacrylamide gel electrophoretic (PAGE) analysis of their genomic RNAs showed the long electropherotype of group A virus with abnormal migration profiles changing considerably from the early to the late phase of illness: (1) The 11th segment became undetectable; (2) the molecular weight of the 6th segment slightly increased; (3) seven to fourteen extra segments appeared; and (4) PAGE patterns of viral genomic RNAs changed every three or four months. These findings suggest that chronic infection with rotavirus accompanied the generation of extra viral genomic segments and their unusual assortments in an immunodeficient host.     

Detection of human cytomegalovirus DNA and viral antigens in tissues of different manifestations of CMV infection

Biopsy and autopsy specimens from 22 patients with cytomegalovirus (CMV) infections were investigated by means of in situ hybridization (ISH) to detect viral DNA and by immunohistochemistry (IHC) to visualize viral proteins. Both methods proved to be valuable tools for histopathology. ISH sometimes recognized cells that did not show typical CMV inclusions. An antiserum against the full spectrum of viral proteins (non-infectious enveloped particles) detected most cytomegalic cells in disseminated and organ-limited infections. An antiserum against a recombinant polypeptide (XP1) was particularly useful in connatal CMV infections and organ-limited infections. We have demonstrated that IHC and ISH studies in parallel are the best approach to the detection of CMV infections in pathological specimens.     

Three-year survey of the epidemiology of rotavirus, enteric adenovirus, and some small spherical viruses including "Osaka-agent" associated with infantile diarrhea

Studies were made by electron microscopy (EM) on the viruses associated with diarrhea of outpatients at a pediatric clinic in Osaka Prefecture during the three year period from 1980 through 1982. The viruses detected by EM by negative staining with phosphotungstic acid (PTA) were classified morphologically into 6 groups: rotavirus, adenovirus and four kinds of small spherical viruses, calicivirus, astrovirus, picornavirus/parvovirus (P/P)-like agent and Osaka-agent. Osaka-agent seems to be a newly identified small virus. It is 35-40 nm in diameter with a fringe of spike-like structures on its surface. Viruses were detected in 181 of the 395 cases of diarrhea (45.8%). Rotavirus was detected in 122 (30.9%) of the total cases and in 67.4% of the virus-positive cases, while other viruses were detected in 15% of the total cases; adenovirus in 23 (6%) and small agents in 36 (9%). Rotavirus infection showed a distinctive seasonal variation, being mainly restricted to cooler months, but infections with other viruses did not show any seasonal variation. The age distribution of patients suggested that infants of 0 to 2 years old are very susceptible to all viruses. Attempts to cultivate these viruses in vitro were successful with only two isolates of adenovirus type 5.     

Astrovirus induces diarrhea in the absence of inflammation and cell death

Astroviruses are a leading cause of infantile viral gastroenteritis worldwide. Very little is known about the mechanisms of astrovirus-induced diarrhea. One reason for this is the lack of a small-animal model. Recently, we isolated a novel strain of astrovirus (TAstV-2) from turkeys with the emerging infectious disease poult enteritis mortality syndrome. In the present studies, we demonstrate that TAstV-2 causes growth depression, decreased thymus size, and enteric infection in infected turkeys. Infectious TAstV-2 can be recovered from multiple tissues, including the blood, suggesting that there is a viremic stage during infection. In spite of the severe diarrhea, histopathologic changes in the intestine were mild and there was a surprising lack of inflammation. This may be due to the increased activation of the potent immunosuppressive cytokine transforming growth factor beta during astrovirus infection. These studies suggest that the turkey will be a useful small-animal model with which to study astrovirus pathogenesis and immunity.     

Herpes B virus replication and viral lesions in the liver of a cynomolgus macaque which died from severe disease with rapid onset

Herpes B virus (BV, Macacine alphaherpesvirus 1) infects macaques asymptomatically, with rare exceptions, but can cause fatal encephalitis in humans. Here, we report disseminated BV infection in a cynomolgus macaque that had died within 12 hour after the onset of unspecific symptoms. Multifocal lesions surrounded by viral antigen were detected in liver while other organs remained inconspicuous, indicating that the liver is a major target. Moreover, high copy numbers of viral DNA were found in feces, underlining the excrements are a potential source of transmission.     

致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用

为探讨致倦库蚊对登革Ⅱ型病毒的中肠感染屏障作用,通过病毒分离、逆转录聚合酶链反应、透射电镜等技术进行了相关研究。结果表明：吸食感染性血液后,登革Ⅱ型病毒能侵染白纹伊蚊中肠上皮细胞并大量复制,但不能侵染致倦库蚊中肠上皮细胞。以上研究证明致倦库蚊对登革Ⅱ型病毒存在中肠感染屏障。     

Detection of human cytomegalovirus DNA and viral antigens in tissues of different manifestations of CMV infection

Biopsy and autopsy specimens from 22 patients with cytomegalovirus (CMV) infections were investigated by means of in situ hybridization (ISH) to detect viral DNA and by immunohistochemistry (IHC) to visualize viral proteins. Both methods proved to be valuable tools for histopathology. ISH sometimes recognized cells that did not show typical CMV inclusions. An antiserum against the full spectrum of viral proteins (non-infectious enveloped particles) detected most cytomegalic cells in disseminated and organ-limited infections. An antiserum against a recombinant polypeptide (XP1) was particularly useful in connatal CMV infections and organ-limited infections. We have demonstrated that IHC and ISH studies in parallel are the best approach to the detection of CMV infections in pathological specimens. Presented in part at the 71st Congress of the German Pathological Society, Salzburg, June 1987     

Roles of the gut microbiota in severe SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. The pathophysiological mechanisms linking gut dysbiosis and severe SARS-CoV-2 infection are poorly understood, although gut microbiota disorders are related to severe SARS-CoV-2 infections. The roles of the gut microbiota in severe SARS-CoV-2 infection were compared with those in respiratory viral infection, which is an easily understood and enlightening analogy. Secondary bacterial infections caused by immune disorders and antibiotic abuse can lead to dysregulation of the gut microbiota in patients with respiratory viral infections. The gut microbiota can influence the progression of respiratory viral infections through metabolites and the immune response, which is known as the gut–lung axis. Angiotensin-converting enzyme 2 is expressed in both the lungs and the small intestine, which may be a bridge between the lung and the gut. Similarly, SARS-CoV-2 infection has been shown to disturb the gut microbiota, which may be the cause of cytokine storms. Bacteria in the gut, lung, and other tissues and respiratory viruses can be considered microecosystems and may exert overall effects on the host. By referencing respiratory viral infections, this review focused on the mechanisms involved in the interaction between SARS-CoV-2 infections and the gut microbiota and provides new strategies for the treatment or prevention of severe SARS-CoV-2 infections by improving gut microbial homeostasis.     

Crystal Structure of the Avian Astrovirus Capsid Spike

Astroviruses are small, nonenveloped, single-stranded RNA viruses that cause diarrhea in a wide variety of mammals and birds. On the surface of the viral capsid are globular spikes that are thought to be involved in attachment to host cells. To understand the basis of species specificity, we investigated the structure of an avian astrovirus capsid spike and compared it to a previously reported human astrovirus capsid spike structure. Here we report the crystal structure of the turkey astrovirus 2 (TAstV-2) capsid surface spike domain, determined to 1.5-Å resolution, and identify three conserved patches on the surface of the spike that are candidate avian receptor-binding sites. Surprisingly, the overall TAstV-2 capsid spike structure is unique, with only distant structural similarities to the human astrovirus capsid spike and other viral capsid spikes. There is an absence of conserved putative receptor-binding sites between the human and avian spikes. However, there is evidence for carbohydrate-binding sites in both human and avian spikes, and studies with human astrovirus 1 (HAstV-1) suggest a minor role in infection for chondroitin sulfate but not heparin. Overall, our structural and functional studies provide new insights into astrovirus host cell entry, species specificity, and evolution.     

Pediatric Virology

Pediatric virology is not an isolàted discipline. Rather, the syndromes associated with viral infection are modified by the unique characteristics of infancy and childhood. Fortunately for the pediatrician, and certainly for children, viral infections in childhood are rarely fatal, and are almost never serious. Future efforts of the pediatrician and virologist should be directed toward increased fetal salvage as with rubella and the prevention of severe, viral lower respiratory tract disease.     

人星状病毒研究进展

人星状病毒是引起婴幼儿病毒性腹泻的重要病原之一,本文回顾了其分子生物学特征、型别、所致疾病特征、致病机制、流行病学特征及检测方法等方面的文献,指出人星状病毒目前存在多种型别,尤其是近年来发现了多种新型的星状病毒,主要的流行型别是血清型1型,不仅引起肠道内感染,还可以引起其他系统疾病,所致疾病特征和其他腹泻相关病毒相似,但是其致病机制的研究尚不清楚,对于几种新发现的星状病毒,其与疾病的相关性研究较少,且未建立完善的实验室检测方法,还需要进一步的研究。     

Identification of viruses in the urine of renal transplant recipients by cytomorphology

This study was designed to reassess the cytomorphology of viral infections in urinary cells obtained from renal transplant patients and to examine the association, if any, between these cytologic changes and the transplant rejection. A total of 2,354 cytologic specimens obtained from 91 renal transplant recipients was evaluated. A combination of techniques, including cellulosic filters, immunofluorescence, hemagglutination inhibition and electron microscopy, was utilized. Cytologic observations were correlated with the patient's clinical history. Thirty-eight patients revealed cytologic evidence of viral infections (herpes simplex, cytomegalovirus and papovavirus). These viral infections had distinct cytomorphology. Cytomegalovirus infection may manifest as intracytoplasmic, orangeophilic inclusions, in addition to the classical intranuclear inclusion. In the majority of renal transplant patients there appeared to be no relationship between the viral infection and the renal transplant rejection episodes.