A critical part of the hepatitis B virus (HBV) life cycle is the packaging of the pregenomic RNA (pgRNA) into nucleocapsids. While this process is known to involve several viral elements, much less is known about the identities and roles of host proteins in this process. To better understand the role of host proteins, we isolated pgRNA and characterized its protein interactome in cells expressing either packaging-competent or packaging-incompetent HBV genomes. We identified over 250 host proteins preferentially associated with pgRNA from the packaging-competent version of the virus. These included proteins already known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to the viral genome, demonstrating the ability of the approach to effectively reveal functionally significant host-virus interactors. Three of these host proteins, AURKA, YTHDF2, and ATR, were selected for follow-up analysis. RNA immunoprecipitation qPCR (RIP-qPCR) confirmed pgRNA-protein association in cells, and siRNA knockdown of the proteins showed decreased encapsidation efficiency. This study provides a template for the use of comparative RNA-protein interactome analysis in conjunction with virus engineering to reveal functionally significant host-virus interactions.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Hepatitis B virus (HBV) is an enveloped, reverse-transcribing DNA virus that is a major cause of liver disease and hepatocellular carcinoma. Subcellular trafficking events underpinning HBV capsid assembly and virion egress remain poorly characterized.

Reliable laboratory testing is of great importance to detect Bartonella bacilliformis infection. We evaluated the sensitivity and specificity of the enzyme-linked immunosorbent assay (ELISA) using recombinant protein Pap31 (rPap31) for the detection of antibodies against B. bacilliformis as compared with immunofluorescent assay (IFA). Of the 302 sera collected between 1997 and 2000 among an at-risk Peruvian population, 103 and 34 samples tested positive for IFA-immunoglobulin G (IgG) and IFA-IgM, respectively. By using Youden's index, the cutoff values of ELISA-IgG at 0.915 gave a sensitivity of 84.5% and specificity of 94%. The cutoff values of ELISA-IgM at 0.634 gave a sensitivity of 88.2% and specificity of 85.1%. Using latent class analysis, estimates of sensitivity and specificity of almost all the assays were slightly higher than those of a conventional method of calculation. The test is proved beneficial for discriminating between infected and non-infected individuals with the advantage of low-cost and high-throughput capability.

Carrion's disease is caused by infection with the alpha-proteobacterium Bartonella bacilliformis. Distribution of the disease is considered coincident with the distribution of its known vector, the sand fly Lutzomyia verrucarum. Recent epidemics of B. bacilliformis infections associated with atypical symptomatology in nonendemic regions have raised questions regarding the historic and present distribution of this bacterium and the scope of disease that infection causes. Phylogenetic relationships and genomic diversity of 18 B. bacilliformis isolates (10 isolates from a region where Carrion's disease is epidemic, Cuzco, Peru, and 8 isolates from a region where Carrion's disease is endemic, Caraz, Peru) were assessed using genomic data generated by infrequent restriction site PCR and gene sequence analysis of the flagellin gltA and ialB genes. A population genetic analysis of the genomic diversity suggests that what was once considered an epidemic region of Peru did not result from the recent introduction of B. bacilliformis.

: Evidence incriminating phlebotomine sand flies as vectors of Bartonella bacilliformis, which causes bartonellosis, has not been proven beyond doubt. Our research was designed to strengthen this hypothesis. In addition, we developed a Real- Time PCR method capable of detecting as little as 100fg of Bartonella DNA in sand flies. Twelve primers were designed using TaqMan Probe/Primer Data and tested. Lutzomyia verrucarum sand flies were collected from an endemic focus of bartonellosis in and around Caraz, Ancash, Peru. Unfed female sand flies were pooled in groups of 5 for DNA extraction. Of 472 pools assayed, 13 tested positive (2.75%). The sensitivity of the test was 100% and specificity was 91.11% based on assays of known samples. We conclude that this method has the necessary sensitivity and specificity to detect B. bacilliformis infection in wild-caught sand flies enabling us to determine the true infection sand fly rates in an endemic area of Peru.

Bartonella bacilliformis has caused debilitating illness since pre-Incan times, but relatively little is known about its epidemiology. A population-based, prospective cohort investigation was conducted in a Peruvian community with endemic bartonellosis. By use of house-to-house and hospital surveillance methods, cohort participants were monitored for evidence of bartonellosis. Of 690 participants, 0.5% had asymptomatic bacteremia at study initiation. After 2 years of follow-up, the incidence of infection was 12.7/100 person-years. The highest rates were in children <5 years old, and there was a linear decrease in incidence with increasing age. Seventy percent of cases were clustered in 18% of households. Age and bartonellosis in a family member were the best predictors of B. bacilliformis infection. There were multiple clinical presentations and significant subclinical infection. A cost-effective control strategy should include vector control and surveillance efforts focused on children and clusters of households with highest endemicity.

Bartonella bacilliformis causes bartonellosis, a potentially life-threatening emerging infectious disease seen in the Andes Mountains of South America. There are no generally accepted serologic tests to confirm the disease. We developed an indirect fluorescence antibody (IFA) test for the detection of antibodies to B. bacilliformis and then tested its performance as an aid in the diagnosis of acute bartonellosis. The IFA is 82% sensitive in detecting B. bacilliformis antibodies in acute-phase blood samples of laboratory-confirmed bartonellosis patients. When used to examine convalescent-phase sera, the IFA is positive in 93% of bartonellosis cases. The positive predictive value of the test is 89% in an area of Peru where B. bacilliformis is endemic and where the point prevalence of infection is 45%.

ABSTRACT Two simple Bartonella bacilliformis immunoblot preparation methods were developed. Antigen was prepared by two different methods: sonication of whole organisms or glycine extraction. Both methods were then tested for sensitivity and specificity. Well-defined control sera were utilized in the development of these diagnostic immunoblots, and possible cross-reactions were thoroughly examined. Sera investigated for cross-reaction with these diagnostic antigens were drawn from patients with brucellosis, chlamydiosis, Q fever, and cat scratch disease, all of whom were from regions where bartonellosis is not endemic. While both immunoblots yielded reasonable sensitivity and high specificity, we recommend the use of the sonicated immunoblot, which has a higher sensitivity when used to detect acute disease and produces fewer cross-reactions. The sonicated immunoblot reported here is 94% sensitive to chronic bartonellosis and 70% sensitive to acute bartonellosis. In a healthy group, it is 100% specific. This immunoblot preparation requires a simple sonication protocol for the harvesting of B. bacilliformis antigens and is well suited for use in regions of endemicity.

To evaluate enteropathogens and other factors associated with severe disease in children with diarrhea, 381 children <5 years of age with diarrhea and moderate to severe dehydration (in-patients) and 381 age-, sex-, and date-of-visit-matched children with mild diarrhea (out-patients) presenting to a hospital in Peru, were studied. Rotavirus was detected in 52% of the in-patients and 35% of the out-patients (odds ratio [OR]=2.3, 95% confidence interval [95% CI]= 1.6-3.2); 95% of the rotaviruses among in-patients were of serotypes G1-G4. The risk of severe diarrhea was particularly great in children who were not exclusively breast-fed in early infancy and who also lacked piped water in their homes (for children with both characteristics OR=6.8, 95% CI=3.6-12.8). The high prevalence of rotavirus and its association with severe diarrhea underscores the need for rotavirus vaccines. Interventions to educate mothers and improve access to safe water should augment the impact of rotavirus vaccines in preventing severe diarrhea.

Sprague-Dawley rats are frequently used in experiments and their normal hematological, serologic and anatomical parameters are not easily available. The aim of this study was to measure these parameters in adult Sprague-Dawley rats. Twenty-four rats (12 males) whose weight ranged between 200 and 300 g, raised at the Instituto de Salud Pública in controlled environmental conditions and fed with pellets were studied. After a 24 h fast, animals were anesthetized with Droperidol and Ketamine, and after obtaining a cardiac blood sample, sacrificed by exsanguination. Most organs were withdrawn and weighed. Mean values for blood glucose, calcium, amylase, total proteins, cholesterol, AST, ALT, bilirubin, alkaline phosphatases, electrolytes and complete blood count were determined. Heart, lung, pancreas, kidney and testicle mean weight was also calculated. Male and female rats had significant differences in packed red cell volume, white blood cell count and hemoglobin.