Changes in small intestinal homeostasis, morphology, and gene expression during rotavirus infection of infant mice

Rotavirus is the most important cause of infantile gastroenteritis. Since in vivo mucosal responses to a rotavirus infection thus far have not been extensively studied, we related viral replication in the murine small intestine to alterations in mucosal structure, epithelial cell homeostasis, cellular kinetics, and differentiation. Seven-day-old suckling BALB/c mice were inoculated with 2 x 10(4) focus-forming units of murine rotavirus and were compared to mock-infected controls. Diarrheal illness and viral shedding were recorded, and small intestinal tissue was evaluated for rotavirus (NSP4 and structural proteins)- and enterocyte-specific (lactase, SGLT1, and L-FABP) mRNA and protein expression. Morphology, apoptosis, proliferation, and migration were evaluated (immuno)histochemically. Diarrhea was observed from days 1 to 5 postinfection, and viral shedding was observed from days 1 to 10. Two peaks of rotavirus replication were observed at 1 and 4 days postinfection. Histological changes were characterized by the accumulation of vacuolated enterocytes. Strikingly, the number of vacuolated cells exceeded the number of cells in which viral replication was detectable. Apoptosis and proliferation were increased from days 1 to 7, resulting in villous atrophy. Epithelial cell turnover was significantly higher (<4 days) than that observed in controls (7 days). Since epithelial renewal occurred within 4 days, the second peak of viral replication was most likely caused by infection of newly synthesized cells. Expression of enterocyte-specific genes was downregulated in infected cells at mRNA and protein levels starting as early as 6 h after infection. In conclusion, we show for the first time that rotavirus infection induces apoptosis in vivo, an increase in epithelial cell turnover, and a shutoff of gene expression in enterocytes showing viral replication. The shutoff of enterocyte-specific gene expression, together with the loss of mature enterocytes through apoptosis and the replacement of these cells by less differentiated dividing cells, likely leads to a defective absorptive function of the intestinal epithelium, which contributes to rotavirus pathogenesis

Spot the Difference-Development of a Syndrome Based Protein Microarray for Specific Serological Detection of Multiple Flavivirus Infections in Travelers

Background The family Flaviviridae, genus Flavivirus, holds many of the world’s most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods. Goal We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray. Results Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses. Conclusion Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses.

The hanta hunting study: Underdiagnosis of puumala hantavirus infections in symptomatic non-travelling leptospirosis-suspected patients in the Netherlands, in 2010 and April to November 2011

Leptospirosis and haemorrhagic fever with renal syndrome (HFRS) are hard to distinguish clinically since these two important rodent-borne zoonoses share hallmark symptoms such as renal failure and haemorrhage. Leptospirosis is caused by infection with a spirochete while HFRS is the result of an infection with certain hantaviruses. Both diseases are relatively rare in the Netherlands. Increased incidence of HFRS has been observed since 2007 in countries that border the Netherlands. Since a similar rise in incidence has not been registered in the Netherlands, we hypothesise that due to overlapping clinical manifestations, hantavirus infections may be confused with leptospirosis, leading to underdiagnosis. Therefore, we tested a cohort of non-travelling Dutch patients with symptoms compatible with leptospirosis, but with a negative diagnosis, during 2010 and from April to November 2011. Sera were screened with pan-hantavirus IgG and IgM enzyme-linked immunosorbent assays (ELISAs). Sera with IgM reactivity were tested by immunofluorescence assay (IFA). ELISA (IgM positive) and IFA results were confirmed using focus reduction neutralisation tests (FRNTs). We found hantavirus-specific IgG and/or IgM antibodies in 4.3% (11/255) of samples taken in 2010 and in 4.1% (6/146) of the samples during the 2011 period. After FRNT confirmation, seven patients were classed as having acute Puumala virus infections. A review of hantavirus diagnostic requests revealed that at least three of the seven confirmed acute cases as well as seven probable acute cases of hantavirus infection were missed in the Netherlands during the study period

Test, trace, isolate:Evidence for declining SARS-CoV-2 PCR sensitivity in a clinical cohort

Real-time reverse transcription-polymerase chain reaction (RT-PCR) on upper respiratory tract (URT) samples is the primary method to diagnose SARS-CoV-2 infections and guide public health measures, with a supportive role for serology. We reinforce previous findings on limited sensitivity of PCR testing, and solidify this fact by statistically utilizing a firm basis of multiple tests per individual. We integrate stratifications with respect to several patient characteristics such as severity of disease and time since onset of symptoms. Bayesian statistical modelling was used to retrospectively determine the sensitivity of RT-PCR using SARS-CoV-2 serology in 644 COVID-19-suspected patients with varying degrees of disease severity and duration. The sensitivity of RT-PCR ranged between 80% − 95%; increasing with disease severity, it decreased rapidly over time in mild COVID-19 cases. Negative URT RT-PCR results should be interpreted in the context of clinical characteristics, especially with regard to containment of viral transmission based on ‘test, trace and isolate’

Etiology of atopy in infancy: the KOALA Birth Cohort Study.

PG - 679-84 AB - The aim of the KOALA Birth Cohort Study in the Netherlands is to identify factors that influence the clinical expression of atopic disease with a main focus on lifestyle (e.g., anthroposophy, vaccinations, antibiotics, dietary habits, breastfeeding and breast milk composition, intestinal microflora composition, infections during the first year of life, and gene-environment interaction). The recruitment of pregnant women started in October 2000. First, participants with 'conventional lifestyles' (n = 2343) were retrieved from an ongoing prospective cohort study (n = 7020) on pregnancy-related pelvic girdle pain. In addition, pregnant women (n = 491) with 'alternative lifestyles' with regard to child rearing practices, dietary habits (organic, vegetarian), vaccination schemes and/or use of antibiotics, were recruited through organic food shops, anthroposophic doctors and midwives, Steiner schools, and dedicated magazines. All participants were enrolled between 14 and 18 wk of gestation and completed an intake questionnaire on family history of atopy and infant care intentions. Documentation of other relevant variables started in the pregnant mother and covered the first and third trimester as well as early childhood by repeated questionnaires at 14-18, 30, and 34 wk of gestation and 3, 7, 12, and 24 months post-partum. A subgroup of participants, including both conventional and alternative lifestyles, was asked to consent to maternal blood sampling, breast milk and a faecal sample of the infant at 1 month post-partum, capillary blood at age 1 yr, venous blood and observation of manifestation of atopic dermatitis during home visits at the age of 2 yr (using the UK working party criteria and the severity scoring of atopic dermatitis index), and buccal swabs for DNA isolation from child-parent trios. From the start, ethical approval and informed consent procedures included gene-environment interaction studies. Follow-up at 3 and 7 months post-partum was completed with high response rates (respectively 90% and 88% in the conventional group, and 97% and 97% in the alternative group). The home visits at 2 yr of age will be completed in 2005. Preliminary results show that we have succeeded in recruiting a large population with various lifestyle choices with a fairly large contrast with regard to dietary habits (including organic foods, vegetarian diet), vaccination schemes and/or use of antibiotics. We have also been able to collect a large number of faecal samples (n = 1176) and capillary blood samples at age 1 yr (n = 956). Furthermore, a large proportion of the participants have consented with genetic studies. Mid 2006 we expect to report our first results on the relationship between the various exposures in early life and childhood atopy. An outline of the focus and design of the KOALA Birth Cohort Study is presented. AD - Department of Epidemiology, Care and Public Health Research Institute (Caphri), Maastricht University, Maastricht, The Netherlands. [email protected]

Remarkable spatial variation in the seroprevalence of Coxiella burnetii after a large Q fever epidemic.

Prior to the 2007-2010 Q fever epidemic in the Netherlands, the seroprevalence of antibodies against Coxiella burnetii in the general population was 1.5%, which is low compared to other countries. We aimed to determine the seroprevalence after the Q fever epidemic among people living in the affected area, compare the seroprevalence with the incidence of Q fever notifications during the 2007-2010 Q fever epidemic, and to identify farm exposures associated with having antibodies against C. burnetii

Spot the Difference—Development of a Syndrome Based Protein Microarray for Specific Serological Detection of Multiple Flavivirus Infections in Travelers

The family Flaviviridae, genus Flavivirus, holds many of the world’s most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods. We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray. Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses

Specialist laboratory networks as preparedness and response tool - the Emerging Viral Diseases-Expert Laboratory Network and the Chikungunya outbreak, Thailand, 2019.

We illustrate the potential for specialist laboratory networks to be used as preparedness and response tool through rapid collection and sharing of data. Here, the Emerging Viral Diseases-Expert Laboratory Network (EVD-LabNet) and a laboratory assessment of chikungunya virus (CHIKV) in returning European travellers related to an ongoing outbreak in Thailand was used for this purpose. EVD-LabNet rapidly collected data on laboratory requests, diagnosed CHIKV imported cases and sequences generated, and shared among its members and with the European Centre for Disease Prevention and Control. Data across the network showed an increase in CHIKV imported cases during 1 October 2018-30 April 2019 vs the same period in 2018 (172 vs 50), particularly an increase in cases known to be related to travel to Thailand (72 vs 1). Moreover, EVD-LabNet showed that strains were imported from Thailand that cluster with strains of the ECSA-IOL E1 A226 variant emerging in Pakistan in 2016 and involved in the 2017 outbreaks in Italy. CHIKV diagnostic requests increased by 23.6% between the two periods. The impact of using EVD-LabNet or similar networks as preparedness and response tool could be improved by standardisation of the collection, quality and mining of data in routine laboratory management systems