The Robert Koch Institute (RKI): main building in Berlin. more
Germany’s Public Health institute RKI monitors the population’s health and wellbeing. more
The Robert Koch Institute is the German government's central institution for the identification, surveillance and prevention of infectious diseases in the Portfolio of the German Ministry of Health. more
The molecular characterization of circulating enteroviruses enables statements on phylogenetic relationships and the detection of infection chains. The methods of nucleic acid amplification and sequencing in different genome regions are used to investigate pathogen variability (antigenic drift) and to compare these with biological properties (e.g. neutralization test). The aim is also to use amplification techniques with subsequent sequencing in the protein-coding region to obtain information on the enterovirus type directly from original clinical material, bypassing virus cultivation.
Examples of picornavirus types currently being processed at the NRZ PE are enterovirus type A71 (EV-A71) and human parechoviruses (HPeV).
Enterovirus A71 (EV-A71)
Enterovirus A71 is widespread worldwide and repeatedly causes large hand-foot-and-mouth (HFMD) epidemics, particularly in Southeast Asia. In Germany, EV-A71 is primarily detected in cases of aseptic meningitis/encephalitis, less frequently in patients with acute flaccid paralysis (AFP) or HFMD.
The EV-A71-positive samples received within the nationwide enterovirus surveillance (EVSurv) are analyzed with molecular and virologic methods at the NRZ PE. The EV-A71-specific PCR in the VP1 region enables not only the determination of the type, but also the assignment to a genogroup. This showed, for example, that in 2011, in addition to the previously dominant C2 genogroup, C4 viruses could also be detected, which showed high homologies to the viruses circulating in Southeast Asia. In 2015, a new variant within the C1 genogroup was detected in Germany. These recombinant viruses caused an outbreak in Spain/Catalonia in 2016 (brain stem encephalitis) and have also been detected in other European countries (e.g. France, the Netherlands, Denmark) and worldwide (e.g. USA, Japan, Australia, Africa).
Surveillance of circulating enteroviruses is of great importance for a better understanding of the epidemiology of enteroviral infections.
As these are not nationally notifiable diseases or pathogens according to the Infection Protection Act (IfSG) (except in cases of suspected poliomyelitis), the NRZ PE relies on the support of the health authorities and hospitals. As part of nationwide enterovirus surveillance and in case of outbreaks (e.g. HFMD), the detection/typing of enteroviruses at the NRZ PE is free of charge.
Human parechoviruses (HPeV)
Parechoviruses generally cause mild infections of the gastrointestinal and respiratory tract. In addition to meningitis/encephalitis but also myocarditis, infections in children under 3 months of age in particular present as neonatal sepsis. Infections with parechoviruses are widespread and occur mainly in the first five years of life. Neonatal infections usually present as sepsis. While parechovirus types 1 and 2 have been known for a long time, at least 15 other parechovirus types have recently been detected using molecular methods. The sequencing of the protein-coding VP1 region and/or entire genome enables molecular investigations into the parechoviruses circulating in Germany.
The European Network for Non-Polio Enteroviruses (ENPEN) was established under the auspices of the European Society of Clinical Virology (ESCV). ENPEN brings together experts from various clinical disciplines (pediatrics, neurology, virology), public health institutions, national reference laboratories and universities from over 20 European countries. The NRZ PE has been actively involved in this network since its inception, the main aim of which is to raise awareness for more effective detection of enterovirus diseases, exchange data on outbreaks and emerging enteroviruses and improve corresponding preventive measures.
Current topics include:
As the WHO/EURO Regional Reference Laboratory for Poliomyelitis, the NRC Poliomyelitis and Enteroviruses is responsible for Central and Western Europe (Poland, Hungary, Spain, Portugal, France, Switzerland, Belgium, Luxembourg, Republic of Moldova). The main task here is the sequencing of the protein-coding VP1 region of the poliovirus isolates sent in. Europe was certified polio-free over 20 years ago (June 2002). However, as global eradication has not yet been achieved, there is a risk of re-introduction of wild polioviruses into polio-free countries. In the final phase of polio eradication, vaccine-derived polioviruses (VDPV) are of particular interest. As experience in recent years has shown, such viruses can circulate in the population with insufficient population immunity (cVDPV) and cause polio epidemics. However, polioviruses can also be shed for many years in people with immunodeficiency (iVDPV).
Therefore, the circulation of polioviruses must continue to be monitored. The WHO requires that all polioviruses (regardless of their origin) be subjected to vaccine/wild virus differentiation. PCR-based methods are used for this (e.g. Sabin vaccine virus-specific PCR or screening of VDPV). The sequencing of polioviruses in different genome regions is also established at the NRZ PE.
In Germany, syndromic surveillance of hospitalized patients with suspected aseptic meningitis/encephalitis or acute flaccid paresis (AFP) (enterovirus surveillance, EVSurv) is currently carried out to monitor the absence of polio. In the final phase of polio eradication, however, virological testing of wastewater samples could also play an important role as an early warning system. In order to be able to react adequately to the perspective changes in WHO requirements, a method for the reliable detection of polioviruses from wastewater samples was established at the NRZ PE as part of a BMG-funded pilot project. This feasibility study is being carried out in close cooperation with the Federal Environment Agency (UBA). Since May 2021, wastewater samples from several sewage treatment plants have been tested for polioviruses on a weekly basis. Cultivation on different cell lines (RD, L20B) with subsequent sequencing of the protein-coding VP1 region provides the most sensitive results.
The aim is to acquire further partners who can also carry out wastewater concentration suitable for poliovirus detection. The high sensitivity and quality of the laboratory methods used are to be ensured through interlaboratory tests.
If the circulation of polioviruses is suspected, wastewater tests at various locations in Germany could therefore help to better assess the extent of the spread of polioviruses.
Date: 15.12.2023
