[Detection of West Nile virus in human samples: follow-up studies during the 2015 seasonal period]. / A nyugat-nílusi vírus kimutatása humán betegmintákból: nyomon követéses vizsgálatok a 2015. évi szezonális idoszakban.

INTRODUCTION: West Nile virus, a mosquito-borne viral zoonosis is responsible for human infections in Hungary. Laboratory diagnosis is based on serological tests, however the application of molecular methods has been appreciated. AIM: The aim of the study was to investigate blood, cerebrospinal-fluid and urine samples of acutely ill patients and to follow-up PCR positive cases to ascertain the length of virus excretion. METHOD: Clinical specimens were examined by indirect-immunofluorescent, haemagglutination-inhibition, two PCR tests and Sanger-sequencing. Virus isolation in case of two patients was successful. RESULTS: A follow-up study could be carried out in case of 5 patients. Viral nucleic acid was detectable in urine even for several weeks after symptom onset and viral RNA was present at higher concentration compared with other samples. CONCLUSIONS: PCR analysis of urine could provide useful epidemiological and diagnostic information. Therefore, it is recommended to collect urine samples in order to supplement the serological diagnosis. Orv Hetil. 2017; 158(20): 791-796.

Detection and sequencing of West Nile virus RNA from human urine and serum samples during the 2014 seasonal period.

West Nile virus, a widely distributed mosquito-borne flavivirus, is responsible for numerous animal and human infections in Europe, Africa and the Americas. In Hungary, the average number of human infections falls between 10 and 20 cases each year. The severity of clinically manifesting infections varies widely from the milder form of West Nile fever to West Nile neuroinvasive disease (WNND). In routine laboratory diagnosis of human West Nile virus infections, serological methods are mainly applied due to the limited duration of viremia. However, recent studies suggest that detection of West Nile virus RNA in urine samples may be useful as a molecular diagnostic test for these infections. The Hungarian National Reference Laboratory for Viral Zoonoses serologically confirmed eleven acute human infections during the 2014 seasonal period. In three patients with neurological symptoms, viral RNA was detected from both urine and serum specimens, albeit for a longer period and in higher copy numbers with urine. Phylogenetic analysis of the NS3 genomic region of three strains and the complete genome of one selected strain demonstrated that all three patients had lineage-2 West Nile virus infections. Our findings reaffirm the utility of viral RNA detection in urine as a molecular diagnostic procedure for diagnosis of West Nile virus infections.

Explosive spread of a neuroinvasive lineage 2 West Nile virus in Central Europe, 2008/2009.

For the first time outside sub-Saharan Africa, a lineage 2 West Nile virus (WNV) emerged in Hungary in 2004. It caused sporadic cases of encephalitis in goshawks (Accipiter gentilis), other predatory birds, and in mammals. As a consequence, a surveillance program was initiated in Hungary and in Austria, which included virological, molecular, serological and epidemiological investigations in human beings, birds, horses, and mosquitoes. The virus strain became endemic to Hungary, however only sporadic cases of infections were observed between 2004 and 2007. Unexpectedly, explosive spread of the virus was noted in 2008, when neuroinvasive West Nile disease (WND) was diagnosed all over Hungary in dead goshawks and other birds of prey (n=25), in horses (n=12), and humans (n=22). At the same time this virus also spread to the eastern part of Austria, where it was detected in dead wild birds (n=8). In 2009, recurrent WND outbreaks were observed in Hungary and Austria, in wild birds, horses, and humans in the same areas. Virus isolates of both years exhibited closest genetic relationship to the lineage 2 WNV strain which emerged in 2004. As we know today, the explosive spread of the lineage 2 WNV in 2008 described here remained not restricted to Hungary and Austria, but this virus dispersed further to the south to various Balkan states and reached northern Greece, where it caused the devastating neuroinvasive WND outbreak in humans in 2010.

Experimental infection of goats with tick-borne encephalitis virus and the possibilities to prevent virus transmission by raw goat milk.

OBJECTIVES: The aim of this work was to study the tick-borne encephalitis virus (TBEV) infection of goats and the possibilities to prevent human milk-borne infections either by immunizing animals or the heat treatment of milk. METHODS: An experiment was conducted with 20 milking goats. Ten goats (half of them immunized) were challenged with live TBEV and 10 were left uninfected. Clinical signs and body temperatures of the animals were recorded and milk samples were collected daily. The presence of viral RNA and infectious virions in milk were detected by RT-PCR and intracerebral inoculation of suckling mice, respectively. Milk samples containing infectious virions were subjected to various heat treatment conditions and retested afterwards to assess the effect on infectivity. RESULTS: The infected goats did not show any clinical signs or fever compared to uninfected ones. Infectious virions were detected for 8-19 days from the milk samples (genome for 3-18 days by PCR) of infected goats. Immunized goats did not shed the virus. After heat treatment of the milk, the inoculated mice survived. CONCLUSIONS: Goats shed the virus with their milk without showing any symptoms. Human milk-borne infections can be avoided both by immunizing goats and boiling/pasteurizing infected milk.

Serologic evidence of West Nile virus infection in patients with exanthema in Hungary.

The presence of WNV in Europe has been well known for decades, although the first human infections and avian outbreaks were diagnosed in Hungary only in 2003. An annual average of 6-8 cases of the neuroinvasive form of WNV infection has been detected in the region since then, but a higher number (17) of WNV associated neuroinvasive disease occurred in 2008. In 2004, a surveillance system was established for monitoring WNV-associated meningo-encephalitis cases in Hungary, but a milder type of illness (with fever, rash and/or influenza like symptoms) is not followed. Fifty-two sera of 45 patients with mild clinical symptoms (fever, exanthema) were tested for anti-WNV antibodies in 2008 in a retrospective study by immunofluorescence test and ELISA. Seven patients had antibodies against WNV, serologic evidence of recent WNV infection was found in 4 out of the 7 patients. Infections could be acquired predominantly in August and in September, which seems to be a risk period for WNV in Hungary. The possibility of a recent WNV infection should be taken into consideration in the occurrence of fever and rush at late summer. Differential diagnosis of exanthematous patients should include WNV serology tests and should be done routinely.

Severe tick-borne encephalitis in a patient previously infected by West Nile virus.

We describe severe tick-borne encephalitis (TBE) in a patient who had previously experienced West Nile fever, another flavivirus infection endemic in Hungary. Previous West Nile virus infection does not develop immunity either against TBE virus infection or the disease, and it does not mitigate its clinical course. The possibility of antibody-dependent enhancement is considered.

A simple spatial model to explain the distribution of human tick-borne encephalitis cases in hungary.

Tick-borne encephalitis (TBE) is a common medical problem in Hungary and throughout much of Europe and Asia. This paper develops a geographic model that helps to predict the distribution of human tick-borne encephalitis cases in Hungary. The model is tested on a dataset of serologically confirmed TBE cases mapped by patients' residences. Case densities (incidence rates) are compared to predicted distributions of TBE derived from digital land-cover data. Maps are analyzed at the county level and on a smaller spatial scale. The analyses identified three major factors that shape the geographic distribution of human TBE cases in Hungary. The most important component is the distribution of forest habitat. TBE incidence correlates positively with the amount of forested habitat in each county. On a finer scale, the amount of forests within a 2500-meter radius of each town and village correlated significantly with TBE incidence rate. Based on these data, about 30% of the variation in TBE incidence is accounted for by the specific distribution of forest habitats in Hungary. Besides the distribution of forests, differences in human land-use practices among regions also affect the distribution of TBE cases. Additionally, because of the low transmission rate of the virus to humans, the perceived distribution of TBE cases is affected by random stochastic events. As a consequence of stochastic variation, meaningful patterns in the distribution of TBE cases can be only recognized when data are analyzed over broader temporal and spatial scales.

Reverse ELISA for IgG and IgM antibodies to detect Lassa virus infections in Africa.

BACKGROUND: Anti-Lassa antibodies are detected by indirect immunofluorescence assay (IFA) or by enzyme-immunoassay (ELISA). Both methods have problems to detect low amounts of specific antibodies. OBJECTIVES: We report here highly sensitive and specific reverse ELISAs to detect Lassa virus IgG and IgM antibodies. Due to the reverse techniques, serum samples could be applied at dilutions of 1:10 without increasing non-specific background reactions. STUDY DESIGN: For IgM antibody detection microtiter plates were coated with anti-IgM antibodies and for IgG antibody detection with rheumatoid factor (RF) (Sachers M, Emmerich P, Mohr H, Schmitz H. Simple detection of antibodies to different viruses using rheumatoid factor and enzyme-labelled antigen (ELA). J Virol Methods 1985;10:99-110). In both assays a tissue culture antigen was used in combination with a labeled anti-Lassa monoclonal antibody (Hufert FT, Ludke W, Schmitz H. Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies. Arch Virol 1989;106(3-4):201-12). RESULTS: The reverse ELISA turned out to detect virus-specific IgG and IgM antibody in all 20 samples of West African patients collected 2-8 weeks after onset of Lassa fever. Moreover, both IFA and reverse ELISA found IgG antibodies in 53 out of 643 samples of healthy West Africans (sensitivity of 100%). Six of the 643 samples were positive by reverse IgG ELISA only. Thus, the specificity compared to IIF was 99.0%, but it may be even higher, because compared to IFA the IgG ELISA was clearly more sensitive in detecting low antibody titers. CONCLUSIONS: In Ghana 3% seropositives were found by IFA, but 4% by the reverse ELISA. The reverse ELISAs can be performed with high sensitivity and specificity under field conditions in Africa.