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Title: Serum-Neutralization Assay Can Efficiently Replace Plaque Reduction Neutralization Test for 1 Detection and Quantitation of West Nile Virus Antibodies in Human and Animal Serum Samples 2

Authors: Annapia Di Gennaro1, Alessio Lorusso1*, Claudia Casaccia1, Annamaria Conte1, Federica Monaco1 3 and Giovanni Savini1 4

Affiliation: 1OIE Reference Laboratory for West Nile disease, Istituto Zooprofilattico Sperimentale 5 dell’Abruzzo e Molise “G. Caporale”, Teramo-Italy 6

*Corresponding Author: Alessio Lorusso, e-mail: a.lorusso@izs.it, Tel: +390861332440, Fax: 7 +390861332251. 8

Running title: Serum-Neutralization for West Nile virus Antibody Titration 9

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CVI Accepts, published online ahead of print on 6 August 2014Clin. Vaccine Immunol. doi:10.1128/CVI.00426-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 30

A serum-neutralization (SN) assay was compared with the official plaque reduction neutralization test for 31

quantitation of West Nile virus antibodies. A total number of 1348 from equids and 38 human sera were 32

tested by both methods. Statistically significant differences were not observed thus supporting the use of 33

SN for routinely purposes. 34

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Manuscript 55

According to the International Committee on Taxonomy of Viruses (ICTV, 2013), 53 virus species are 56

included in the genus Flavivirus within the Family Flaviviridae. Some of them are important mosquito-borne 57

pathogens with severe impact on human and animal health. Together with important human flaviviruses, 58

the West Nile virus (WNV) and Usutu virus (Usuv) - the most widespread mosquito-borne flaviviruses in 59

Europe (1, 2) - form the Japanese encephalitis virus sero-group (3, 4). Accordingly flaviviruses may also 60

cross react affecting the specificity of many serological tests. The plaque reduction neutralization test 61

(PRNT) is the most specific serological test for proper serological identification (3, 5, 6). The most stringent 62

PRNT test which is represented by the 90% endpoint PRNT (PRNT90) is considered as the gold standard 63

protocol for the sero-diagnosis of flavivirus infections. In differential PRNT, in which specimens were tested 64

simultaneously for two or more viruses, a neutralizing titer of fourfold or higher for one virus compared to 65

those of the others was considered virus specific (5). However, possible occurrence of co-infections could 66

not be discarded in case of similar titers against two or more flaviviruses. PRNT still presents some 67

drawbacks. Firstly, it takes around one week to be completed. Secondly, it is expensive and requires highly 68

trained staff. Thirdly, PRNT using wild type flaviviruses can be technically difficult, as many flaviviruses grow 69

slowly and have pinpoint-sized plaque phenotypes. Indeed, viruses may be comprised of mixed 70

populations, or quasispecies, which grow at different rates and produce plaques of various sizes. Last but 71

not least it requires a quantity of serum difficult to obtain when dealing with wild animals and particularly 72

with some species of wild birds. 73

In order to test the consistency of the serum-neutralization (SN) in quantitating specific WNV antibodies, 74

we tested by SN (7) and PRNT90 (OIE, World Organization for Animal Health, 2012) 1348 serum samples 75

from equids (horses and donkeys) collected during the surveillance programme for West Nile neuroinvasive 76

disease (WNND) organized by the Italian Ministry of Health and 38 sera of human patients collected during 77

the first outbreak of West Nile virus neuroinvasive disease in humans in Croatia during 2012 (8). 78

Furthermore, 25 serum samples from horses previously showed to be positive for Usuv, were tested at the 79

same time by SN and PRNT90 using Usuv and WNV as antigens. The serum samples were inactivated at 56°C 80

for 30 min. Starting from 1:5, serial 2-fold dilution were made in microtitre plates, and 100 TCID units of 81

antigen were added to each dilution. Thereafter, the mixtures were incubated at 37°C for 1 h and 105 Vero 82

cells were added to each well. Plates were incubated at 37°C for 5 days. Starting from the third day after 83

incubation, plates were checked for cytopathic effect (cpe) and the antibody titre was defined as the 84

reciprocal of the highest dilution of the serum which showed 100% neutralization. Positive and negative 85

control sera were included in each plate. Sera with a titre of 1:10 were considered positive. Prior to the SN 86

and PRNT90, Usuv and WNV antigens (USUV strain 939/01 and WNV strain Eg-101, respectively) were 87

titrated by TCID50 using Vero cells. After 4 days, the titre was determined by using the Reed and Muench 88

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formula. In order to compare SN and PRNT90 assays, two different comparisons were performed for 89

resulting titres and positive/negative result values: titres were compared by Wilcoxon non parametric test 90

for dependent samples; positive/negative results were compared using McNemar χ2 test for dependent 91

samples and the level of agreement was evaluated by the k-Cohen coefficient (9). All the analyses were 92

performed in SPSS Statistics 17.0. 93

As for serum samples from equids, the two diagnostic tests were not statistically different in terms of titres 94

and positive/negative results. Contingency table (Tab 1) shows the level of agreement between the two 95

tests. The McNemar χ2 test gave a value of 0.83 (p>0.05) showing a not significant difference between the 96

two methods and the k-Choen, equal to 0.78 (p<0.05), gave a good level of agreement beyond the chance. 97

Also titres were not significantly different (Wilcoxon test = -1.69, p>0.05). Similar results were also obtained 98

when human sera were tested as the two tests were not statistically different in terms of titres and 99

positive/negative results. Contingency table (Tab 2) shows the level of agreement between the two tests 100

for human samples. The McNemar χ2 test gave a value of 1.33 (p>0.05) showing a not significant difference 101

between the two methods and the k-Choen, equal to 0.81 (p<0.05), gave a good level of agreement beyond 102

the chance. Also titres were not significantly different (Wilcoxon test = -1.67, p>0.05). The 25 samples from 103

horses previously demonstrated to be positive for Usuv, were shown to be positive for WNV and Usuv in 104

both tests with a broad range of titres (Tab 3). Results from serum samples from horses 6, 8, 18, 19, 21 and 105

25 suggest the presence of antibodies for both viruses as results of a double infection whereas serum from 106

horse number 10 shows specific antibodies only for Usuv. The remaining serum samples seemed to be 107

positive only for WNV. Moreover, horses 6-10, 12, 23 and 25 showed a recent infection by WNV as 108

suggested by the IgM Elisa test (Idexx IgM WNV). 109

In the present manuscript, we showed on a large number of serum samples, that SN can efficiently replace 110

PRNT at quantitating WNV antibodies. The two diagnostic tests were not statistically different in terms of 111

titres and positive/negative results thus supporting the use of SN during the daily activities of diagnostic 112

laboratories dealing with WNV serological investigation. SN is definitely faster (up to five days) and requires 113

less labor when compared to PRNT and, reasonably, its use can be translated for serological investigation 114

for other flaviviruses. Cross-reactivity does exist between WNV and Usuv when both serological methods 115

are used. However, specific antibodies can be attributed to one or the other virus as there is a neutralizing 116

titer of fourfold or higher for one virus compared to the other independently in both serological assays. 117

Nevertheless, both viruses contemporarily circulate in the same areas (10) thus leading to the presence of 118

sero-reactors for both viruses as it seems to be the case for horses 6, 8, 18, 19, 21 and 25. However, horses 119

19 and 21 showed a fourfold or greater difference in titres with both the SN and PRNT for WNV as 120

compared to Usuv although with significant high titres for this latter. In this case, it is difficult to exclude 121

the presence of a double infection. Definitely, an efficient and an up-to-date epidemiological picture of 122

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circulation of flaviviruses in a given area help to address the confusing results potentially coming from both 123

assays. Importantly, even in the case of double infection, SN performed as PRNT thus confirming the 124

feasibility of this test for serological routinely purposes. 125

Acknowledgements 126

Funding was provided by the Italian Ministry of Health. 127

Table 1. Contingency table between PNTR and SN for equid serum samples 128

129 PNTR

positive negative total

SN positive 1010 54 1064

negative 45 239 284 total 1055 293 1348 130

Table 2. Contingency table between PNTR and SN for human serum samples 131 PNTR

positive negative total

SN positive 10 0 10

negative 3 25 28 total 13 25 38 132

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Table 3. Serum samples from horses tested by SN and PRNT using WNV and Usuv as antigens 144

Horse n USUV, titres WNV titres IgM WNV

SN PNRT SN PNRT 1 1:20 1:10 1:160 1:80 - 2 1:10 1:5 1:160 1:80 - 3 1:10 1:20 1:40 1:40 - 4 1:10 1:20 1:80 1:40 - 5 1:10 1:20 1:40 1:40 - 6 1:20 1:20 1:20 1:20 + 7 1:20 1:20 1:40 1:80 + 8 1:20 1:20 1:40 1:20 + 9 1:10 1:20 1:40 1:20 + 10 1:40 1:20 1:5 1:5 + 11 1:5 1:5 1:320 1:320 - 12 1:5 1:10 1:40 1:20 + 13 1:5 1:5 1:40 1:20 - 14 1:10 1:10 1:80 1:80 - 15 1:40 1:20 1:640 1:320 - 16 1:80 1:40 1:640 1:640 - 17 1:20 1:20 1:320 1:160 - 18 1:80 1:80 1:160 1:80 - 19 1:160 1:80 1:640 1:320 - 20 1:10 1:10 1:320 1:320 - 21 1:80 1:40 1:320 1:160 - 22 1:10 1:20 1:640 1:320 - 23 1:10 1:10 1:160 1:320 - 23 1:80 1:80 1:640 1:320 + 25 1:320 1:160 1:160 1:80 +

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