1 recovirus neutralizing antibody prevalence in
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RECOVIRUS NEUTRALIZING ANTIBODY PREVALENCE IN HUMAN SERUM SAMPLES. 1
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Tibor Farkasa,b#, Cindy Wong Ping Luna*, 3
Cincinnati Children’s Hospital Medical Centera and University of Cincinnati College of Medicine, 4
Cincinnati, Ohiob 5
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Running title: ReCV neutralizing antibodies in humans 7
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#Address correspondence to Tibor Farkas, [email protected] 9
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*Present address: Cindy Wong Ping Lun, University of Cincinnati, Cincinnati, OH 11
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Word count abstract: 48 15
Word count text: 1853 16
Tables: 0 17
Figures: 2 18
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JCM Accepts, published online ahead of print on 4 June 2014J. Clin. Microbiol. doi:10.1128/JCM.01187-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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ABSTRACT 21
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To investigate recovirus infections and association with zoonosis, virus neutralizing antibody 23
prevalence against three recovirus serotypes was tested in the general population and in 24
zookeepers. Neutralizing antibodies were detected in significantly higher number of zookeepers 25
than in the general population but with significantly lower titers than in macaques. 26
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Norovirus, Sapovirus, Lagovirus, Vesivirus and Nebovirus are the 5 established genera 32
within the family Caliciviridae. “Recovirus”, “Valovirus” and “chicken calicivirus” are three new 33
proposed genera (1). The Tulane virus is the prototype recovirus (ReCV) strain (2). 34
Caliciviruses (CV) cause enteric, respiratory, vesicular and hemorrhagic diseases in 35
animals, while human CVs, including noro- and sapoviruses, are one of the leading causes of 36
acute gastroenteritis in both developed and developing countries (1). 37
One of the major obstacles of human CV research is the inability to grow human CVs in cell 38
culture. ReCVs not only can be grown in cell culture but evolutionarily and biologically are 39
closely related to human noroviruses (NoV) and are a promising surrogate for human NoV 40
research (3,4,5). 41
ReCVs are endemic in captive rhesus macaque colonies and based on serological surveys 42
they also infect other primate species, including humans (3, 6). Human ReCV infections were 43
first suggested by the detection of Tulane virus neutralizing antibodies in archived serum 44
samples collected from animal caretakers at the Tulane National Primate Research Center (3). 45
Recently, the molecular detection of a novel ReCV strain in stool samples of Bangladeshi 46
patients exhibiting clinical symptoms of acute gastroenteritis was reported (7). Since the human 47
ReCV strain genetically is significantly different from any of the known rhesus isolates, the 48
zoonotic origin of these human cases is not clear. 49
Recently, the characterization of ten cell culture-adapted genogroup 1 ReCV isolates, 50
including the Tulane virus, revealed the existence of at least four serotypes (4). This presented 51
the opportunity for further investigation of ReCV infections in humans and their possible 52
zoonotic origin by evaluating seroprevalence against different serotypes and in different human 53
populations. To represent the general population without exposure to non-human primate 54
contact, 100 randomly selected serum samples collected in 2013 were obtained from the 55
Biobank of Cincinnati Children’s Hospital Medical Center (CCHMC). These samples 56
represented male (n=53) and female (n=47) patients, between 1 and 61 years of age (average 57
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12.2), 77% Caucasian, 16% African-American, 2% Hispanic and 5% Non-Hispanic other. 58
Written informed consents were obtained from the patients or from the parent or legal guardian 59
of a minor. In addition, one hundred serum samples collected from zookeepers in several large 60
North-American zoos, including animal caretakers, veterinarians, and veterinary- and laboratory 61
technicians who were working with non-human primates, were kindly provided by Dr. William 62
Switzer. These samples had been previously collected by Antibody Systems Inc. (Fort Worth, 63
TX, USA), for research purposes. No unique identifiers or information on the age, race and sex 64
of these individuals were available. The work performed in this study with human serum 65
samples did not meet the definition of human subject research because all the samples were 66
de-identified. The institutional IRB deemed it as exempt from IRB review. 67
One hundred serum samples, collected between September 2009 and February 2010 during a 68
previous study from animals between 1.5 and 26.5 years old (average 5.9), both males (n=45) 69
and females (n=69) housed at the Tulane National Primate Research Center, and provided by 70
Dr. Karol Sestak, were used to establish seroprevalence in rhesus macaques (Macaca mulatta) 71
(6). 72
Heat-inactivated (56°C, 30 min) serum samples were tested in a cytopathic effect (CPE)-73
based virus neutralization assay to inactivate 100 TCID50 of ReCVs as described in our previous 74
studies (3, 6). Briefly, virus/serum mix was incubated for 1 hour at 37 °C and transferred into 75
duplicate wells of 96 well tissue culture plates seeded with 104 LLC-MK2 cells/well. Virus and 76
mock inoculated control wells were included. Plates were stained with crystal violet when all 77
cells in the virus control wells were rounded and detached (~72 hours post inoculation). All sera 78
were tested at 1:10 dilution. Positive samples were end titrated. Neutralization was established 79
when the cell monolayer was >50% intact in both of the wells for a given dilution. Statistical 80
significance of antibody prevalence between the sample populations and between the mean 81
titers of positive samples was evaluated by the Fisher’s exact test and one way analysis of 82
variance (ANOVA), respectively. 83
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The prevalence of neutralizing antibodies against the three ReCV serotypes tested in this 84
study ranged between 96-99% (≥1:10 dilution) in the rhesus macaques. Interestingly the human 85
samples collected from zookeepers exhibited a significantly higher prevalence of virus 86
neutralizing antibodies (P<0.0001) against all serotypes (28-100%) than samples collected from 87
the normal population (3-18%) (Fig 1). Among the Cincinnati samples, where demographic 88
information was available, the presence or level of VN antibodies was independent of age, sex 89
or race. The marked difference in the prevalence of ReCV neutralizing antibodies between the 90
two human populations is consistent with a zoonotic transmission of ReCVs. However, this was 91
not supported by the antibody titers of the positive samples. The mean titers of virus neutralizing 92
antibodies in both human populations were significantly lower (P<0.05) than in the macaques. 93
No statistically significant difference was detected between the mean titers of the two human 94
populations (Fig 2). 95
The possibility that ReCVs infect humans has been previously indicated by the detection of 96
Tulane virus neutralizing antibodies in serum samples collected from animal caretakers and 97
more importantly by the molecular detection of a novel ReCV strain in stool samples of 98
Bangladeshi patients (3, 7). These findings also raised the question about the zoonotic potential 99
of ReCVs, which could be supported by the close genetic relationship of the non-human primate 100
host and humans and by the evolutionary relatedness and shared biological features of ReCVs 101
and human NoVs, including the role of HBGA binding in susceptibility. 102
It is well established that chimpanzees and perhaps other non-human primate species can 103
experimentally be infected with human NoVs (8). However, whether human NoV transmission to 104
non-human primates occurs under natural circumstances is unknown. The interspecies 105
transmission of several other viral agents between different primate species is well documented, 106
including Cercopithecine Herpes Virus I, Herpes simplex 1 and 2, measles, hepatitis A and C 107
and the Ebola viruses. The zoonotic transmission of several animal CVs, including NoVs that 108
are genetically closely related to human NoVs (e.g. G2 swine and G3 bovine NoVs) has been 109
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suggested previously (9), but even with decades of worldwide surveillance of human NoV 110
infections the detection of swine or bovine NoVs in human samples has not yet been reported. 111
On the other hand human NoVs can replicate in gnotobiotic pigs, which indicates the possibility 112
of the emergence of swine-human recombinant NoVs or that swine could serve as reservoir for 113
human NoVs. Recently, the detection of human GII3, GII4 and GII13 NoVs that were associated 114
with human outbreaks in the same year has been reported in swine in Japan (10). However, 115
the low copy numbers of these viruses compared to swine NoV strains raises questions about 116
whether the human NoVs replicated in the pigs or were the result of environmental 117
contamination. 118
The interspecies transmission or zoonotic potential of several CVs, including human and 119
animal NoVs was also suggested previously based on serological studies. Since a cell culture 120
system is not available for most of these viruses, seroprevalence studies mostly relied on VLP 121
based immunoassays. For example, the interspecies transmission of human NoVs was 122
suggested based on the detection of ELISA antibodies against human NoVs in pigs (11). 123
Widdowson and colleagues reported higher prevalence of ELISA antibodies against bovine 124
NoVs in veterinarians (28%) than in the general population (20%), indicating the possible 125
zoonotic transmission of bovine NoVs (19). However, the existence of cross-reactive epitopes 126
between human and swine or bovine NoVs has also been reported (11, 13). This makes the 127
detection of anti-human NoV ELISA antibodies in different animal species or vice versa, difficult 128
to interpret. 129
In our study, not ELISA but neutralizing antibodies against three ReCV serotypes were 130
evaluated in individuals with known exposure to non-human primates and in the general 131
population. The prevalence of ReCV neutralizing antibodies was significantly higher against all 132
serotypes investigated in the samples collected from the zookeepers than in the samples 133
collected in Cincinnati (Fig 1). In the human but not in the rhesus samples, lower 134
seroprevalence rates were observed against FT285 ReCV compared to the other two strains. 135
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This might be explained by the restricted HBGA binding of FT285 (binds only to type B HBGA; 136
T. Farkas, unpublished) and the significant difference between the distribution of major HBGA 137
types in humans and in the macaque population studied (3). 138
The higher prevalence of ReCV neutralizing antibodies in the human population with 139
documented exposure to non-human primates points to the zoonotic potential of ReCVs. 140
However, the significantly lower levels of neutralizing antibodies in the human samples 141
compared to that in the macaques (Fig 2) raises several questions that need to be further 142
investigated. The low level of neutralizing antibody titers in the human samples could be 143
explained by a restricted infection and replication of the non-human primate ReCVs in the 144
human host, resulting in weaker humoral immune responses. The existence of cross-145
neutralizing antibodies between non-human primate and human host specific ReCVs, human 146
NoVs or other primate CVs that are able to infect humans cannot be excluded. However, cross-147
neutralization with human host specific CVs, including NoVs and sapoviruses, would not explain 148
the difference between the prevalence rates among the two human populations in this study. 149
Furthermore, in our previous study multivalent anti-NoV hyperimmune sera did not neutralize 150
the Tulane virus, although the number of NoVs included was limited. While the zoonotic nature 151
of most animal CVs is not clearly established, vesicular disease of a laboratory worker caused 152
by San Miguel sea lion virus (SMSV-5) has been described (14). Vesiviruses have been isolated 153
from a wide variety of domestic and wild animals, including non-human primates. Cross reactive 154
neutralizing antibodies generated by a zoonotic vesivirus, with lower affinity to ReCVs could 155
explain the higher seroprevalence in zookeepers and the lower antibody titers in the human 156
samples. Due to the unavailability of vesivirus serotyping reagents and the large number of 157
vesivirus serotypes, excluding cross-neutralization between ReCVs and vesiviruses will be 158
challenging. However, since clear serotypic differences were observed even between closely 159
related ReCVs (4), cross-neutralization between genetically distant CVs is less likely. 160
Non-specific neutralization is more likely to occur at lower serum dilutions. The highest VN 161
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antibody titer detected in the human samples was 1:320, while it was 1:1,280 in the macaques. 162
The prevalence of individuals with ≥ 1:80 VN antibody levels against any of the three ReCVs 163
was 84% in the macaques, 30% in the zookeepers and 9% in the Cincinnati population, still 164
indicating an association with non-human primate exposure. To clearly establish the zoonotic 165
nature of ReCVs, detection of ReCV strains endemic in non-human primates is needed in 166
human populations. This could be achieved in a targeted cohort study of animal caretakers 167
working at non-human primate centers. 168
Close to 60% of modern emerging infectious diseases are considered to have zoonotic 169
origin. Furthermore, over half of the shared pathogens listed as emerging in humans are 170
viruses, and a large number of them have been isolated from wild NHPs (15). Due to the high 171
biological relatedness of ReCVs and human NoVs and their hosts the risk of cross species 172
adaptation by genetic mutations or the emergence of recombinant viruses with unknown 173
pathogenic potential should be considered high. Our findings together with the recent detection 174
of ReCVs in human stool samples (7) call for detailed investigations of ReCV infections in both 175
non-human primate and human populations. 176
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ACKNOWLEDGEMENTS 178
This study was supported by the Infectious Diseases Scholar Fund of CCHMC to T.F. 179
We thank Dr. William Switzer (CDC) and Dr. Karol Sestak (TNPRC) for providing human and 180
macaque serum samples, respectively. We are grateful to Dr. Mekibib Altaye (CCHMC) for 181
assistance with the statistical analysis, and to Dr. Peter Dickie (CCHMC) for critical reading of 182
the manuscript. 183
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FIGURE LEGENDS 230
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Figure 1. Prevalence of ReCV neutralizing antibodies. Percentage of samples with ≥10 VN 232
titer is shown. ** indicates statistically significant difference between the Cincinnati samples and 233
samples obtained from zookeepers (P<0.0001). 234
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Figure 2. Virus neutralization antibody titers. Mean and SEM of positive samples is shown. * 236
indicates statistically significant difference between macaque and human samples (P<0.05). No 237
statistically significant difference was detected between the two human populations. 238
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