Experimental Infection and Response to Rechallenge of Alpacas with Middle East Respiratory Syndrome Coronavirus

We conducted a challenge/rechallenge trial in which 3 alpacas were infected with Middle East respiratory syndrome coronavirus. The alpacas shed virus at challenge but were refractory to further shedding at rechallenge on day 21. The trial indicates that alpacas may be suitable models for infection and shedding dynamics of this virus.


M iddle East respiratory syndrome coronavirus
(MERS-CoV) was first reported in September 2012 (1); since then, >1,600 confirmed cases have been reported to the World Health Organization (http://www.who.int/csr/ don/29-february-2016-mers-saudi-arabia/en). The role of domestic animals as an intermediate host for humans was initially suggested by case histories of infected patients who had visited farms or tended sick animals shortly before onset of infection (2). This suggestion was given credence by a study of camel serum samples that showed high levels of neutralizing antibodies in disparate camel populations (3); the findings were subsequently confirmed by virus detection and sequencing (4).
Infection trials in camels have been limited (5,6), mainly because of difficulties in housing and handling the animals in a high-containment facility, which is necessary because the virus has a Biosafety Level 3 classification (7). However, the alpaca, a close relative within the Camelidae family, may provide a temperamentally suitable and valuable animal model for MERS-CoV infection, particularly for developing and testing vaccine candidates for camels. We sought to assess whether alpacas could be infected by means of a natural (oronasal) route, to determine whether viral shedding occurred after reinfection, and to evaluate the development of serologic markers of protection.

The Study
We obtained 3 adult female alpacas (Vicugna pacos) from a commercial supplier in Victoria, Australia, and housed them in the Biosafety Level 3 containment facility at the CSIRO Australian Animal Health Laboratory. Before experiments, the alpacas were allowed to acclimatize for 6 days; during this time, intrauterine temperature data loggers were implanted according to a previously published procedure (8). We found no previous MERS-CoV challenge trial reported in alpacas, so we chose a preliminary dose and rechallenge time on the basis of our experience with other virus infection trials for other emerging infectious diseases (8).
The animals remained clinically healthy except for a reduced condition score that occurred by day 18 in 1 animal (alpaca 2); no signs of upper or lower respiratory tract disease appeared in any animal. Increased temperature was noted in alpaca 2 during days 17-20, but fever (rectal temperature >39°C) was not recorded. Gross abnormalities at postmortem examination were found only in alpaca 2 and comprised extensive adhesions of the caudal sac of compartment 1 of the stomach to the umbilicus; clinical findings in this animal were attributed to this lesion.
RNA extraction and real-time PCR were performed by following specimen-handling procedures established for Hendra virus (8) and were used to identify shedding patterns after each challenge. After initial challenge,  (Table 1).

Experimental Infection and Response to Rechallenge of Alpacas with Middle East Respiratory Syndrome Coronavirus
Virus isolation was undertaken with Vero cells by using published protocols (9) and was successful for all 3 animals from all types of samples. Virus recovery was successful from oral and superficial nasal swab samples through day 12; deep nasal swab samples were positive only through day 10. All urogenital and rectal swab samples were negative by both real-time PCR and virus isolation. After rechallenge, viral RNA was not detected with confidence from any sample (Figure).
Serum samples were assessed for immunologic responses by using a virus neutralization test (VNT) and a Luminex bead assay to the nucleocapsid protein. We used in-house assays modeled after those previously developed to assess the serologic status of feral camels in central Australia (10). All animals were seronegative by both Luminex and VNT before challenge. Antibody was first detected by Luminex on day 10 or day 12 in each animal ( Table 2); neutralizing antibody titers were 1:20 to 1:40 in alpaca 2 from day 10. Neutralizing antibody titers of 1:10 to 1:20 were detected in alpaca 1 from day 21 on but not in alpaca 3 at any time during the study. For controls, we used MERS-CoV positive and negative serum samples from Egypt and Australia (online Technical Appendix Table, http://wwwnc.cdc.gov/EID/article/22/6/16-0007-Techapp1.pdf).

Conclusions
Our study confirms that alpacas are susceptible to MERS-CoV infection; this finding is consistent with a previous report showing that alpaca kidney cell lines possessing the dipeptidyl peptidase-4 receptor could be infected in vitro (11). Our challenge/rechallenge trial was planned as a first stage in the assessment of the alpaca as a potential surrogate for camels for MERS-CoV vaccine testing. Consequently, the trial was not designed for direct comparison with 2 previous MERS-CoV challenge trials reported in camels (5,6). Our trial used a lower challenge dose and a different timeframe for observation; nevertheless, some preliminary comparative observations may be useful. In the previous studies, as in ours, the animals were inoculated   by the oronasal route, and live virus was detected through day 7 postinfection. Similarly, neutralizing antibodies were detected beginning 7-8 days postinfection. However, findings in the trials with camels differed considerably from findings in our trial. The trials with camels detected live virus from nasal washes at days 1-3, a nasal discharge, and transient temperature rises; viral RNA was detected by real-time PCR for an extended period. Furthermore, the VNT titers for camels were much higher than those for the alpacas in our study. These differences possibly represent underlying dissimilarities in immune responses to MERS-CoV for the 2 species but may also result from the higher infecting dose (10 7 50% tissue culture infective dose) used in the camel studies.
Our study showed that alpacas secreted live virus after oronasal infection and that the immune response to the initial infection prevented further excretion following reinfection. An underlying assumption in our trial is that the initial infection equates to natural vaccination and that the lack of viral excretion thus follows an induced immune memory response. However, our results indicate that this immunologic response is complex; although a strong serologic response developed in only 1 alpaca, all 3 alpacas were refractory to reinfection.
This study has several limitations. First, it was a preliminary study with only 3 animals and functioned more as proof of concept than a definitive study of the use of alpacas as a model for studying infection dynamics of MERS-CoV in camelids. Second, our observation period of 21 days before rechallenge is informative but does not provide complete information on duration of protective immunity. Future studies should have a larger sample and a longer period of study postinoculation. Third, our study did not seek to understand the pathogenesis of infection; we did not conduct histopathology or immunohistochemistry to understand the site of initial viral replication and the role of mucosal immunity in mounting an effective immune response upon infection.
Notwithstanding these limitations, we believe that the alpaca might be a useful model that could greatly facilitate the development and testing of vaccine candidates. We recommend further research and trials to substantiate this potential. Experimental Infection of Alpacas with MERS-CoV