An attenuated Machupo virus with a disrupted L-segment intergenic region protects guinea pigs against lethal Guanarito virus infection

Machupo virus (MACV) is a New World (NW) arenavirus and causative agent of Bolivian hemorrhagic fever (HF). Here, we identified a variant of MACV strain Carvallo termed Car91 that was attenuated in guinea pigs. Infection of guinea pigs with an earlier passage of Carvallo, termed Car68, resulted in a lethal disease with a 63% mortality rate. Sequencing analysis revealed that compared to Car68, Car91 had a 35 nucleotide (nt) deletion and a point mutation within the L-segment intergenic region (IGR), and three silent changes in the polymerase gene that did not impact amino acid coding. No changes were found on the S-segment. Because it was apathogenic, we determined if Car91 could protect guinea pigs against Guanarito virus (GTOV), a distantly related NW arenavirus. While naïve animals succumbed to GTOV infection, 88% of the Car91-exposed guinea pigs were protected. These findings indicate that attenuated MACV vaccines can provide heterologous protection against NW arenaviruses. The disruption in the L-segment IGR, including a single point mutant and 35 nt partial deletion, were the only major variance detected between virulent and avirulent isolates, implicating its role in attenuation. Overall, our data support the development of live-attenuated arenaviruses as broadly protective pan-arenavirus vaccines.


Results
MACV strain Car 68 and Chic are lethal in guinea pigs, while strain Car 91 is attenuated. We previously reported 36 that a MACV strain Carvallo variant (Car 91 ) does not produce acute disease in Hartley guinea pigs (SFig. 1). Because the failure of Car 91 to cause lethality was unexpected, we produced another stock of virus derived from an early passage of strain Carvallo (produced in 1968) and designated it Car 68 . The virulence of Car 68 was examined in Hartley guinea pigs to determine if, contrary to Car 91 , this variant produced acute disease. As a control, one group of animals were infected with Chicava (Chic), a MACV strain known to cause lethal disease in this model 37 . Groups of eight animals were infected with the indicated strains and survival, weight and fever were monitored for 30 days (Fig. 1). All animals infected with strain Chic begin to lose weight between days 8-20 (Fig. 1B), but only one animal developed high fever ( > 41.0 °C) (Fig. 1C). All Chic-infected animals succumbed to infection by day 24. Animals infected with Car 68 displayed weight loss between days 9-21, but none of the animals developed high fever ( > 41.0 °C). Car 68 also produced a lethal disease in guinea pigs; however three animals survived infection (~63% mortality rate). Distinct from Chic, three Car 68 infected animals developed paralysis starting with the hind-limb and were euthanized on day 21. The three surviving Car 68 infected animals began to rapidly increase in weight after a period of weight loss, and by day 30 they exceeded their starting weight by ~3-20%. The mean time to death (MTD) for Car 68 and Chic was 23.5 and 22 days, respectively. Confirming our earlier observations (SFig. 1), animals infected with Car 91 survived infection without displaying signs of disease (Fig. 1A). Survival differences between Car 68 and Chic infected animals were not significant (logrank; p = 0.1331); however differences in survival between Car 91 versus Car 68 were highly significant (log-rank; p = 0.0082). Additionally, weight loss between Car 68 and Chic were significant compared to Car 91 for several days (Two-way ANOVA; p < 0.05). Viremia was detected in all four Chic-infected animals euthanized due to disease severity with GMT titers of 1,088 pfu/ml ( Fig. 2A). Only one Car 68 -infected animal had detectable viremia (166 pfu/ml), and viremia was undetected in the Car 91 -infected group.
To gain insight into the interaction of Car 91 and Car 68 within infected guinea pigs, two additional groups of three animals each were infected with Car 68 and Car 91 as above and on day 14 viremia and hematology were evaluated. No viremia was detected for either strain. However, significant differences in white blood cells (WBC), lymphocyte numbers (LYMPH), and platelet (PLT) levels were observed between the avirulent and virulent Carvallo strains (SFig. 2). The avirulent strain had elevated WBCs, LYMPH and PLT values compared to animals infected with Car 68 and uninfected control animals. The pathogenic Car 68 strain had WBC and LYMPH levels equal to that of control animals, but reduced PLT values. Additionally, two of three Car 68 infected guinea pigs had increased levels (but not statistically significant) of large unstained cells (LUCs), which is indicative of an acute viral infections 38 . Similar results were obtained for Chic (data not shown). Overall, our findings demonstrated that contrary to Car 91 , the Car 68 strain variant can produce an acute and lethal disease in guinea pigs.
We next evaluated the serum from Car 91 -challenged guinea pigs for the presence of binding and neutralization antibodies 30 days post-challenge. ELISA titers were determined using VSVΔG particles pseudotyped with glycoproteins from the MACV strain Carvallo as antigen. Six of eight guinea pigs had detectable antibodies against MACV glycoprotein with a log 10 GMT of 2.8 (Fig. 2B). MACV neutralizing antibody was detected in all but two infected animals with PRNT50 and PRNT80 GMTs of 269 and 59.5, respectively (Fig. 2C). The same two guinea pigs had no detectable PRNT50 or ELISA titers.
Genomic analysis of Car 91 and Car 68 . The genomes of Car 91 and Car 68 were sequenced to determine the genetic variation(s) that may contribute to virus attenuation. Sequencing revealed five changes between Car 68 and Car 91 in the L-segment (Fig. 3A). Three changes resulted in undisruptive silent nt substitutions in the polymerase protein sequence. Another nt change in the Car 91 IGR at position 399 (C → U) was detected that matched the reference strain Carvallo sequence (Genbank accession #NC005079). We also identified a 35 nt deletion in the IGR of strain Car 91 (Fig. 3B). No changes in the S segment were identified between Car 91 and Car 68 .
Scientific RepoRts | 7: 4679 | DOI:10.1038/s41598-017-04889-x Defective interfering (DI) particles are viral particles that carry mutations in the genome (typically deletions or rearrangements) that render the genome non-viable. During some infections, DI particles can accumulate to high levels through co-infection and disrupt the replication of viruses with viable genomes, leading to attenuation 39 . To examine whether a difference in the relative abundance of DI particles between Car 91 and Car 68 could be responsible for the attenuated phenotype of Car 91 , we examined the Illumina sequencing dataset in two ways. First, we looked for sequence coverage depth variation between the two isolates. Since DI particles often contain internal deletions, extreme levels of DI particles will skew sequencing coverage towards the ends of the genome fragments. Comparison of the patterns of sequence coverage depth between Car 68 and Car 91 did not reveal any obvious differences. For a more sensitive assay, we looked for evidence of chimeric reads, which would also be indicative of DI particle abundance. Chimeric reads are reads that span a deletion or rearrangement breakpoint, resulting in the 5′ and 3′ ends of the read aligning to non-adjacent regions of a reference genome. We detected 0.66% chimeric reads for Car 68 and 0.43% chimeric reads for Car 91 . Additionally, the pattern of deletions across the genome segments (by size and location) is similar between the two viruses (SFig. 3). Since the proportion of chimeric reads is higher in Car 68 , indicating that Car 68 may actually have a higher proportion of DI particles, and the pattern of deletions across the genome is qualitatively similar, it is unlikely that DI particles are contributing to the attenuated phenotype of Car 91 .
Altogether, these findings demonstrated that Car 91 has a significantly altered L-segment IGR relative to the earlier passaged Car 68 , including a 35 nt partial deletion. This disruption resulted in a predicted IGR structure with a ΔG value of −16.2 kcal/mol compared to −51.4 kcal/mol of Car 68 (Fig. 3C). Thus, the Car 68 structure is predicted to be more thermodynamically stable compared to Car 91 by 3.2-fold. In vitro characterization of Car 91 , Car 68 and Chic. To begin to address the basis of the attenuation of Car 91 , we examined both Carvallo variants Car 91 and Car 68 , and strain Chic for differences in particle-to-pfu ratios and replication kinetics. Car 91 had the highest particle-to-pfu ratio with a geometric mean of 369, compared to Car 68 and Chic whose GMT ratios were 26 and 13, respectively (Fig. 4A). These findings indicate that compared to Car 68 , Car 91 has a ~14-fold increase in the particle to pfu ratio. However, these differences were not statistically significant (T-test; p > 0.05).
The growth kinetics of both strain Carvallo variants and strain Chic were investigated in Vero cells, 104CL guinea pig fibroblasts and Human umbilical vein cells (HUVECs). Cells were infected with Car 91 , Car 68 or Chic and replication was assayed at 24, 48 and 72 h post-infection (hpi) (Fig. 4B). After 24 h, Car 68 grew to the highest levels in both Vero and HUVEC. 24 h growth for all three viruses in 104CL cells was markedly lower than Vero and HUVECs, however Car 91 titers were the lowest in these cells. At 48 hpi, Car 91 replication was still reduced compared to the other viruses in 104Cl and Vero cells, however in HUVECs Car 91 and Car 68 had similar titers. After 72 h, Chic had the highest titers in Vero and HUVECS. Titers of Car 68 and Chic were similar in 104CL cells at this time point. Overall, Car 91 replicated the poorest in all cell types tested with titers several fold lower than those of Car 68 and Chic. The replication differences between Car 91 and Car 68 were statistically significant (two-way ANOVA; p < 0.05) at 72 h in HUVECs and 104CL cells, but not in Vero cells. Growth titers were also significantly different between Car 91 and Chic at 72 hpi in all cell types (two-way ANOVA; p < 0.05). Together these findings indicated that Car 91 does not replicate as efficiently in cell culture as the virulent Car 68 and Chic strains.
MACV strain Car 91 protects guinea pigs against lethal infection by GTOV. Because Car 91 was highly attenuated in guinea pigs but produced detectable immune responses in 10/12 guinea pigs (SFig. 1 and Fig. 4), we hypothesized that it might function as an attenuated vaccine. Therefore, we examined the ability of Car 91 to protect guinea pigs against GTOV, a distantly related human pathogenic NW arenavirus species and causative agent of Venezuelan hemorrhagic fever 34,40,41 . Eight guinea pigs were challenged with GTOV 45 days after exposure to Car 91 (Fig. 5). As a control for acute infection, a group of six weight-matched naïve guinea pigs were also infected with GTOV. Animals were monitored for survival, weight loss and fever over 25 days (Fig. 5A-C). Consistent with previous findings 36, 41 , control animals began to lose weight starting around day 6 with concomitant fever. All control animals succumbed to infection with a MTD of 16 days. All but one animal The presence of binding antibody against MACV, JUNV and GTOV was evaluated by ELISA using sera from Car 91 -exposed (vaccinated) guinea pigs collected prior to and 30 days after GTOV challenge (Fig. 6A). Prior to GTOV challenge, six of eight animals infected with MACV strain Car 91 had detectable antibodies against MACV with a log 10 GMT of 2.5. These responses increased following GTOV challenge to 3.6, but this increase was not significant (T-test; p = 0.1461). Antibody titers against GTOV prior to GTOV challenge were low or below detection. However, antibody titers against GTOV rose significantly (T-test; p = 0.0002) after GTOV challenge to a log 10 GMT of 2.5. Antibody titers against JUNV were also detected prior to GTOV challenge (log 10 GMT 2.0) in all but one animal and these responses significantly increased after GTOV challenge (T-test; p = 0.0242) with a log 10 GMT of 3.0. Animal#4, which succumbed to GTOV infection despite receiving Car 91 , had undetectable ELISA titers against MACV, GTOV and JUNV and an undetectable PRNT50 titer against MACV. Animal#1 survived GTOV challenge despite having no detectable humoral responses against GTOV and MACV, and a low response against JUNV ELISA antigen.
The PRNT titers against MACV significantly increased subsequent to GTOV challenge, with GMT PRNT80 titers rising from 59 to 320 (T-test; p = 0.0112) (Fig. 6B). Despite the presence of MACV, GTOV and JUNV IgG antibody in all animals surviving GTOV challenge, we did not detect any neutralizing activity against GTOV or JUNV (Fig. 6C). These findings demonstrated that guinea pigs inoculated with Car 91 are protected against heterologous challenge by GTOV. However, this protection occurred in the absence of detectable neutralizing antibody responses against the challenge virus.

Discussion
A major goal of arenavirus vaccine design is to develop a pan-arenavirus vaccine that protects against heterologous species within either the OW and NW complexes, or more broadly. Previous work has shown that JUNV, MACV and TACV can cross-protect against each other in animal models 24,28 . However, we and others have found that JUNV and MACV to be much more serologically related (based on cross-neutralization and GP1 cross-binding) compared to GTOV 36 . Accordingly, to thoroughly gauge the level of heterologous protection against other NW arenaviruses, we purposely challenged Car 91 -vaccinated guinea pigs with the more genetically and serologically distant GTOV 36,40 . We also delayed challenge for 45 days after the initial inoculation with Car 91 to avoid any transient innate immune effects that may have enhanced protection. The single animal succumbing to GTOV challenge failed to produce detectable antibodies against even MACV. We predict a higher dose (>10-fold) of Car 91 would have elicited more robust and protective immune responses and thus resulted in 100% protection. This prediction is based on the fact that the dose of the JUNV attenuated vaccine Candid#1 in humans is 40,000 pfu 20 , whereas here we used Car 91 at 1,000 pfu. Curiously, neutralizing antibody against GTOV was not detected in any animal even after GTOV challenge. Other studies demonstrate that TACV protects against JUNV infection in the absence of neutralizing antibody responses targeting the challenge virus 28 . These data support a model whereby non-neutralizing antibody and/or cytotoxic T-cell responses may play essential roles in protection against heterologous challenges. More work will be needed to fully address the correlates of protection however; our findings clearly indicate that attenuated MACV strains can produce cross-protective immune responses against distantly related arenaviruses at least within the same complex. While not tested in our study, it is likely Car 91 -inoculated animals would have been protected against challenge by a virulent strain of MACV (i.e. Car 68 or Chic). JUNV strain candid#1 protects against JUNV in infected animals, and this correlates with neutralizing antibody responses 21 .
We previously reported that treatment of guinea pigs with anti-MACV neutralizing antibodies significantly reduces the humoral immune responses against Car 91 , suggesting the avirulent isolate had to replicate to some extent within infected animals to produce adequate immune responses 36 . This is supported by hematology data (SFig. 2) showing that the numbers of WBC/LEUKO are elevated over control animals two weeks after inoculation with Car 91 indicating an active immune response against the avirulent strain. Interestingly, WBC/LEUKO values after Car 68 (SFig. 2) and Chic (data not shown) exposure were similar to the controls, which is consistent with the ability of virulent strains of arenaviruses to cause suppression of the leukocyte responses 34 . Similarly, PLT values were also decreased for virulent strains consistent with the ability of arenaviruses to cause thrombocytopenia in infected hosts 34 . Importantly, Car 91 did not cause any apparent signs of disease such as weight loss or fever and LUC values of exposed animals, which are indicative of acute viral infections 38 , were not elevated, but were for the virulent Car 68 isolate.
Arenavirus IGRs are situated between each encoded ORF on both L and S segments 8 , and play important roles in transcription and production of infectious progeny virions 42 . Mechanistically, IGRs fold into single or double stem-loop structures and are essential for transcription termination. Because the tertiary structure of the IGR is critical for mRNA transcription termination, modifications can significantly impact the efficiency of replication by disrupting protein synthesis. For example, truncation of the LUJV L-segment IGR produces a virus that replicates less efficiently in vitro due to inefficient gene transcription 43 . Our findings strongly suggest that spontaneous alteration of the L-segment IGR is chiefly responsible for the loss of virulence of the Car 91 variant in guinea pigs. High levels of DI particles in the Car 91 stock could also potentially cause attenuation, but examination of the sequencing data does not support the presence of higher levels of DI particles in the Car 91 stock compared to the Car 68 stock. The three silent mutations detected in the L protein open reading frame do not impact amino acid coding, making it unlikely they could contribute largely to attenuation. Thus, the only substantial difference between the avirulent Car 91 and virulent Car 68 is the L-segment IGRs. However, future studies using available MACV reverse genetic systems 44 will be needed to fully determine if the 35 nt partial IGR deletion alone is solely responsible for the attenuation.
It is unclear how a partial L-segment IGR deletion arose during passage of strain Carvallo. The available evidence indicates that Car 91 was passaged two additional times in VeroE6 cells compared to Car 68 . Some work has shown that MACV can be attenuated by cell culture passage 23 , but these studies did not report if attenuation involved IGR modification. Curiously Yun, et al., reported that the JUNV strain XJ was apathogenic in guinea pigs after additional passaging in mouse brains 45 . Other variants of XJ are known to be virulent in the guinea pig model [46][47][48] . It was not reported if this attenuation was the result of an IGR mutation or some other factor(s). The spontaneous loss of virulence of different mammalian arenaviruses as a result of propagation underscores the need to obtain sequencing data from clinical isolates as soon as they emerge to prevent the deleterious effects of cell culture or animal adaptation of the viral genome.
Car 91 had reduced replication fitness in vitro compared to Car 68 , including reduced replication in primary endothelial cells, which are in vivo targets of the virus 49 . However, these growth defects were relatively modest. Notably, the 1.5 log reduction in growth was similar to that observed for the LUJV containing the partial L-segment IGR deletion 43 . Whether the Car 91 replication deficiency alone results in the attenuation in vivo is not clear. It is possible that reduction in the size of the IGR and the resultant bearing on its tertiary structure may impact innate immune signaling pathways within infected cells, and this could play a critical role in attenuation. Hyperstimulation of the innate immune response as evidenced by interferon (IFN) stimulated gene expression has been observed in cell culture for attenuated JUNV vaccine strain Candid#1 50 . Future studies will be needed to fully elucidate the mechanism(s) by which partial IGR deletion produces apathogenic arenaviruses in vivo, with particular emphasis on activation of IFN stimulated gene products. Such analysis will benefit from the development of transcriptomic platforms specific to guinea pig gene expression 51 .
Recent work by Iwasaki, M. et al. has focused on exploiting alterations in the IGR as a means of producing rational whole-virus vaccines against arenaviruses. Addition of synthetic (non-viral S-IGR like) sequence or Figure 6. Binding and neutralizing antibody responses in guinea pigs infected with GTOV. (A) Antibody binding titers were determined by coating 96-well plates with the indicated PsVs and incubating them with serially diluted antiserum samples from before (circles/PRE) or after (squares/POST) challenge with GTOV. The dashed line denotes the limit of detection. The red circle denotes the single animal (Animal #4) that succumbed to infection. The blue symbols denote the same animal before and after GTOV challenge. Note that titers against MACV prior to GTOV challenge are also depicted in Fig. 4B. (B) PRNT80 titers against MACV (Car 68 ) prior to and after challenge with GTOV were determined as above. Note that PRNT80 titers prior to GTOV challenge are also depicted in Fig. 4C. (C) PRNT50 titers against JUNV, MACV and GTOV were determined as in Fig. 2. Titers were determined as described above. The dashed line indicates the limit of detection. For all panels, asterisks denote statistical significance.
swapping the IGRs of the L-and S-segments produces attenuated viruses that can protect mice against secondary challenge with wild-type LCMV 52,53 . Our work indicates that modification of the L-segment IGR, including a 35 nt deletion, can also produce an attenuated virus that functions as a vaccine. Whether substitution of the IGR, incorporation of a synthetic IGR or deletion of the IGR is the best approach in live-attenuated arenavirus vaccine development remains to be determined. One advantage to deletion of multiple nucleotides is that reversion to wild-type is improbable. Work involving the arenavirus IGR as a vaccine strategy has focused exclusively on OW arenaviruses, specifically LCMV. Our study advances these vaccine strategies by supporting alteration of the IGRs as a powerful means of producing an attenuated live-virus vaccine against NW arenaviruses.
Many human pathogenic arenaviruses are endemic and well-described, such as LASV which causes >100,000 infections annually, in addition to several of the South American arenaviruses including JUNV, MACV and GTOV 54 . However, novel human pathogenic arenaviruses emerge at unpredictable rates in both the Americas and Africa 2, [13][14][15][16]55 , the most recent being Lujo virus in Africa. Accordingly, any vaccine-based countermeasure should be designed to protect broadly protect against known and unknown arenaviruses. Our work and the work of others 52, 53 support the use of IGR-modification as a strategy for pan-arenavirus vaccine development. With the advent of arenavirus reverse genetics 56 , other attenuation strategies such as codon deoptimization 57, 58 could be combined with IGR-modification to produce rationally-designed vaccines that are both highly-attenuated, yet replication competent and safe for human use.

Viruses and cells. GTOV strain INH95551, MACV strain Chicava and two MACV strain variants of
Carvallo from passages dated 1968 (Car 68 ) and 1991 (Car 91 ) were propagated as previously reported 36 . All viruses were twice plaque purified prior to use. Car 68 was passaged twice in sucking hamster brains and once in VeroE6 cells. Car 91 was passaged an addition two times in VeroE6 cells. 239 T cells and 104CL guinea pig fibroblasts (ATCC) were maintained in MEM or RPMI containing 10% heat-inactivated fetal bovine serum (FBS), 1% antibiotics (100 U/ml penicillin, 100 μg/ml of streptomycin, respectively. HUVECs were purchased from a commercial source (Lonza) and maintained in endothelial growth medium.

Challenge of Hartley guinea pigs.
Female Hartley guinea pigs (300-400 g) were implanted with IPTT-3000 identification chips to monitor temperature (BMDS INC; Seaford, DE). Animals were challenged with the indicated MACV strains (1,000 pfu) or GTOV (2,000 pfu) diluted in a total volume of 0.5 ml PBS by intraperitoneal (i.p.) injection. Animals were weighed and monitored for fever. All animal studies were conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles state in the Guide for the Care and Use of Laboratory Animals, National Research Council 59 . All animal experimental protocols were approved by a standing internal institutional animal care and use committee (IACUC). The facilities where this research was conducted are fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. Animals meeting criteria were humanly euthanized.

Plaque reduction and neutralization tests (PRNTs).
PRNTs were performed as previously described 60 using guinea pig serum serially diluted two-fold starting at 1:40. Percent neutralization was calculated relative to the number of plaques in the presence of negative control serum. Titers represent the reciprocal of the highest dilution resulting in a 50% reduction in the number of plaques. Data were plotted using Graphpad Prism software.
Growth kinetics. Vero, 104CL and HUVECs were seeded at a density of 1 eql × 10 5 cells per well in 24-well plates and infected at an MOI of 0.1 with the indicated viruses diluted in culture medium. Virus growth at 24, 48 and 72 h was determined by plaque assay on Vero cell monolayers. All samples were run in two independent replicates and plotted as the mean + / − standard deviation (SD) using Graphpad Prism software.
Particle-to-PFU ratio. Particle counts were determined with a Virocyt machine (Virocyt, Boulder, CO) using the manufacture's protocol. The particle-to-pfu ratios were determined by dividing particle counts by the amount of infectious virus. Four independent virus preparations per strain were used in the calculations. Genome sequencing. RNA was extracted from Trizol homogenates of MACV, converted to cDNA, and subjected to sequence-independent, single primer amplification (SISPA) 61 . The products of these reactions were used to generate libraries that were sequenced on an Illumina MiSeq. Sequencing reads were assembled using DNAStar SeqMan NGen. Predicted secondary structures of the IGRs were determined using DNAstar Genequest.
Identification of defective genomes. To look for putatively defective vNiral genomes (i.e., genomes with large deletions or rearrangements), we identified chimeric reads from the Illumina dataset. Chimeric reads were defined as reads with 1) two distinct, non-overlapping alignments to different regions of the MACV genome and with 2) ≥99% of read bases aligned to the reference when considering both alignments together. To prevent bleed through between multiplexed samples, we used non-overlapping dual indexes and we filtered out any reads with index base qualities less than Phred 20, on average. Only the first read from each pair was used to avoid double counting. Illumina and SISPA adaptors were clipped using Cutadapt v1.9.dev1 62 and Prinseq-lite v0.20.3 63 was used to 1) remove 6 nt from the beginning and end of each read ("-trim_right 6 -trim_left 6"; to remove random hexamers), 2) trim low quality bases from the 3′ ends of the reads ("-trim_qual_right 30 -trim_qual_type min -trim_qual_window 5"), 3) remove reads <40nt in length or with a mean quality score less than Phred 20 ("-min_len 40 -min_qual_mean 20"), 4) remove reads with low complexity ("-lc_method dust -lc_threshold 3") and 5) remove exact duplicates ("-derep 14"). Reads were then aligned to a reference sequence (Car 68 GenBank: KM198592.1 and KM198593.1; Car 91 sequences have been uploaded to GenBank) using BWA mem v.0.7.12 with default parameters 64 . Chimeric reads were identified and characterized using a custom script, chimeric_reads. py v3.5.4 (https://github.com/jtladner/Scripts/tree/master/chimeric_reads). Note that the S-segment consensus sequence for Car 91 exactly matches that of Car 68 (KM198592.1).
Pseudovirion neutralization assay (PsVNA) and ELISA. The pseudovirion neutralization assay (PsVNA) has been described in detail elsewhere 36,65 . Briefly, a vesicular stomatitis virus backbone with a luciferase reporter gene (PsV) was used to produce particles decorated with glycoproteins from MACV, JUNV and GTOV. These particles were subsequently incubated with the indicated serially diluted sera in triplicate and the geometric mean PsVNA80 titers (GMT) plotted. The use of PsV as solid phase antigen in ELISAs has been previously described in detail 36 . Statistical analysis. Two-way ANOVA with the Bonferroni correction was used to analyze both weight and viral replication (in vitro). Log-rank test was performed for statistical analysis of survival. The statistical significance of PRNTs was determined using an unpaired two-tailed Student's t test. Significance levels were set at a p value less than 0.05. All analyses were performed using Prism software. Data Availability. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files) with the exception of the genomic data for Car 91 . The sequencing information for Car 91 has been uploaded to Genbank.