# On the interactions between virulent bacteriophages and bacteria in the gut.

Damien Maura, Laurent Debarbieux

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Damien Maura, Laurent Debarbieux. On the interactions between virulent bacteriophages and bacteria in the gut.. Bacteriophage, 2012, 2 (4), pp.e23557. 10.4161/bact.23557. pasteur-01539087

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# On the interactions between virulent bacteriophages and bacteria in the gut

Damien Maura & Laurent Debarbieux

To cite this article: Damien Maura & Laurent Debarbieux (2012) On the interactions between virulent bacteriophages and bacteria in the gut, Bacteriophage, 2:4, e23557, DOI: 10.4161/bact.23557

To link to this article: http://dx.doi.org/10.4161/bact.23557

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# On the interactions between virulent bacteriophages and bacteria in the gut

Damien Maura¹,²,³ and Laurent Debarbieux⁴,\*

¹Department of Surgery; Harvard Medical School and Massachusetts General Hospital; Boston, MA USA; ²Department of Microbiology and Immunobiology; Harvard Medical School; Boston, MA USA; ³Shriners Hospitals for Children Boston; Boston, MA USA; ⁴Institut Pasteur; Molecular Biology of the Gene in Extremophiles Unit; Department of Microbiology; Paris, France

We recently described the targeting of O104:H4 *Escherichia coli* in mouse gut by several virulent bacteriophages, highlighting several issues relating to virus-host interactions, which we discuss further in this addendum to the original publication.

## Background

Recent metagenomic studies have confirmed that the digestive tract is a complex environment in which bacteria and bacteriophages coexist.¹ However, little is known about the nature of the interactions between these two antagonist populations in this very particular environment. Given the constant exposure to bacteriophages resulting from food consumption, the overall effect of bacteriophages in the human gut might be thought to be negligible as, to our knowledge, no effect of bacteriophages has ever been reported. However, temperate bacteriophages have long been known to play an important role in the transfer of genetic information between hosts, and it has been shown that, as expected, such transfer can occur in vivo in mouse gut.² These findings confirmed that prophages, like other mobile genetic elements, participate in genomic shuffling within the gut microflora.³⁻⁵ However, what do we know about the impact of virulent bacteriophages on the gut micobiota? Evidence for an active role of virulent bacteriophages dates back to the first paper to coin the term "bacteriophage," published by Félix d'Herelle, who isolated the bacteriophages from patients

recovering from dysentery and suggested that these organisms played an active role in disease recovery. This observation formed the cornerstone of a new field, phage therapy, which is now returning to the fore as a possible solution for treating bacterial infections due to multidrug-resistant pathogens.⁶,⁷ Several studies have investigated the use of virulent bacteriophages to treat intestinal infections.⁸,⁹ Possibly the most surprising result of these studies was the finding that no common pattern could be deciphered for either bacteria or bacteriophages. Differences in bacteria, bacteriophages and colonization or infection models may account for this, but this situation contrasts strongly with observations in other animal models of infection (in the skin, lungs, etc.) for which different bacteria and bacteriophages have also been evaluated but for which highly effective curative action has frequently been reported.¹⁰ It is therefore possible that interactions between bacteriophages and bacteria are much more complex in the gut than in other organs. Our recent article provides a detailed illustration of this view, and goes one step further, by investigating the limiting factors for these virus-host interactions in the intestine.¹¹

## The Bacteriophages

We isolated three bacteriophages, each belonging to one of the three families of the Caudovirales order, from sewage. These bacteriophages (CLB_P1, CLB_P2 and CLB_P3) were individually

**Keywords:** bacteriophage infection, gut, permissivity, pseudolysogeny, phage therapy

Submitted: 09/03/12

Revised: 12/10/12

Accepted: 01/07/13

http://dx.doi.org/10.4161/bact.23557

\*Correspondence to: Laurent Debarbieux; Email: laurent.debarbieux@pasteur.fr

Addendum to: Maura D, Galtier M, Le Bouguénec C, Debarbieux L. Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine. Antimicrob Agents Chemother 2012; 56:6235-42; PMID:23006754; http://dx.doi.org/10.1128/AAC.00602-12
<figure><img src="image_11.png" /><figcaption>**Figure 1.** Changes in fecal bacteriophage concentration over time in mice colonized with the O104:H4 55989 *E. coli* strain. Four groups of 4 to 5 mice were colonized with *E. coli* 55989Str on day 0. Three days later, they received normal drinking water or drinking water containing one of the bacteriophages studied (closed circles, CLB_P1; squares, CLB_P2; triangles, CLB_P3) or a cocktail of the three bacteriophages (dashed line) at a concentration of 1 × 10⁵ pfu/ml each. On day 30, 20 individual colonies from feces were individually assessed for sensitivity to bacteriophages. The concentration of bacteria in the feces over the 30-d period was stable at about 1 × 10⁸ cfu/ml.</figcaption></figure>

characterized in vitro on planktonic cul-
tures and on biofilms.¹² The T4-like
bacteriophage CLB_P2 had the best char-
acteristics in vitro, in terms of the lack of
bacteriophage-resistant clones in plank-
tonic conditions and efficacy on biofilms.
However, a cocktail of the three bacterio-
phages was more effective against biofilms
than individual bacteriophages, suggest-
ing a synergistic effect potentially due to
the combined action of multiple cell wall-
degrading enzymes or a capacity to infect
multiple physiological stages of the bact-
erium within the biofilm. Surprisingly,
CLB_P1, a T7-like bacteriophage, was the
least efficient at reducing biofilms, despite
the bacteriophage T7 having previously
been identified as a good candidate for
therapeutic applications on the basis of in
vivo experiments.¹³ It strongly indicated
that given the high genomic variability of
bacteriophages generalization of their in
vivo efficacy cannot be drawn only from
bacteriophage morphology/classification.
Finally CLB_P3, a close relative of bacte-
riophage T1, is, to our knowledge, the first
T1-like bacteriophage ever to be tested in
an in vivo model.

The Bacteria

A colonization model based on the entero-
aggregative *E. coli* strain 55989 was the
starting point of our study. This serotype
O104:H4 strain was initially isolated from
an AIDS patient suffering from diarrhea.

A very closely related clone of the 55989
strain recently acquired a prophage encod-
ing the Shiga toxin stx2, leading to the
emergence of highly virulent O104:H4
stx2+ clones, which caused an outbreak of
diarrhea and hemolytic uremic syndrome
affecting 4000 people in Western Europe
in June 2011.¹⁴ Interestingly, CLB_P1 and
CLB_P2 but not CLB_P3 bacteriophages
isolated with 55989 strain also infected
efficiently several of these epidemic
clones.¹²

The Animal Model

*E. coli* strains are not normally consid-
ered to be resident in the mouse intestinal
microbiota, although there have been spo-
radic reports of their presence.⁹,¹⁵ We used
an intestinal carriage model in which
high levels of *E. coli* are maintained over
several weeks through the addition of an
antibiotic to drinking water. This antibi-
otic eliminates part of the natural flora,
allowing stable colonization by *E. coli*. In
the absence of antibiotic pressure, *E. coli*
levels decrease over the course of a few
hours to a few days, depending on the
strain used.¹⁶

Histological analyses of the gut tissues
of mice after three days of colonization
with strain 55989 revealed no damage,
demonstrating that this model mimics,
in both the small and large intestines, the
state of asymptomatic carriage found in
humans for enteroaggregative *E. coli*.

The Interactions
of Bacteriophages
and Bacteria in Animals

Contact between virulent bacteriophages
and their bacterial hosts triggers an “arms
race” between the two species. It has been
reported, in both in vitro and field stud-
ies, that bacteriophage-resistant mutants
emerge, at various rates, and that variants
of the bacteriophages subsequently infect
these new clones.¹⁷,¹⁸ However, no such
long-term study has yet been performed in
the gut environment. Our model, involv-
ing the addition of bacteriophages to the
drinking water of colonized mice for a
period of 24 h, leading to the continuous
replication of bacteriophages within the gut
over a period of several weeks, provides us
with an opportunity to address such ques-
tions in vivo, within the digestive tract.
No bacteriophage resistant mutants were
observed after three weeks in mice treated
with the cocktail of three bacteriophages.¹²
We also analyzed the three bacteriophages
individually: CLB_P1 was rapidly cleared
(< 4 d), CLB_P3 persisted for more than
twice as long (< 11 d) and CLB_P2 per-
sisted in the gut for at least 30 d without
selecting for the growth of predominantly
bacteriophage-resistant mutants (Fig. 1).
The absence of such mutants in the gut
was surprising, as we know that bacte-
riophages infect bacteria in this organ,
exerting a selective pressure on their hosts
that should lead to the selection of such
mutants. However, the gut environment
may not be favorable to the selection of
bacteriophage-resistant mutants, as such
mutants may have no selective advantage
and may be rapidly outcompeted by the
wild-type strain. This would result in the
mutants being eliminated from the gut as
soon as they appeared.

Several hypotheses may be put forward
to explain why the infection of bacteria
with bacteriophages did not lead to the
growth of resistant bacterial clones. First,
random collisions between bacteriophages
and bacteria can be artificially increased
in test tubes, by shaking, whereas such
collisions are probably much less frequent
in vivo. Nevertheless, we showed that a
dose-dependent decrease in the number
of bacteria in the gut could be obtained
in vivo, suggesting that the vast majority
(over 90%) of bacteria could be infected by bacteriophages. However, this was not enough to trigger the complete elimination of the bacterial host, suggesting that some bacteria may be physically protected against bacteriophage infection. Such protection might result from the localization of bacteria in particular sites inaccessible to bacteriophages, such as crypts or villi. This population never coming into contact with bacteriophages may be the source of the wild-type bacteria responsible for the renewal of the sensitive bacterial population. In contrast to bacteriophages that rely on passive diffusion to move from one site to another, bacteria actively move within the intestine and colonize inaccessible niches giving them the possibility to escape bacteriophage predation.¹⁹ Alternatively, the bacteria may be physiologically protected against bacteriophage infection. Our ex vivo experiments on homogenized colonized gut samples revealed that, in this case, the bacteria were less permissive to bacteriophage infection than bacteria grown in LB and subsequently added to homogenized non colonized gut samples. This was true for bacterial cells isolated from the mouse colon, but not from the ileum, reflecting the removal of *E. coli* by bacteriophages in the ileum. The metabolism of bacterial cells clearly differs between the small and the large intestine, due mainly to differences in nutrient availability, and we confirmed in vitro that this *E. coli* strain had a lower permissivity to bacteriophage infection in stationary phase than in the exponential growth phase. The antibiotic used to maintain the level of bacterial colonization in the gut may counteract the effect of the bacteriophages. It is possible that the selection pressure imposed by the antibiotic favors colonization with this *E. coli* strain not only directly, due to its resistance, but also indirectly due to a lack of metabolic competitors eliminated by the antibiotic.

Overall, the low permissivity to bacteriophage infection and lack of bacteriophage-resistant mutants are consistent with the hypothesis put forward by Reyes et al. that predator-prey relationships are scarce in the intestine.² These authors suggested that the low coevolution dynamics in the gut might be due to the predominance of

prophages in this organ. Our work suggests that bacteria with a low permissivity to virulent bacteriophage infections may also play a role in this phenomenon. There may be several reasons for the low permissivity to infection observed. For example, the localization or expression of the bacteriophage receptor at the cell surface may be modified in the gut. This has been shown for bacteriophages infecting the intestinal pathogen *Vibrio cholerae*, as the pattern of expression of the CTXφ and JF9 bacteriophage receptors, TCP and CRP, respectively, is modified by environmental conditions along the length of the intestine.²⁰⁻²² Phase variation has also been implicated in bacteriophage receptor availability at the cell surface.²³

In addition to host responses, the viral cycle in the gut environment may itself be affected. As suggested by several authors, pseudolysogeny may play a significant role in virus-host interactions in specific environmental conditions.²⁴,²⁵ Pseudolysogeny is characterized by a “pause” in the classical lytic cycle. The viral genome is maintained in the bacterial cytoplasm in a plasmid-like form until favorable conditions for the resumption of the lytic cycle occur. Unlike lysogenic phages, pseudolysogenic phages do not integrate into the bacterial chromosome.²⁶ Pseudolysogeny seems to be entirely dependent on host physiology, as no viral gene has yet been shown to be involved. This hypothesis is supported by our findings that the infection of *E. coli* in vivo in the intestine could not be inhibited (with DNA damaging agents, bacteriophage washout and phage-receptor interaction competitor agents) whereas such inhibition was possible in vitro when the strain was grown in LB. Indeed, the presence of pseudolysogens in the bacterial cytoplasm, where they have access to the host DNA repair system, may enable them to survive dsDNA breaks (caused by damaging agents). Moreover, decreasing extracellular bacteriophage concentration, either by washout or through the use of competitor agents, would not change the fate of host cells harboring these viral pseudolysogens, as they would by lysed when favorable conditions for lytic cycle resumption were encountered, such as during the plating of samples in Petri dishes. This led us to

develop a molecular approach (qPCR) to quantifying bacteria from samples containing excess bacteriophages (note that a DNase treatment step was added prior to DNA extraction to eliminate DNA originating from lysed cells). This excess may have been reached because the bacteria contained pseudolysogens, making it difficult to obtain accurate counts by direct plating methods. This suggests that some published studies may have overestimated bacteriophage efficacy, due to the absence of bacterial colonies on plates for samples containing bacteriophages.

The potential role of pseudolysogeny in the maintenance of bacteriophages in the gut ecosystem raises many questions about interactions of bacteriophages and bacteria in animals. If bacterial host cells can divide while harboring a pseudolysogen, as suggested by Los et al., it would be interesting to determine whether the pseudolysogen persists only in the parental cell or whether it is transmitted to the daughter cells.²⁷ If such transmission occurs, does pseudolysogen replication involve host factors only, or is a subset of viral proteins involved? If viral genes are expressed during the pseudolysogenic stage, do they confer a selective advantage on the bacterial host, leading to pseudolysogen maintenance? Conversely, if the presence of the pseudolysogen is a disadvantage how does the host cell eliminate it? This might result in abortive infections, which are thought to play a role in CRISPR evolution, by providing viral spacer sequences.²⁸ During pseudolysogeny, allelic exchanges might also occur between pseudolysogens and bacterial genomes via homologous recombination. Extending this hypothesis to the level of the intestinal microbiota highlights the potential role of pseudolysogeny in horizontal gene transfer, which may contribute to microbiota dynamics. Undoubtedly, experimental assessments of the role of pseudolysogeny should now be further developed.

## The Lessons to be Learned

First, our results highlight the need to develop molecular approaches for the accurate quantification of bacteria. Even the simplest determination of bacterial counts on Petri dishes was less simple
than initially anticipated, revealing unexpected facets to the gut environment. As in all scientific disciplines, reproducibility is essential for appropriate comparisons and the techniques used must therefore be reliable. The development of molecular approaches, as demonstrated by the modest efforts made in our study, would certainly increase the reliability of results.

Second, we provided evidence that not all bacteriophages active in vitro performed as well in vivo. This will undoubtedly lead to future studies, but it raises the question of how to select the best bacteriophages for therapeutic applications? Our results clearly indicate that CLB_P2 was the most suitable of the three bacteriophages tested for therapeutic usage, but the molecular properties underlying the better performance of this bacteriophage remain to be determined. We found that activities in vitro on biofilms and in vivo in animals were well correlated. For this

particular model, the use of in vitro biofilms before in vivo testing might improve predictions of bacteriophage efficacy. However, other in vitro models may be required to mimic different settings in vivo. As it will not be possible to test all therapeutic candidates in a pertinent in vivo system on a large scale, the search for molecular determinants involved in virus-host interactions in vivo remains essential. Such determinants common to all bacteriophages are not likely to be identified however, we may expect some of them to be conserved among genetically closely related bacteriophages.

In recent years, microbiology has moved from single-celled model organisms to more complex environmental systems (e.g., lakes, oceans and sites of infection), but we need to bear in mind that bacteria do not live alone, there are always bacteriophages around... and that is as good as it gets. Many aspects remain to be elucidated to increase our understanding of the

interactions between bacteriophages and bacteria in the gut. Studies of these aspects will improve our ecological understanding of this complex environment, potentially enhancing the use of bacteriophages to treat gut infections, which is dependent on such knowledge but also on academic and industrial supports as recently highlighted by Harald Brüssow.²⁹ However, so far laboratory studies focused on the tip of an iceberg, the submerged part corresponding to what is likely to be discovered in studying bacteriophages in a relevant ecological niche. In conclusion, even when taking everything we know about interactions between the well-known bacterium *E. coli* and its bacteriophages into account, attempts to study these interactions in the digestive tract, the real environment of *E. coli*, are likely to modify our current knowledge of these interactions, reflecting how much we still have to learn about the gut microbiome and the complex interactions among its components.

## References

1. Reyes A, Semenkovich NP, Whiteson K, Rohwer F, Gordon JJ. Going viral: next-generation sequencing applied to phage populations in the human gut. Nat Rev Microbiol 2012; 10:607-17; PMID:22864264; http://dx.doi.org/10.1038/nrmicro2853.

2. Reyes A, Haynes M, Hanson N, Angly FE, Heath AC, Rohwer F, et al. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 2010; 466:334-8; PMID:20631792; http://dx.doi.org/10.1038/nature09199.

3. Doucet-Populaire F, Tieu-Cuot P, Dosbaa I, Andremont A, Courvalin P. Inducible transfer of conjugative transposon Tn1545 from Enterococcus faecalis to Listeria monocytogenes in the digestive tracts of gnotobiotic mice. Antimicrob Agents Chemother 1991; 35:185-7; PMID:1849709; http://dx.doi.org/10.1128/AAC.35.1.185.

4. Andremont A, Gerbaud G, Tancrède C, Courvalin P. Plasmid-mediated susceptibility to intestinal microbial antagonisms in Escherichia coli. Infect Immun 1985; 49:751-5; PMID:3897062.

5. Duerkop BA, Clements CV, Rollins D, Rodrigues JL, Hooper LV. A composite bacteriophage alters colonization by an intestinal commensal bacterium. Proc Natl Acad Sci U S A 2012; 109:17621-6; PMID:23045666; http://dx.doi.org/10.1073/pnas.1206136109.

6. Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM. Phage treatment of human infections. Bacteriophage 2011; 1:66-85; PMID:22334863; http://dx.doi.org/10.4161/bact.1.2.15845.

7. Burrowes B, Harper DR, Anderson J, McConville M, Enright MC. Bacteriophage therapy: potential uses in the control of antibiotic-resistant pathogens. Expert Rev Anti Infect Ther 2011; 9:775-85; PMID:21905786; http://dx.doi.org/10.1586/eri.11.90.

8. Brüssow H. Phage therapy: the Escherichia coli experience. Microbiology 2005; 151:2133-40; PMID:16000704; http://dx.doi.org/10.1099/mic.0.27849-0.

9. Letarov A, Kulikov E. The bacteriophages in human- and animal body-associated microbial communities. J Appl Microbiol 2009; 107:1-13; PMID:19239553; http://dx.doi.org/10.1111/j.1365-2672.2009.04143.x.

10. Saussereau E, Debarbieux L. Bacteriophages in the experimental treatment of Pseudomonas aeruginosa infections in mice. Adv Virus Res 2012; 83:123-41; PMID:22748810; http://dx.doi.org/10.1016/B978-0-12-394438-2.00004-9.

11. Maura D, Galtier M, Le Bouguénec C, Debarbieux L. Virulent bacteriophages can target O104:H4 enteroaggregative Escherichia coli in the mouse intestine. Antimicrob Agents Chemother 2012; 56:6235-42; PMID:23006754; http://dx.doi.org/10.1128/AAC.00602-12.

12. Maura D, Morello E, du Merle L, Bomme P, Le Bouguénec C, Debarbieux L. Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice. Environ Microbiol 2012; 14:1844-54; PMID:22118225; http://dx.doi.org/10.1111/j.1462-2920.2011.02644.x.

13. Weiss M, Denou E, Bruttin A, Serra-Moreno R, Dillmann ML, Brüssow H. In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli. Virology 2009; 393:16-23; PMID:19699505; http://dx.doi.org/10.1016/j.virol.2009.07.020.

14. Muniesa M, Hammerl JA, Hertwig S, Appel B, Brüssow H. Shiga toxin-producing Escherichia coli O104:H4: a new challenge for microbiology. Appl Environ Microbiol 2012; 78:4065-73; PMID:22504816; http://dx.doi.org/10.1128/AEM.00217-12.

15. Kasman LM. Barriers to coliphage infection of commensal intestinal flora of laboratory mice. Virol J 2005; 2:34; PMID:15833115; http://dx.doi.org/10.1186/1743-422X-2-34.

16. Foucault ML, Thomas L, Goussard S, Branchini BR, Grillot-Courvalin C. In vivo bioluminescence imaging for the study of intestinal colonization by Escherichia coli in mice. Appl Environ Microbiol 2010; 76:264-74; PMID:19880653; http://dx.doi.org/10.1128/AEM.01686-09.

17. Gómez P, Buckling A. Bacteria-phage antagonistic coevolution in soil. Science 2011; 332:106-9; PMID:21454789; http://dx.doi.org/10.1126/science.1198767.

18. Kunisaki H, Tanji Y. Intercrossing of phage genomes in a phage cocktail and stable coexistence with Escherichia coli O157:H7 in anaerobic continuous culture. Appl Microbiol Biotechnol 2010; 85:1533-40; PMID:19763563; http://dx.doi.org/10.1007/s00253-009-2230-2.

19. Butler SM, Camilli A. Going against the grain: chemotaxis and infection in Vibrio cholerae. Nat Rev Microbiol 2005; 3:611-20; PMID:16012515; http://dx.doi.org/10.1038/nrmicro1207.

20. Zahid MS, Waise TM, Kamruzzaman M, Ghosh AN, Nair GB, Mekalanos JJ, et al. The cyclic AMP (cAMP)-cAMP receptor protein signaling system mediates resistance of Vibrio cholerae O1 strains to multiple environmental bacteriophages. Appl Environ Microbiol 2010; 76:4233-40; PMID:20472740; http://dx.doi.org/10.1128/AEM.00008-10.

21. Waldor MK, Tschäpe H, Mekalanos JJ. A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139. J Bacteriol 1996; 178:4157-65; PMID:8763944.

22. Nielsen AT, Dolganov NA, Rasmussen T, Otto G, Miller MC, Felt SA, et al. A bistable switch and anatomical site control Vibrio cholerae virulence gene expression in the intestine. PLoS Pathog 2010; 6:e1001102; PMID:20862321; http://dx.doi.org/10.1371/journal.ppat.1001102.

23. Wegrzyn G, Thomas MS. Modulation of the susceptibility of intestinal bacteria to bacteriophages in response to Ag43 phase variation -- a hypothesis. Med Sci Monit 2002; 8:HY15-8; PMID:12070443.
24. Clokie MR, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage 2011; 1:31-45; PMID:21687533; http://dx.doi.org/10.4161/bact.1.1.14942.

25. Ripp S, Miller RV. Dynamics of the pseudolysogenic response in slowly growing cells of Pseudomonas aeruginosa. Microbiology 1998; 144:2225-32; PMID:9720044; http://dx.doi.org/10.1099/00221287-144-8-2225.

26. Miller RV, Day MJ. Contribution of lysogeny, pseudolysogeny, and starvation to phage ecology. In: Abedon S, ed. Bacteriophage Ecology: Cambridge University Press, 2008:114-44.

27. Los M, Wegrzyn G, Neubauer P. A role for bacteriophage T4 rl gene function in the control of phage development during pseudolysogeny and in slowly growing host cells. Res Microbiol 2003; 154:547-52; PMID:14527655; http://dx.doi.org/10.1016/S0923-2508(03)00151-7.

28. Abedon ST. Facilitation of CRISPR adaptation. Bacteriophage 2011; 1:179-81; PMID:22164352; http://dx.doi.org/10.4161/bact.1.3.16709.

29. Brüssow H. What is needed for phage therapy to become a reality in Western medicine? Virology 2012; 434:138-42; PMID:23059181; http://dx.doi.org/10.1016/j.virol.2012.09.015.