Quantitative conversion of biomass in giant DNA virus infection

Bioconversion of organic materials is the foundation of many applications in chemical engineering, microbiology and biochemistry. Herein, we introduce a new methodology to quantitatively determine conversion of biomass in viral infections while simultaneously imaging morphological changes of the host cell. As proof of concept, the viral replication of an unidentified giant DNA virus and the cellular response of an amoebal host are studied using soft X-ray microscopy, titration dilution measurements and thermal gravimetric analysis. We find that virions produced inside the cell are visible from 18 h post infection and their numbers increase gradually to a burst size of 280–660 virions. Due to the large size of the virion and its strong X-ray absorption contrast, we estimate that the burst size corresponds to a conversion of 6–12% of carbonaceous biomass from amoebal host to virus. The occurrence of virion production correlates with the appearance of a possible viral factory and morphological changes in the phagosomes and contractile vacuole complex of the amoeba, whereas the nucleus and nucleolus appear unaffected throughout most of the replication cycle.


A.1. X-ray microscopy of virus infections
We present all additional x-ray micrographs of non-infected ( Fig. S1-S4) and virus-infected ( Fig.  S5-S13) amoebae that were used to study the viral replication cycle. The infection times range from 6 h (Fig. S5) up to 70 h (Fig. S13). All images were acquired using the Stockholm laboratory x-ray microscope with exposure times of mainly 60 s. For comparison, transmission electron microscopy of sectioned, stained, resin-embedded, amoeba cells are shown for noninfected (Fig. S14a) and virus-infected (Fig. S14b-c) amobae.

A.2. Relationship between conversion of biomass and energy consumption
Our experimental measure of conversion of biomass based on the high carbon-to-water contrast present in the water window can be related to the energy consumption needed to produce the virions inside the host cell. Following the same approach as Mahmoudabadia et al.
(1), which is based on the equations presented by Lynch and Marinov (2), we assume that the basal metabolic requirement of a cell scales with cell volume as whereas metabolic requirement for cellular growth scales similarly as This results in that the total energy budget of a cell during the cell division time t [h] is Since the cell metabolism is slowed due to the incomplete medium without glucose, we estimate the cell division time to be approximately the same as the last infection time, i.e. t = 70 h. The virus consumption of the host's energy budget is thus E V /E C , given that the energetic cost of the virus E V can be estimated. On the other hand, the cell also has energy stored in its cytoplasm and organelles, which must also correspond to E G so that the total energy bound in the cell is Assuming that our x-ray contrast is proportional to the number of glucose molecules supplied to build the cell, which in turn is proportional to E T , we can write conversion of biomass from cell to virus as Thus, the virus consumption of the host's energy budget becomes Eq. (S6) relates conversion of biomass to virus consumption of the host's energy budget as defined in Mahmoudabadia et al.
(1). Given that the amoebae have an average volume of 3000 µm 3 (3) and the cell division time is assumed to be 70 h, the virus consumption of the host's energy budget will be 67% larger than the measured conversion of biomass.

B. Supplementary figures
Supplementary Figure S1. Laboratory cryogenic x-ray microscopy of healthy, non-infected amoebae imaged 4 h after transfer to incomplete PPYG medium (without glucose). Scale bar is 10 µm and is valid for all images.
Supplementary Figure S2. Laboratory cryogenic x-ray microscopy of healthy, non-infected amoebae imaged 24 h after transfer to incomplete PPYG medium (without glucose). Note that the center image (b) consists of two images that have been stitched together. All scale bars are 10 µm.
Supplementary Figure S3. Laboratory cryogenic x-ray microscopy of healthy, non-infected amoebae imaged 48 h after transfer to incomplete PPYG medium (without glucose). Scale bar is 10 µm and is valid for all images.
Supplementary Figure S4. Laboratory cryogenic x-ray microscopy of healthy, non-infected amoebae imaged 72 h after transfer to incomplete PPYG medium (without glucose). Scale bar is 10 µm and is valid for all images.
Supplementary Figure S5. Laboratory cryogenic x-ray microscopy of virus-infected amoebae imaged 6 hpi. Scale bar is 10 µm and is valid for all images.
Supplementary Figure S6. Laboratory cryogenic x-ray microscopy of virus-infected amoebae imaged 12 hpi. Scale bar is 10 µm and is valid for all images.
Supplementary Figure S8. Laboratory cryogenic x-ray microscopy of virus-infected amoebae imaged 18 hpi. Scale bar is 10 µm and is valid for all images.

C. Supplementary methods
The source code used to analyze the raw data presented in the main article is comprised of 7 MATLAB (R2018a) scripts available in a separate zip-file and whose input and docstrings are presented below.

C.1. Marked example images for viral particle identification
Three example images used as input for viral particle identification using MATLAB.