Quantitative conversion of biomass in giant DNA virus infection

Abstract

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.

Figure 1

Electron microscopy of virions of Lurbovirus. (a) Scanning electron micrograph of isolated virions of Lurbovirus using a scanning transmission electron microscope (STEM) detector in high-angle annular dark-field mode. (b) Transmission electron micrograph of a cryo-frozen virion of Lurbovirus. The double cork-like structures indicate morphological similarity to Cedratviruses. (c) Transmission electron micrograph of a sectioned, stained, resin-embedded, virus-infected amoeba cell imaged 10 hpi (at 32 °C in medium containing 20 mM glucose). Scale bars are 1 µm, 500 nm and 10 µm, respectively.

Figure 2

Laboratory full-field water-window cryo-microscopy of non-infected and infected amoeba. X-ray micrographs of a typical (a) non-infected amoeba (n = 14) and (b) infected amoeba imaged 54 hpi (n = 5). The images to the right show enlargements of the square insets. The appearance of numerous large (~ 1-µm length) ovoid virions in the infected cell clearly distinguishes it from the non-infected cell, which contains smaller (typically < 500 nm) and more circular objects, probably mitochondria or storage granules. A possible viral factory (VF) in the vicinity of the nucleus is indicated by the dashed white line. In order to clearly show the difference between the virions and the smaller X-ray absorbing objects, three examples in each image have been marked by arrows and a white cross indicating their length and width. Another striking difference is the typical vacuolated structure (v) of the non-infected cell, which is less prominent in the infected cell. Note that the nucleus (N) including the darker nucleolus (n) and the nuclear envelope (ne) appears to be intact in both cells. Scale bars are 10 µm in the whole images and 2 µm in the enlargements.

Figure 3

Virus infection series, ranging from non-infected cells to cells 70 hpi, visualized using laboratory cryogenic X-ray microscopy. (a) Non-infected amoeba (n = 5) that is included for comparison and has been treated identically as the infected amoebae 30 hpi aside from not adding the virus. The infection times in the displayed images are (b) 6 h (n = 5), (c) 12 h (n = 6), (d) 15 h (n = 5), (e) 18 h (n = 6), (f) 24 h (n = 5), (g) 30 h (n = 5), (h) 54 h (n = 5) and (i) 70 h (n = 4), respectively. In the images up to 15 hpi (a–d) no clearly distinguishable virions were detected. At 18 hpi (e) a few virions can be seen, e.g. in a vacuole in the right part of the cell as well as to the lower left of the nucleus. At longer infection times (f–i) the virions take up an increasing part of the cell volume, as well as the surrounding medium, until they appear densely packed throughout the cell. Possible viral factories, like the one marked in Fig. 2, can be seen close to the nuclei at 18–30 hpi (e–g). The vacuolated structure of the healthy cells seems to disappear as the production of virions takes off, while the cell nucleus can be seen in all images up to 54 hpi (a–h). Scale bar is 10 µm and valid for all images.

Figure 4

TGA thermograms of virus and cells. Solid line represents cells and dashed line represents virus. Measurements (n = 2) were performed under inert atmosphere. The residual mass after pyrolysis consists mostly of char, and was used to estimate the relative carbon content of virus (68%) and amoeba (28%). The error bars represent the standard error of the mean.

Figure 5

Virion production and conversion of biomass (C_1.3 + N_1.9 + O_0.1) as function of infection time. (a) The number of virions were counted in 52 different cells with infection times ranging from non-infected (0 h) to 70 h. The data at 70 hpi should be seen as a more approximate count and is therefore displayed in red. Each blue or red ring corresponds to an individual amoeba, while the solid line shows the average at each infection time. An estimated uncertainty (“Materials and methods”) was calculated for each data point and is indicated by the shadowed area. In addition, the result of end-point dilution titrations (n = 15) is shown at > 100 hpi (black square), including a 95% confidence interval. (b) The conversion of biomass from host to virus is estimated by measuring the X-ray absorption in the virions compared to the whole cell, in 32 X-ray images with sufficient quality. A high estimate (dashed line) is calculated by summing the absorption in the areas in the images marked as virions. A more moderate estimate (solid line) is given by multiplying the average number of counted virions by the typical absorption in a single virion. The conversion given by the end-point dilution titrations, together with TGA, is shown at > 100 hpi. Note that end-point dilution titrations and TGA were performed at 32 °C in PPYG medium containing 20 mM glucose, whereas quantitative analysis of X-ray micrographs were performed at room temperature in PPYG medium containing no glucose.