Gino Fornaciari
University of Pisa, Italy

The mummy of a two year old child, who died in Naples about the middle of the sixteenth century, revealed a widespread vesiculopustular exanthema. The macroscopic aspects, regional distribution, and light microscopy suggested the possibility of a case of smallpox.  Electron microscopy showed the presence of many egg shaped dense structures (250 nm x 150 nm), composed of a central dense region surrounded by a zone of lower density. After incubation with rabbit anti variola virus antiserum, followed by protein A-gold complex immunostaining, some of these particles
were completely covered by the protein A-gold, although others were only partially labeled. The controls were completely negative. It is therefore evident that the child was affected by a severe form of smallpox. The excellent preservation of the antigenic structure of some viral particles is probably due to dry microclimatic conditions and to treatment with some substances  (aqua vitae, lye) used for the embalming.

The Basilica of San Domenico Maggiore, which dates back to the beginning of the 14th century, is one of the largest and most important churches in Naples.  The humanist Giovanni Pontano and the philosophers Tommaso Campanella and Giordano Bruno studied in this Abbey.  Saint Thomas Aquinas taught in the annexed convent of the Dominicans. 
The impressive Sacristy of San Domenico Maggiore, in a suspended gateway close to the vault, contains 38 wooden coffins, or “arks”, with the bodies of 10 Aragonese princes and other Neapolitan nobles, who died in the 15th and 16th centuries. 
The sarcophagi, richly dressed in doxycycline generic clothes made of silk, brocade and other material, are distributed in two rows, one above the other.  The smaller coffins of the lower row are generally of anonymous individuals, while the larger coffins of the upper row are identified by the coats-of-arms and the names of the personages buried inside.  In particular they include the Aragonese kings Alfonso I (who died in 1458), Ferrante I (1494), Ferrante Il (1496), Queen Giovanna IV (1518), the Marquis of Pescara Ferdinando d’Avalos, who won the famous battle of Pavia against the French King François I in 1525 and Isabella d’Aragona, Duchess of Milan (1525), who had Leonard da Vinci at her court.
The majority of the individuals had been embalmed and this is certainly not surprising, considering the high social class of the individuals buried in San Domenico.  From the physician Ulisse Aldrovandi we know that during the Renaissance “the European kings and great personages used to entrust embalming of their bodies to their doctors and surgeons” (Aldrovandi 1602). The very complex evisceration and embalming methods indicate long-practiced and diffused customs but some well preserved individuals show no apparent signs of embalming. In this case the natural mummification of the bodies can probably be attributed to the very dry microclimatic conditions of the Basilica10. 

The mummies of San Domenico Maggiore are unique in Italy not only for the antiquity and excellent state of preservation of the bodies, but also for the fame of the personages, whose lives and causes of death are well known.  King Ferrante II, for example, died of malaria, while the Marquis of Pescara died of pulmonary tuberculosis.  The possibility of comparing the paleopathological with the historical data provided extremely interesting results

The sarcophagi of San Domenico Maggiore were carefully examined from 1984 to 1987 by a team of the Institute of Pathology of Pisa University.
Anthropological, radiological  and autoptic examinations of the mummies were carried out on site; later, the laboratory studies were performed in Pisa. 

Among these bodies, one of the most interesting from the paleopathological point of view was the artificial mummy of an anonymous  2 year old boy (2 years±8 months) (Ubelaker 1978), who died about the middle of the sixteenth century (radiocarbon dating is 1569± 60) (Fig. 1).


Fig. 1 The mummy of the boy, still dressed
    in the monastic clothe of the Dominican order.

The skin of this mummy showed a widespread vesiculopustular skin eruption. The scalp, the face with the palpebral regions and the lips, the posterior trunk, the arms and the glutei were the most involved regions. The forearms, including the palms of the hands, and the legs were also affected.


  Fig.2 The mummy, characterized by an enormous abdomen,
showed a widespread vesiculo-pustular skin eruption.


Fig. 3 The eruption of the face,
     the palpebral regions and the lips.

The vesicles and pustules (size 10-20  mm), completely dry, with crystalline content, showed a sort of central umbilication and a pale, peripheral halo. In some regions (skull and ribs) the lesions were so deep that even the periosteum was affected. The clinical appearance and distribution of the skin lesions, in particular palm involvement, excluded varicella.


Fig. 4 Some pustules (size 10-20  mm), completely dry,
showing a crystalline content and a sort of central
umbilication with a pale, peripheral halo.

Macroscopic aspects, regional distribution and the apparently simultaneous developmental stage of the lesions suggested a case of smallpox.

Samples of skin with vesicles or pustules, rehydrated according to Sandison
(1955), embedded in paraffin, were stained with Van Gieson for light microscopy.  
By light microscopy, at low magnification, the vesicles showed an evident separation of superficial layers of the epidermis from the basal layer, with the dermis still containing sebaceous glands and hair follicles.  
Histological patterns, mainly the dermal involvement, were again highly suggestive of smallpox.
The possibility of a case of smallpox was confirmed by indirect immunofluorescence with anti-variola virus antibody.

We decided to look for the virus in the cutaneous lesions of this well preserved mummy.   Myxovirus-like particles were demonstrated in a Peruvian mummy dated around 950 A.D. (Dalton et al. 1976), but to date the only attempt to identify smallpox virus in the mummy of Ramses V has failed (Hopkins 1983).

After dilution in distilled water of the crystalline content of some vescicles, the supernatant was collected and mixed with a similar quantity of 3% fosfotungstic acid at 6.4 pH, according the technique of negative staining with osmium tetroxide for viruses.
Ultrastructural observation of the pustule content with a Siemens Elmiskop 1A transmission electron microscope at the negative staining showed many tetrahedral viral-like particles (so-called “brick-like” particles), morphologically very similar to smallpox viruses.
Small fragments (1 mm) taken from the bottom of the pustules were rehydrated as described above, fixed with 2.5’è buffered glutaraldehyde, postfixed in OsO4, and prepared for electron microscopy.   In some cases, epon-embedded ultrathin sections were treated for 30 minutes with a saturated solution of sodium metaperiodate as described by Bendayan (1983), and incubated overnight at 4°C in an anti-variola virus antiserum diluted 1:30.   After repeated washings, sections were exposed to protein A gold complex for one hour at room temperature.
Preparation and absorption of colloidal gold to protein A has been described (Horisberger 1979, Roth et al. 1978).   The sections were then washed,  stained with uranyl acetate and lead citrate, and observed with a Siemens Elmiskop 1A electron microscope.

The skin was largely acellular, the surviving composition consisting predominantly of bands of collagen fibers and elastic tissue. Some areas showed pycnotic nuclei or membrane remains with rare, intact desmosomes.
Among the connective fibers, mainly in superficial areas, many egg shaped, dense structures (250 nm x 200 nm) could be seen. They were composed of a central dense region, or core, surrounded by a lower density area.


Fig. 5  Some egg shaped, dense structures (250 nm x 200 nm),
with a central dense region, or core, surrounded by a lower density area (inset).

Following incubation with human anti-variola virus antiserum, after protein A-gold complex immunostaining, some of these particles were completely covered by protein A-gold, although some others were partially labelled, only very few gold grains were scattered over the connective tissue.


Fig. 6 Virus-like particles, completely (a) or partially (b, c) covered
by protein A-gold, following incubation with human anti-variola
virus antiserum, after protein A-gold complex immunostaining.

As a specific control for our study, we performed the following tests:
1) Incubation with protein A gold complex alone;
2) Immunolabelling (with the same antiserum and the protein A-gold) of the skin of an unaffected mummy of Saint Domenico Maggiore.  
In the second case, we could not see any virus-like particles. In both controls the density of gold grains was very low.

These results showed that the antigenic structure of the viral particles was well preserved and that this Neapolitan child died of a severe form of smallpox some four centuries ago (Fornaciari and Marchetti 1986a, 1986b, 1986c, 1986d, 1986e; Fornaciari et al. 1989i). Their identification appeared to be about as certain as current technology will had permitted, short of cultural isolation of the virus.

It was impossible at that time to express an opinion on the maintenance of biological activity of the virus in mummy.
We pointed out only the excellent preservation of the antigenic structure of some viral particles. This was probably because of the very dry microclimatic condition of the Sacristy, and also partly because of the substances used in embalming.
In Naples, as described in an ancient embalming handbook (Lanzoni 1693), the bodies were washed with aqua vitae and lye after being eviscerated, and finally stuffed with resinous substances. It is possible that these procedures produced a true histological fixation of the tissue.
At that time various opinions have been expressed about the ability of smallpox virus to survive in its victims postmortem (Zuckerman 1984, Hopkins 1985, Meers 1985), and hence about the potential risk to archaeologists who had disturbed their remains.

An attempt of cultivation, required in particular by the excellent preservation of the virus and carried out at the Centers for Infectious Diseases of Atlanta in Georgia, resulted totally negative.

After almost 20 years we can definitely affirm that this worry was not realistic; actually paleo-molecular studies demonstrated that ancient DNA is extremely fragmented, in pieces of 100-150 base pairs; therefore virus, also if very well preserved from morphological point of view, was totally devoid of biological activity

Molecular study of ancient viral DNA, at present performed at the Laboratory of Molecular Archeo-Anthropology of the University of Camerino directed by prof. Franco Rollo, not only can further confirm the diagnosis of smallpox based on morphology and immunohistochemistry but, with the sequencing of some viral genes, could pave the way for further research on the secular evolution of these viruses.


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