Characterization of <i>Monkeypox virus</i> infection in African rope squirrels (<i>Funisciurus sp</i>.)

<div><p>Monkeypox (MPX) is a zoonotic disease endemic in Central and West Africa and is caused by <i>Monkeypox virus</i> (MPXV), the most virulent <i>Orthopoxvirus</i> affecting humans since the eradication of <i>Variola virus</i> (VARV). Many aspects of the MPXV transmission cycle, including the natural host of the virus, remain unknown. African rope squirrels (<i>Funisciurus spp</i>.) are considered potential reservoirs of MPXV, as serosurveillance data in Central Africa has confirmed the circulation of the virus in these rodent species [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005809#pntd.0005809.ref001" target="_blank">1</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005809#pntd.0005809.ref002" target="_blank">2</a>]. In order to understand the tissue tropism and clinical signs associated with infection with MPXV in these species, wild-caught rope squirrels were experimentally infected via intranasal and intradermal exposure with a recombinant MPXV strain from Central Africa engineered to express the luciferase gene. After infection, we monitored viral replication and shedding via <i>in vivo</i> bioluminescent imaging, viral culture and real time PCR. MPXV infection in African rope squirrels caused mortality and moderate to severe morbidity, with clinical signs including pox lesions in the skin, eyes, mouth and nose, dyspnea, and profuse nasal discharge. Both intranasal and intradermal exposures induced high levels of viremia, fast systemic spread, and long periods of viral shedding. Shedding and luminescence peaked at day 6 post infection and was still detectable after 15 days. Interestingly, one sentinel animal, housed in the same room but in a separate cage, also developed severe MPX disease and was euthanized. This study indicates that MPXV causes significant pathology in African rope squirrels and infected rope squirrels shed large quantities of virus, supporting their role as a potential source of MPXV transmission to humans and other animals in endemic MPX regions.</p></div

Progression of disease of rope squirrels infected intradermally with MPXV-Congo/Luc+.

<p>The days post infection (pi) of various clinical signs are shown for the group, above the time scale. The days of luminescence are shown in green. Days of shedding of virus in nasal, ocular, oral and rectal mucosal secretions, as detected by TCID₅₀ assay, are shown below the time scale. Colors coordinate between clinical signs and route of shedding: nasal shedding and respiratory disease is shown in red. Ocular lesions and ocular shedding are shown in yellow. Oral lesions and oral shedding are shown in blue. Rectal shedding, for which no corresponding clinical observations were made, is shown in purple.</p

Ventral bioluminescent images of African rope squirrels intradermally infected with MPXV-Congo/Luc+ on days 3, 6, 8, and 11 post infection (pi).

<p>Color scale shows the intensity of luminescence in counts, with purple representing the lowest intensity, green the mid-range and red the highest intensity. Animals were infected on the dorsal scapular area. Luminescence in the oral and nasal areas, which begins on day 3 pi, is the result of replication of the virus distant from the initial site of infection. RS12 died on day 8 pi.</p

Ventral bioluminescent images of African rope squirrels intranasally infected with MPXV-Congo/Luc+ on days 13, 15, 18, and 20 post infection (pi).

<p>Color scale shows the intensity of luminescence, with purple representing the lowest intensity, green the mid-range and red the highest intensity. After day 13 pi, RS13 was found dead. RS16 was euthanized after imaging on day 13 pi. RS11 had a large amount of luminescence in the oral and nasal areas on day 13 pi, but decreased throughout the remainder of the days. By day 20 pi, luminescence is not visible in bioluminescent overlay images.</p

Pre-infection neutralizing activity of serum of rope squirrels against MPXV/Congo/Luc+.

<p>Positive controls were serum from a human vaccinated with vaccinia smallpox vaccine (positive control 1) and serum from a Gambian pouched rat (<i>Cricetomys gambianus</i>) that survived MPXV infection (positive control 2). Serum from an uninfected Gambian pouched rat was used as a negative control. RS14 and RS19 had a slightly greater than 50% reduction in viral luminescence at 1:40 dilution. However, this was not considered a protective titer. Neither of these animals survived infection with MPXV.</p

Frequency and distribution of histologic lesions in African rope squirrels infected intradermally (ID) or intranasally (IN) with MPXV-Congo/Luc+.

<p>(+) indicates a lesion was found in that individual animal within that tissue. (−) indicates that no lesion was seen in the sections collected and examined microscopically. The frequency of the lesion was calculated for all 9 animals, regardless of route of infection.</p

Ventral bioluminescent images of African rope squirrels intradermally infected with MPXV-Congo/Luc+ on days 13, 15, 18, and 20 post infection (pi).

<p>Color scale shows the intensity of luminescence, in counts, with purple representing the lowest intensity, green the mid-range and red the highest intensity. Luminescence, indicative of viral replication, in the oral and nasal areas continued until day 18 pi. Distal limbs and the tail were common sites of replication. These sites often corresponded to lesions on the feet.</p

Epidemiologic and Ecologic Investigations of Monkeypox, Likouala Department, Republic of the Congo, 2017

Monkeypox, caused by a zoonotic orthopoxvirus, is endemic in Central and West Africa. Monkeypox has been sporadically reported in the Republic of the Congo. During March 22–April 5, 2017, we investigated 43 suspected human monkeypox cases. We interviewed suspected case-patients and collected dried blood strips and vesicular and crust specimens (active lesions), which we tested for orthopoxvirus antibodies by ELISA and monkeypox virus and varicella zoster virus DNA by PCR. An ecologic investigation was conducted around Manfouété, and specimens from 105 small mammals were tested for anti-orthopoxvirus antibodies or DNA. Among the suspected human cases, 22 met the confirmed, probable, and possible case definitions. Only 18 patients had available dried blood strips; 100% were IgG positive, and 88.9% (16/18) were IgM positive. Among animals, only specimens from Cricetomys giant pouched rats showed presence of orthopoxvirus antibodies, adding evidence to this species’ involvement in the transmission and maintenance of monkeypox virus in nature

Disease progression and shedding of rope squirrels infected intranasally with MPXV-Congo/Luc+.

<p>The days post infection (pi) of various clinical signs are shown for the group, above the time scale. The days of luminescence are shown in green. Days of shedding of virus in nasal, ocular, oral and rectal mucosal secretions, as detected by TCID₅₀ assay, are shown below the time scale. Colors coordinate between clinical signs and route of shedding: nasal shedding and respiratory disease is shown in red. Ocular lesions and ocular shedding are shown in yellow. Oral lesions and oral shedding are shown in blue. Rectal shedding, for which no corresponding clinical observations were made, is shown in purple.</p

Shedding of MPXV by the sentinel African rope squirrel, RS14, following mock intranasal infection.

<p>The animal was housed in its own cage in the same room as the study animals, and was sampled and imaged following the same schedule as the other study animals. Swabs were taken from oral, ocular, nasal and rectal mucosa then tested by TCID₅₀ assay to determine the concentration of virus shed in PFU/mL. Titer for all swabs increased from day 13 pi (oral) or day 15 pi (nasal, ocular, rectal) until day 18 pi, when the animal was euthanized. Oral shedding was the highest, with a final titer greater than 1.3 x 10⁸ PFU/mL on day 18 pi.</p