Anaplasma platys-Like Infection in Goats, Beijing, China.

As one of the important tick-borne zoonotic pathogens, Anaplasma has both veterinary and public health significance. Here, we performed a survey of Anaplasma infection in the goats from a farm in Beijing, China, and found 44.6% (41/92) were infected with Anaplasma capra, and 22.8% (21/92) were infected with Anaplasma sp. This Anaplasma sp. bacterium was close to a recently emerging Anaplasma platys strain based on gltA and groEL gene phylogenetic analysis. As to further understand the characteristics of Anaplasma sp., we raised a couple of positive goats (n = 2) in the laboratory with tick-free settings. We observed inappetence, vomiting, high fever, and weakness of limbs in the goat's offspring (n = 3). In addition, the blood samples from all offspring were all positive of this Anaplasma spp. We did not see any intracellular morulae in neutrophils, monocytes, and erythrocytes, but we identified some in the platelets of the blood smears from the positive goats by light microscopy. We named it A. platys-like and suggested it may infect platelets and be transmitted vertically through the placenta of goats. These findings deserve further evaluation.

Development of a chimeric Zika vaccine using a licensed live-attenuated flavivirus vaccine as backbone.

The global spread of Zika virus (ZIKV) and its unexpected association with congenital defects necessitates the rapid development of a safe and effective vaccine. Here we report the development and characterization of a recombinant chimeric ZIKV vaccine candidate (termed ChinZIKV) that expresses the prM-E proteins of ZIKV using the licensed Japanese encephalitis live-attenuated vaccine SA14-14-2 as the genetic backbone. ChinZIKV retains its replication activity and genetic stability in vitro, while exhibiting an attenuation phenotype in multiple animal models. Remarkably, immunization of mice and rhesus macaques with a single dose of ChinZIKV elicits robust and long-lasting immune responses, and confers complete protection against ZIKV challenge. Significantly, female mice immunized with ChinZIKV are protected against placental and fetal damage upon ZIKV challenge during pregnancy. Overall, our study provides an alternative vaccine platform in response to the ZIKV emergency, and the safety, immunogenicity, and protection profiles of ChinZIKV warrant further clinical development.

Intranasal infection and contact transmission of Zika virus in guinea pigs.

Zika virus (ZIKV) is primarily transmitted to humans through mosquito bites or sexual contact. The excretion and persistence of contagious ZIKV in various body fluids have been well documented in ZIKV patients; however, the risk of direct contact exposure remains unclear. Here, we show that guinea pigs are susceptible to ZIKV infection via subcutaneous inoculation route; infected guinea pigs exhibit seroconversion and significant viral secretion in sera, saliva, and tears. Notably, ZIKV is efficiently transmitted from infected guinea pigs to naïve co-caged animals. In particular, intranasal inoculation of ZIKV is fully capable of establishing infection in guinea pigs, and viral antigens are detected in multiple tissues including brain and parotid glands. Cynomolgus macaques also efficiently acquire ZIKV infection via intranasal and intragastric inoculation routes. These collective results from animal models highlight the risk of exposure to ZIKV contaminants and raise the possibility of close contact transmission of ZIKV in humans.

Characterization of a 2016 Clinical Isolate of Zika Virus in Non-human Primates.

Animal models are critical to understand disease and to develop countermeasures for the ongoing epidemics of Zika virus (ZIKV). Here we report a non-human primate model using a 2016 contemporary clinical isolate of ZIKV. Upon subcutaneous inoculation, rhesus macaques developed fever and viremia, with robust excretion of ZIKV RNA in urine, saliva, and lacrimal fluid. Necropsy of two infected animals revealed that systematic infections involving central nervous system and visceral organs were established at the acute phrase. ZIKV initially targeted the intestinal tracts, spleen, and parotid glands, and retained in spleen and lymph nodes till 10days post infection. ZIKV-specific immune responses were readily induced in all inoculated animals. The non-human primate model described here provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccine and therapeutics.

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.

OBJECTIVE: LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. METHODS: A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. RESULTS: Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. CONCLUSION: The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

[Evaluation of immunization protection efficacy of plague subunit vaccine].

OBJECTIVE: To evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study. METHODS: Groups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization. RESULTS: The immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively. CONCLUSION: BALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.