African and Asian Zika Virus Isolates Display Phenotypic Differences Both In Vitro and In Vivo.

Zika virus (ZIKV) is a mosquito-borne member of the genus Flavivirus that has emerged since 2007 to cause outbreaks in Africa, Asia, Oceania, and most recently, in the Americas. Here, we used an isolate history as well as genetic and phylogenetic analyses to characterize three low-passage isolates representing African (ArD 41525) and Asian (CPC-0740, SV0127-14) lineages to investigate the potential phenotypic differences in vitro and in vivo. The African isolate displayed a large plaque phenotype (∼3-4 mm) on Vero and HEK-293 cells, whereas the Asian isolates either exhibited a small plaque phenotype (∼1-2 mm) or did not produce any plaques. In multistep replication kinetics in nine different vertebrate and insect cell lines, the African isolate consistently displayed faster replication kinetics and yielded ∼10- to 10,000-fold higher peak virus titers (infectious or RNA copies) compared with the Asian isolates. Oral exposure of Aedes aegypti mosquitoes with the African isolate yielded higher infection and dissemination rates compared with the Asian isolates. Infection of Ifnar1-/- mice with the African isolate produced a uniformly fatal disease, whereas infection with the Asian isolates produced either a delay in time-to-death or a significantly lower mortality rate. Last, the African isolate was > 10,000-fold more virulent than the Asian isolates in an interferon type I antibody blockade mouse model. These data demonstrate substantial phenotypic differences between low-passage African and Asian isolates both in vitro and in vivo and warrant further investigation. They also highlight the need for basic characterization of ZIKV isolates, as the utilization of the uncharacterized isolates could have consequences for animal model and therapeutic/vaccine development.

Retrospective use of next-generation sequencing reveals the presence of Enteroviruses in acute influenza-like illness respiratory samples collected in South/South-East Asia during 2010-2013.

BACKGROUND: Emerging and re-emerging respiratory pathogens represent an increasing threat to public health. Etiological determination during outbreaks generally relies on clinical information, occasionally accompanied by traditional laboratory molecular or serological testing. Often, this limited testing leads to inconclusive findings. The Armed Forces Research Institute of Medical Sciences (AFRIMS) collected 12,865 nasopharyngeal specimens from acute influenza-like illness (ILI) patients in five countries in South/South East Asia during 2010-2013. Three hundred and twenty-four samples which were found to be negative for influenza virus after screening with real-time RT-PCR and cell-based culture techniques demonstrated the potential for viral infection with evident cytopathic effect (CPE) in several cell lines. OBJECTIVE: To assess whether whole genome next-generation sequencing (WG-NGS) together with conventional molecular assays can be used to reveal the etiology of influenza negative, but CPE positive specimens. STUDY DESIGN: The supernatant of these CPE positive cell cultures were grouped in 32 pools containing 2-26 supernatants per pool. Three WG-NGS runs were performed on these supernatant pools. Sequence reads were used to identify positive pools containing viral pathogens. Individual samples in the positive pools were confirmed by qRT-PCR, RT-PCR, PCR and Sanger sequencing from the CPE culture and original clinical specimens. RESULTS: WG-NGS was an effective way to expand pathogen identification in surveillance studies. This enabled the identification of a viral agent in 71.3% (231/324) of unidentified surveillance samples, including common respiratory pathogens (100/324; 30.9%): enterovirus (16/100; 16.0%), coxsackievirus (31/100; 31.0%), echovirus (22/100; 22.0%), human rhinovirus (3/100; 3%), enterovirus genus (2/100; 2.0%), influenza A (9/100; 9.0%), influenza B, (5/100; 5.0%), human parainfluenza (4/100; 4.0%), human adenovirus (3/100; 3.0%), human coronavirus (1/100; 1.0%), human metapneumovirus (2/100; 2.0%), and mumps virus (2/100; 2.0%), in addition to the non-respiratory pathogen herpes simplex virus type 1 (HSV-1) (172/324; 53.1%) and HSV-1 co-infection with respiratory viruses (41/324; 12.7%).

Disease Burden of Dengue in the Philippines: Adjusting for Underreporting by Comparing Active and Passive Dengue Surveillance in Punta Princesa, Cebu City.

AbstractDengue virus (DENV) is a serious threat to public health. Having reliable estimates of the burden of dengue is important to inform policy and research, but surveillance systems are not designed to capture all symptomatic DENV infections. We derived the rate of reporting of dengue by comparing active surveillance of symptomatic DENV infections in a prospective community-based seroepidemiological cohort study (N = 1008) of acute febrile illness in Punta Princesa, Cebu City, Philippines, with passive surveillance data from the Cebu City Health Department. Febrile episodes detected in a weekly follow-up of participants were tested for serotype-specific DENV by hemi-nested reverse transcription-polymerase chain reaction (nested RT-PCR) and acute/convalescent blood samples tested by dengue IgM/IgG enzyme immunoassay. We estimated the burden of dengue in the Philippines in disability-adjusted life years (DALYs), and conducted a probabilistic sensitivity analysis using Monte-Carlo simulations to address uncertainty. The results showed a 21% cumulative reporting rate of symptomatic DENV infections, equivalent to an expansion factor of 4.7 (95% certainty level [CL]: 2.2-15.1). Based on surveillance data in the Philippines for 2010-2014, we estimated 794,255 annual dengue episodes (95% CL: 463,000-2,076,000) and a disease burden of 535 (95% CL: 380-994) DALYs per million population using age weights and time discounting and 997 (95% CL: 681-1,871) DALYs per million population without age and time adjustments. Dengue imposes a substantial burden in the Philippines; almost 10 times higher than estimated for rabies, about twice the burden of intestinal fluke infections, and about 10% of the burden of tuberculosis. Our estimates should inform policy makers and raise awareness among the public.

Economic cost and burden of dengue in the Philippines.

Dengue, the world's most important mosquito-borne viral disease, is endemic in the Philippines. During 2008-2012, the country's Department of Health reported an annual average of 117,065 dengue cases, placing the country fourth in dengue burden in southeast Asia. This study estimates the country's annual number of dengue episodes and their economic cost. Our comparison of cases between active and passive surveillance in Punta Princesa, Cebu City yielded an expansion factor of 7.2, close to the predicted value (7.0) based on the country's health system. We estimated an annual average of 842,867 clinically diagnosed dengue cases, with direct medical costs (in 2012 US dollars) of $345 million ($3.26 per capita). This is 54% higher than an earlier estimate without Philippines-specific costs. Ambulatory settings treated 35% of cases (representing 10% of direct costs), whereas inpatient hospitals served 65% of cases (representing 90% of direct costs). The economic burden of dengue in the Philippines is substantial.

Clinical and epidemiological features of human rabies cases in the Philippines: a review from 1987 to 2006.

BACKGROUND: Rabies viral infection causes a fatal encephalomyelitis. In humans, classic features include hydrophobia, aerophobia, hypersalivation, agitation, and neurological symptoms. In the Philippines, canine rabies contributes to a significant burden of human disease. METHODS: We retrospectively reviewed the medical records of 1839 patients admitted to San Lazaro Hospital, Manila, Philippines between 1987 and 2006, with a clinical diagnosis of rabies. We used the World Health Organization case definition for clinical rabies, which is defined by the presence of hydrophobia. RESULTS: Male patients outnumbered females by 2.2 to 1 and twice the number of adults were affected compared with children. Most patients were indigent. Dog bites occurred more than cat bites (97.1% vs. 2.9%) and most cases were caused by a single bite (86.2%), compared to multiple bites (8.7%). Bites to the face, head, and neck led to shorter incubation times, yet the incubation period varied, with most cases (42.7%) occurring in the bracket of 91-365 days post-exposure. Clinical symptoms included hydrophobia in all cases, as per our case definition, and aerophobia in 95.5%; only 9.4% had fever, 9.2% exhibited restlessness, and 6.7% exhibited hypersalivation. Localized neurological symptoms included pain (4.1%), numbness (2.6%), and itching (2.3%). None of the patients received appropriate post-exposure prophylaxis (PEP). CONCLUSIONS: This study examines the largest cohort of rabies patients reported to-date. Better understanding of clinical disease manifestations may help in salvage efforts to save patients with rabies. Knowledge of epidemiological factors will improve preventative efforts to reduce suffering from rabies.

Evaluation of a dengue IgG indirect enzyme-linked immunosorbent assay and a Japanese encephalitis IgG indirect enzyme-linked immunosorbent assay for diagnosis of secondary dengue virus infection.

To establish a new method for the diagnosis of dengue secondary infection, 187 serum samples from the patients with dengue secondary infection, 40 serum samples from the patients with dengue primary infection, and 44 serum samples from the healthy volunteers were tested using the dengue IgG indirect enzyme-linked immunosorbent assay (DEN IgG ELISA). The results of the test were compared with those from the dengue hemagglutination inhibition (DEN HI) test, which has been recommended as the gold standard by the World Health Organization (WHO, 1997). Japanese encephalitis IgG indirect ELISA (JE IgG ELISA) was also performed to measure anti-flavivirus IgG, which cross-reacts with the Japanese encephalitis virus, to test the possibility of an alternative to DEN IgG ELISA. The results of DEN IgG and JE IgG ELISAs were highly correlated with those of the DEN HI test. In the DEN IgG ELISA, a titer of 1:29,000 was the cut-off value for the diagnosis of dengue secondary infection (91.5% accuracy [95% confidence interval, CI], 90.9% sensitivity [95%CI], and 92.9% specificity [95%CI]). A titer of 1:52,000 was the cut-off value for dengue secondary infection using JE IgG ELISA (95.6% accuracy [95%CI], 98.9% sensitivity [95%CI], and 88.1% specificity [95%CI]). In conclusion, this study confirmed that the results of both DEN IgG and JE IgG ELISAs were highly correlated with the results of DEN HI test. Thus, these ELISAs are simple, rapid, sensitive, and quantitative tests that can be used in the determination of dengue secondary infection.