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Wider availability of the live, attenuated SA 14-14-2 Japanese encephalitis (JE) vaccine has facilitated introduction or expansion of immunization programs in many countries. However, information on their impact is limited. In 2006, Nepal launched a JE immunization program, and by 2009, mass campaigns had been implemented in 23 districts. To describe the impact, we analyzed surveillance data from 2004 to 2009 on laboratory-confirmed JE and clinical acute encephalitis syndrome (AES) cases. The post-campaign JE incidence rate of 1.3 per 100,000 population was 72% lower than expected if no campaigns had occurred, and an estimated 891 JE cases were prevented. In addition, AES incidence was 58% lower, with an estimated 2,787 AES cases prevented, suggesting that three times as many disease cases may have been prevented than indicated by the laboratory-confirmed JE cases alone. These results provide useful information on preventable JE disease burden and the potential value of JE immunization programs.

Despite the recent discovery of genetically divergent hantaviruses in shrews of multiple species in widely separated geographic regions, data are unavailable about the genetic diversity and phylogeography of Thottapalayam virus (TPMV), a hantavirus originally isolated from an Asian house shrew (Suncus murinus) captured in southern India more than four decades ago. To bridge this knowledge gap, the S, M, and L segments of hantavirus RNA were amplified by reverse transcription polymerase chain reaction from archival lung tissues of Asian house shrews captured in Nepal from January to September 1996. Pair-wise alignment and comparison revealed approximately 80% nucleotide and > 94% amino acid sequence similarity to prototype TPMV. Phylogenetic analyses, generated by maximum likelihood and Bayesian methods, showed geographic-specific clustering of TPMV, similar to that observed for rodent- and soricid-borne hantaviruses. These findings confirm that the Asian house shrew is the natural reservoir of TPMV and suggest a long-standing virus–host relationship.

Vector-borne infections (VBI) are defined as infectious diseases transmitted by the bite or mechanical transfer of arthropod vectors. They constitute a significant proportion of the global infectious disease burden. United States (U.S.) Department of Defense (DoD) personnel are especially vulnerable to VBIs due to occupational contact with arthropod vectors, immunological naivete to previously unencountered pathogens, and limited diagnostic and treatment options available in the austere and unstable environments sometimes associated with military operations. In addition to the risk uniquely encountered by military populations, other factors have driven the worldwide emergence of VBIs. Unprecedented levels of global travel, tourism and trade, and blurred lines of demarcation between zoonotic VBI reservoirs and human populations increase vector exposure. Urban growth in previously undeveloped regions and perturbations in global weather patterns also contribute to the rise of VBIs. The Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) and its partners at DoD overseas laboratories form a network to better characterize the nature, emergence and growth of VBIs globally. In 2009 the network tested 19,730 specimens from 25 sites for Plasmodium species and malaria drug resistance phenotypes and nearly another 10,000 samples to determine the etiologies of non-Plasmodium species VBIs from regions spanning from Oceania to Africa, South America, and northeast, south and Southeast Asia. This review describes recent VBI-related epidemiological studies conducted by AFHSC-GEIS partner laboratories within the OCONUS DoD laboratory network emphasizing their impact on human populations.

Capacity-building initiatives related to public health are defined as developing laboratory infrastructure, strengthening host-country disease surveillance initiatives, transferring technical expertise and training personnel. These initiatives represented a major piece of the Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) contributions to worldwide emerging infectious disease (EID) surveillance and response. Capacity-building initiatives were undertaken with over 80 local and regional Ministries of Health, Agriculture and Defense, as well as other government entities and institutions worldwide. The efforts supported at least 52 national influenza centers and other country-specific influenza, regional and U.S.-based EID reference laboratories (44 civilian, eight military) in 46 countries worldwide. Equally important, reference testing, laboratory infrastructure and equipment support was provided to over 500 field sites in 74 countries worldwide from October 2008 to September 2009. These activities allowed countries to better meet the milestones of implementation of the 2005 International Health Regulations and complemented many initiatives undertaken by other U.S. government agencies, such as the U.S. Department of Health and Human Services, the U.S. Agency for International Development and the U.S. Department of State.

The Bartonella-associated illnesses are spread world-wide and involve a broad spectrum of signs and symptoms in humans. Several Bartonella species have been shown to be responsible for cases of febrile illnesses. Little information exists on distribution of Bartonella species and their role in human diseases in Nepal. Our preliminary study, a retrospective serological survey of archived specimens, suggests that Bartonella antibodies are prevalent among febrile patients in the Kathmandu Valley of Nepal.

Bartonellae were detected in a total of 152 (23.7%) of 642 tissues from 108 (48.4%) of 223 small mammals trapped in several urban areas of Nepal. Based on rpoB and gltA sequence analyses, genotypes belonging to seven known Bartonella species and five genotypes not belonging to previously known species were identified in these animals.

The geographic distribution of leptospirosis is widespread but no national surveillance program exists in Nepal to establish the incidence of leptospirosis or the disease burden. This study reports the incidence of symptomatic leptospirosis in military personnel participating in an efficacy study of a hepatitis E virus vaccine in Nepal. Among the 1566 study volunteers who completed follow-up, we evaluated 271 illnesses over 2.2 years for the presence of leptospira IgM antibodies by ELISA. Positive ELISA results were confirmed by the microscopic agglutination test. The annual incidence of disease was between 3.5 and 6.1 cases/1000. The prevalence of confirmed leptospirosis was 9% among hepatitis cases and 8% among febrile cases. The most reactive serovars were Bratislava, Autumnalis, Icterohaemorrhagiae, and Sejroe. Leptospirosis should be considered in the differential diagnosis of febrile illnesses and icteric syndromes in Nepal. Additional studies are needed to establish the broader distribution and the spectrum of disease in Nepal.

Worldwide emergence of variant viruses has prompted a change in the 2005–2006 H3N2 influenza A vaccine strain., In July 2004, an outbreak of influenza A (H3N2) was detected at 3 Bhutanese refugee camps in southeastern Nepal. Hemagglutination inhibition showed that ≈40% of the viruses from this outbreak were antigenically distinct from the A/Wyoming/3/03 vaccine strain. Four amino acid differences were observed in most of the 26 isolates compared with the A/Wyoming/3/2003 vaccine strain. All 4 substitutions are located within or adjacent to known antibody-binding sites. Several isolates showed a lysine-to-asparagine substitution at position 145 (K145N) in the hemagglutinin molecule, which may be noteworthy since position 145 is located within a glycosylation site and adjacent to an antibody-binding site. H3N2 viruses continue to drift from the vaccine strain and may remain as the dominant strains during the 2005–2006 influenza season. Thus, the 2005–2006 Northern Hemisphere vaccine strain was changed to A/California/7/2004, a virus with all 4 amino acid substitutions observed in these Nepalese isolates.

A cohort of 62 Nepalese adults with acute hepatitis E was identified and total Ig as well as IgM levels to hepatitis E virus (HEV) capsid protein were determined using the Walter Reed Army Institute of Research (WRAIR) immunoassay. An antibody profile was constructed from serial serum specimens collected up to 14 months following the onset of illness. The decline in total Ig was rapid for the first 3 months. There followed a slow, sustained decline, but antibodies remained above the seropositive level of 20 WRAIR units. The decline of IgM was steeper than total Ig for the first 3 months, but IgM remained detectable after 14 months in 25% of cases. Study data contribute to an understanding of the pathophysiology of human hepatitis E and set an antibody response pattern to be targeted as a part of HEV vaccine development.

To the Editor: Nepal is situated on the southern slopes of the Himalayas, surrounded by India on 3 sides and China to the north. Nepal’s altitude ranges from 8,848 m in the Himalayas to 90 m in the Terai, the southern, low, flatland bordering India. Nepal is a disease-endemic area for many vector-borne diseases, including malaria, kala-azar, Japanese encephalitis, and lymphatic filariasis. Because of the porous border between Nepal and India, social, cultural, and economic activities in cross-border areas are common. Dengue is an emerging disease in Nepal; presumably transmission is moving north from India into the Terai (1–5). The first report of dengue virus isolation or RNA (serotype 2 with nucleotide homology closest to a dengue virus type 2 isolate from India) was in 2008 involving a Japanese patient returning from Nepal in October 2004 (5). Entomologic investigations from the 1980s showed Aedes albopictus in the Terai plains, but Ae. aegypti has not been previously reported. After Indian outbreaks now known to include all 4 dengue serotypes (6), a team from the Epidemiology and Disease Control Division, Kathmandu, investigated suspected cases of dengue fever during September–October 2006 in Banke, the district bordering Uttar Pradesh, India. The team collected blood samples from persons in Banke and, subsequently, from persons in a number of other districts and sent them to the National Public Health Laboratory in Kathmandu or the Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand for analysis with ELISA, reverse transcription–PCR, (RT-PCR), or both. Case definitions for dengue fever were adopted based on World Health Organization guidelines (7). Blood samples were obtained from patients with an acute febrile illness of 2–7 days’ duration and with >2 of the following manifestations: headache, retro-orbital pain, muscular or joint pain, and rash. If laboratory tests were positive, cases were confirmed. Results were confirmed by ELISA performed at the Armed Forces Research Institute of Medical Sciences as previously described (8). Positive results were immunoglobulin (Ig) M >40 units or IgG >100 units. RT-PCR was performed by extracting RNA from 140 μL of each serum sample using QIAGEN Viral RNA Extraction Kit per manufacturer’s instructions (QIAGEN, Germantown, MD, USA). RT-PCR and nested PCR were conducted according to the Lanciotti protocol (9) with the following modifications. Reverse transcriptase from avian myeloblastosis virus (Promega, Madison, WI, USA) was used in the first round RT-PCR. The concentrations of the primers used in the RT-PCR and nested reactions were reduced from 50 pmol to 12.5 pmol per reaction, and the number of nested PCR amplification cycles was increased to 25. Serum specimens were obtained from 70 suspected case-patients from 16 districts from October 13 through December 3, 2006; 25 confirmed cases (13 by ELISA, 10 by RT-PCR, and 2 by both tests) came from 9 districts (Table). The average age was 29 years (range 5–65 years); 80% of the case-patients were men. Three patients had a history of travel to India, but clusters of dengue fever cases reported in October (Banke and Dang districts) indicated local transmission was occurring among patients with no travel history. The Terai districts accounted for 80% of cases. Entomologic collections done indoors and outside at 5 different sites reporting suspected cases identified Ae. albopictus and Ae. aegypti in all 5 districts. Table Dengue laboratory test results, National Public Health Laboratory, Nepal, and AFRIMS, Bangkok, 2006* These clinical and laboratory test results confirmed the presence of all 4 dengue serotypes. Notably, patients from the Dang district had no travel history outside the Dang valley. Because Aedes spp. have been identified in Dang, the data strongly suggest the existence of an endemic cycle of dengue. Underreporting is expected in the absence of diagnostic facilities at the field level. It is unclear whether the predominance of male patients is indicative of greater outdoor as opposed to indoor transmission. Of note, Ae. albopictus has been found in the country since the 1980s; in this study, we found Ae. aegypti in Nepal. Men typically wear short-sleeved clothes due to hot and humid conditions and, therefore, are frequently exposed to mosquito bites. However, men may also access the healthcare system more frequently. The ages of case-patients point to a relative lack of dengue immunity among the older population, and this finding is consistent with a new introduction of dengue. Because dengue hemorrhagic fever appears when >1 serotype becomes endemic to an area (10), the presence of all 4 serotypes portends the emergence of more severe dengue disease in Nepal.

A cornerstone of effective disease surveillance programs comprises the early identification of infectious threats and the subsequent rapid response to prevent further spread. Effectively identifying, tracking and responding to these threats is often difficult and requires international cooperation due to the rapidity with which diseases cross national borders and spread throughout the global community as a result of travel and migration by humans and animals. From Oct.1, 2008 to Sept. 30, 2009, the United States Department of Defense’s (DoD) Armed Forces Health Surveillance Center Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) identified 76 outbreaks in 53 countries. Emerging infectious disease outbreaks were identified by the global network and included a wide spectrum of support activities in collaboration with host country partners, several of which were in direct support of the World Health Organization’s (WHO) International Health Regulations (IHR) (2005). The network also supported military forces around the world affected by the novel influenza A/H1N1 pandemic of 2009. With IHR (2005) as the guiding framework for action, the AFHSC-GEIS network of international partners and overseas research laboratories continues to develop into a far-reaching system for identifying, analyzing and responding to emerging disease threats.

Eight serotypes of human astroviruses (the classic human astroviruses) are causative agents of diarrhea. Recently, five additional astroviruses belonging to two distinct clades have been described in human stool, including astroviruses MLB1, MLB2, VA1, VA2 and VA3. We report the discovery in human stool of two novel astroviruses, astroviruses MLB3 and VA4. The complete genomes of these two viruses and the previously described astroviruses VA2 and VA3 were sequenced, affording seven complete genomes from the MLB and VA clades for comparative analysis to the classic human astroviruses. Comparison of the genetic distance, number of synonymous mutations per synonymous site (dS), number of non-synonymous mutations per non-synonymous site (dN) and the dN/dS ratio in the protease, polymerase and capsid of the classic human, MLB and VA clades suggests that the protease and polymerase of the classic human astroviruses are under distinct selective pressure.

Background Acute febrile illness is common among those seeking medical care and is frequently treated empirically with the underlying illness remaining undiagnosed in resource-poor countries. A febrile illness study was conducted 2009-2010 to identify known and unknown pathogens circulating in Nepal. Method Study methods included diagnostic testing and preliminary ELISA screening of acute and convalescent samples for diseases both known and unknown to be circulating in Nepal, including West Nile virus (WNV). The molecular assays including Polymerase Chain Reaction (PCR), Sanger sequencing and ultra deep sequencing on MiSeq Illumina Platform were conducted to further confirm the presence of WNV. Results The study enrolled 2,046 patients presenting undifferentiated febrile illness with unknown etiology. Sera from 14 out of 2,046 patients were tested positive for west nile virus (WNV) by nested Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Only two out of 14 cases were confirmed for the presence of WNV by sequencing and identified as WNV lineage 1 phylogentically. The two patients were adult males with fever and no neurological symptoms from Kathmandu and Bharatpur, Nepal. Conclusion Two out of 2,046 serum samples contained fragments of WNV genome resembling WNV lineage 1, which is evidence of the continued spread of WNV which should be considered a possible illness cause in Nepal. Electronic supplementary material The online version of this article (doi:10.1186/s12879-014-0606-0) contains supplementary material, which is available to authorized users.

The Armed Forces Health Surveillance Center’s Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) supports and oversees surveillance for emerging infectious diseases, including respiratory diseases, of importance to the U.S. Department of Defense (DoD). AFHSC-GEIS accomplishes this mission by providing funding and oversight to a global network of partners for respiratory disease surveillance. This report details the system’s surveillance activities during 2009, with a focus on efforts in responding to the novel H1N1 Influenza A (A/H1N1) pandemic and contributions to global public health. Active surveillance networks established by AFHSC-GEIS partners resulted in the initial detection of novel A/H1N1 influenza in the U.S. and several other countries, and viruses isolated from these activities were used as seed strains for the 2009 pandemic influenza vaccine. Partners also provided diagnostic laboratory training and capacity building to host nations to assist with the novel A/H1N1 pandemic global response, adapted a Food and Drug Administration-approved assay for use on a ruggedized polymerase chain reaction platform for diagnosing novel A/H1N1 in remote settings, and provided estimates of seasonal vaccine effectiveness against novel A/H1N1 illness. Regular reporting of the system’s worldwide surveillance findings to the global public health community enabled leaders to make informed decisions on disease mitigation measures and controls for the 2009 A/H1N1 influenza pandemic. AFHSC-GEIS’s support of a global network contributes to DoD’s force health protection, while supporting global public health.