Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing.

OBJECTIVE: To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR (M-RTPCR), for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing (NGS) platform. METHODS: Virus genome copy was quantified and serially diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. RESULTS: The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing low-titer virus. CONCLUSION: The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Phylogenetic and Molecular Analysis of an H7N7 Avian Influenza Virus Isolated in East Dongting Lake in 2012.

OBJECTIVE: In March 2012, an H7N7 subtype avian influenza virus (AIV) named A/wild goose/Dongting/PC0360/2012 (H7N7) (DT/PC0360) was recovered from a wild goose in East Dongting Lake. We performed whole-genome sequencing of the isolate, and analyzed the phylogenetic and molecular characterization. METHODS: RNA was extracted from environment samples (including fecal samples from wild bird or domestic ducks, and water samples) for detecting the presence of Influenza A Virus targeting Matrix gene, using realtime RT-PCR assay. The positive samples were performed virus isolation with embryonated eggs. The subtype of the isolates were identified by RT-PCR assay with the H1-H16 and N1-N9 primer set. The whole-genome sequencing of isolates were performed. Phylogenetic and molecular characterizations of the eight genes of the isolates were analyzed. RESULTS: Our results suggested that all the eight gene segments of DT/PC0360 belonged to the Eurasian gene pool, and the HA gene were belonged to distinct sublineage with H7N9 AIV which caused outbreaks in Mainland China in 2013. The hemagglutinin cleavage site of HA of DT/PC0360 showed characterization of low pathogenic avian influenza virus. CONCLUSION: Strengthening the surveillance of AIVs of wild waterfowl and poultry in this region is vital for our knowledge of the ecology and mechanism of transmission to prevent an influenza pandemic.

H5N1 Avian Influenza Pre-pandemic Vaccine Strains in China.

OBJECTIVE: To prepare the 4 candidate vaccine strains of H5N1 avian influenza virus isolated in China. METHODS: Recombinant viruses were rescued using reverse genetics. Neuraminidase (NA) and hemagglutinin (HA) segments of the A/Xinjiang/1/2006, A/Guangxi/1/2009, A/Hubei/1/2010, and A/Guangdong/1/2011 viruses were amplified by RT-PCR. Multibasic amino acid cleavage site of HA was removed and ligated into the pCIpolI vector for virus rescue. The recombinant viruses were evaluated by trypsin dependent assays. Their embryonate survival and antigenicity were compared with those of the respective wild-type viruses. RESULTS: The 4 recombinant viruses showed similar antigenicity compared with wild-type viruses, chicken embryo survival and trypsin-dependent characteristics. CONCLUSION: The 4 recombinant viruses rescued using reverse genetics meet the criteria for classification of low pathogenic avian influenza strains, thus supporting the use of them for the development of seeds and production of pre-pandemic vaccines.

A novel reassortant H2N3 influenza virus isolated from China.

OBJECTIVE: To analyze the genetic composition of a novel H2N3 virus isolate identified from a duck cage swab in a live poultry market (LPM) in 2009 in Guangdong province of China. METHODS: PCR-positive specimens were inoculated into embryonated chicken eggs and subtyped by conventional RT-PCR. All segments of the virus A/environment/Guangdong/2/2009 were sequenced, and phylogenetic trees were constructed and analyzed. RESULTS: The genes of this virus belong to Eurasian-lineage avian viruses. The virus is a reassortant with the HA gene from an H2N2 virus and the NA gene from an H5N3 virus. The PB1, PB2, and NP genes were from an H4N6 virus, the PA was from an H3N8 virus, the M gene was from an H1N3 virus, and the NS gene was from an H10N6 virus. CONCLUSION: A novel avian-origin reassortant H2N3 influenza virus was detected in a live poultry market. Its potential impacts and evolution should be closely monitored.

High levels of virus-specific CD4+ T cells predict severe pandemic influenza A virus infection.

RATIONALE: T-cell responses have been implicated in control and exacerbation of lung injury during influenza A virus (IAV) infection. OBJECTIVES: To examine the breadth and magnitude of influenza-specific CD4(+) and CD8(+) T-cell responses during acute phase of infection. METHODS: Influenza-specific T-cell response to the entire pandemic H1N1/09 IAV proteome and T cell-related cytokine levels were measured in blood from previously healthy individuals with mild (n = 32) and severe (n = 16) IAV infection during the 2009 influenza pandemic. Virus-specific T-cell response in lung and blood was also performed in two acutely infected, severely ill patients using fluorescent-conjugated pdmH1N1/09 Matrix-MHC-I tetrameric complexes. MEASUREMENTS AND MAIN RESULTS: Strong and broad CD4(+) but not CD8(+) T-cell responses were observed in the blood, and were higher in those with severe disease. Antigen-specific CD8(+) T cells in the lungs were on average 45-fold higher compared with blood in severely ill patients. Paradoxically, in patients with severe disease, IL-17, IL-2, IL-4, and IFN-γ levels were significantly decreased. CONCLUSIONS: High levels of circulating virus-specific CD4(+) T cells to two viral internal proteins (nucleoprotein and matrix) in the first phase of infection are associated with subsequent development of severe IAV infection. This finding could be an early and specific marker for ensuing clinical deterioration. Contrasting levels of antigen-specific CD8(+) T cells in lungs and blood have implications on design and analysis of clinical trials for T-cell vaccines because measurements of T cells in the periphery may not reflect events in the lungs.

A novel reassortant H3N8 influenza virus isolated from drinking water for duck in a domestic duck farm in Poyang Lake area.

OBJECTIVE: To conduct a full genome sequence analysis for genetic characterization of an H3N8 influenza virus isolated from drinking water of a domestic duck farm in Poyang Lake area in 2011. METHODS: The virus was cultivated by specific pathogen free (SPF) chicken embryo eggs and was subtyped into hemagglutinin (HA) and neuraminidase (NA) by real-time PCR method. Eight gene segments were sequenced and phylogenetic analysis was conducted. RESULTS: The NA gene of this virus belongs to North American lineage; other seven genes belong to Eurasian lineage. Compared with the viruses containing NA gene, the PB2 and PB1 gene came from different clades. And this indicates that the virus was a novel reassortant genotype. The HA receptor binding preference was avian-like and the cleavage site sequence showed a low pathogenic feature. There was no drug resistance mutation of M2 protein. The mutations of Asn30Asp, and Thr215Ala of the M1 protein implied the potential of pathogenicity increase in mice. CONCLUSION: The finding of novel genotype of H3N8 virus in drinking water in this duck farm near Poyang Lake highlighted the importance of strengthening the surveillance of avian influenza in this region, which could contribute to pinpointing the influenza ecological relations among avian, swine, and human.

[Development of a real-time reverse transcriptase PCR assay for detection of E119V amino acid change in neuraminidase of influenza A (H3N2) using the TaqMan-MGB probe].

OBJECTIVE: To develop a rapid duplex Real-time reverse transcription PCR (rRT-PCR) method to detect E119V mutation on neuraminidase (NA) of influenza A(H3N2) subtype with drug resistance to oseltamivir. METHODS: Twenty-six NA genes of influenza A(H3N2) virus between 2000 and 2012 in GenBank database were selected as the target genes, and specific TaqMan-MGB probe was designed to target the E119V amino acid change in neuraminidase protein. rRT-PCR was then performed and evaluated for the sensitivity, specificity and reproducibility using virus with E119V mutation and clinical samples. RESULTS: This study described the validation of a highly sensitive and specific duplex rRT-PCR for detection of substitutions leading to the E119V amino acid change in NA protein of influenza A(H3N2). Fluorescence signals could be detected even when diluted a A (H3N2) virus (HA = 8) into 10(-5) and linear correlation between the logarithm of the viral titer with the Ct values was observed. In addition, the assay was highly specific in that there was no cross-react with other respiratory viruses, nor did two TaqMan-MGB probes. E119V substitution in quasispecies with both sensitive and resistant viruses could be detected as well. The limit of detection was 5% for quasispecies with high concentrations and 50% for quasispecies with low concentrations. The average coefficient of variation (CV) for within-run assays was 2.32% and 0.57% for H3N2-119E and H3N2-119V primer/probe sets separately, 1.77% and 0.97% for average CV of between-run assays, which exhibited good repeatability. Sequence analysis of twenty NA genes verified glutamic acid (E) at amino acid site 119, which was in consistent with the results from our rRT-PCR method. CONCLUSION: The assay developed in this study is highly sensitive and specific, and easy to operate; thereby it could be used for identification of A(H3N2) virus with E119V amino acid change in NA protein.

Individual bat virome analysis reveals co-infection and spillover among bats and virus zoonotic potential.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within individual bats, and hence the frequency of virus co-infection and spillover among them. We characterize the mammal-associated viruses in 149 individual bats sampled from Yunnan province, China, using an unbiased meta-transcriptomics approach. This reveals a high frequency of virus co-infection (simultaneous infection of bat individuals by multiple viral species) and spillover among the animals studied, which may in turn facilitate virus recombination and reassortment. Of note, we identify five viral species that are likely to be pathogenic to humans or livestock, based on phylogenetic relatedness to known pathogens or in vitro receptor binding assays. This includes a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV and SARS-CoV-2. In vitro assays indicate that this recombinant virus can utilize the human ACE2 receptor such that it is likely to be of increased emergence risk. Our study highlights the common occurrence of co-infection and spillover of bat viruses and their implications for virus emergence.

Identification of dual receptor-binding specific strains of human H5N1 viruses in China.

OBJECTIVE: Both the 2, 6 linkage and its topology on target cells are critical for the recognition by human influenza virus. The binding preference of avian flu virus H5N1 HA to the 2, 3-linked sialylated glycans is considered the major factor limiting its efficient infection and transmission in humans. To monitor potential adaptation of H5N1 virus in human population, the surveillance of receptor-binding specificity was undertaken in China. METHODS: The binding specificity of 32 human H5N1 virus strains isolated from 2003 to 2009 was tested by 2, 3-specific sialidase-treated chicken red blood cell (CRBC) agglutination assay and a solid-phase direct binding assay with synthetic sialylglycopolymers. RESULTS: Dual binding preference to 2, 3 and 2, 6-glycans were found in two strains: A/Guangdong/1/06 (A/GD/1/06) and A/Guangxi/1/08 (A/GX/1/08). Though minor effect of short-2, 6-binding was detected in A/GX/1/08 at a low virus titer, both showed high affinity to the oligosaccharide at a high load. Notably both are of the long-2, 6-recognition, with the same topology as that of human H1N1 and H3N2 viruses. CONCLUSION: The findings suggest that human H5N1 virus in China likely acquired the potential human-adaptation ability. Further research and surveillance on receptor-binding specificity of H5N1 viruses are required.

Individual bat viromes reveal the co-infection, spillover and emergence risk of potential zoonotic viruses.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.

Influenza A virus replication induces cell cycle arrest in G0/G1 phase.

Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G(0)/G(1)-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G(0)/G(1) phase into S phase. Consistent with the G(0)/G(1)-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G(1) into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G(0)/G(1)-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G(0)/G(1) phase were observed compared to those in either unsynchronized cells or cells synchronized in the G(2)/M phase. G(0)/G(1)-phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G(0)/G(1)-phase cell cycle arrest in infected cells.

Distribution of avian influenza viruses according to environmental surveillance during 2014-2018, China.

BACKGROUND: Recurrent infections of animal hosts with avian influenza viruses (AIVs) have posted a persistent threat. It is very important to understand the avian influenza virus distribution and characteristics in environment associated with poultry and wild bird. The aim of this study was to analyze the geographic and seasonal distributions of AIVs in the 31 provinces, municipalities and autonomous region (PMA) of China, compare the AIVs prevalence in different collecting sites and sampling types, analyze the diversity of AIVs subtypes in environment. METHODS: A total of 742 005 environmental samples were collected from environmental samples related to poultry and wild birds in different locations in the mainland of China during 2014-2018. Viral RNA was extracted from the environmental samples. Real-time RT-PCR assays for influenza A, H5, H7 and H9 subtypes were performed on all the samples to identify subtypes of influenza virus. The nucleic acid of influenza A-positive samples were inoculated into embryonated chicken eggs for virus isolation. Whole-genome sequencing was then performed on Illumina platform. SPSS software was used to paired t test for the statistical analysis. ArcGIS was used for drawing map. Graphpad Prism was used to make graph. RESULTS: The nucleic acid positivity rate of influenza A, H5, H7 and H9 subtypes displayed the different characteristics of geographic distribution. The nucleic acid positivity rates of influenza A were particularly high (25.96%-45.51%) in eleven provinces covered the Central, Eastern, Southern, Southwest and Northwest of China. The nucleic acid positivity rates of H5 were significantly high (11.42%-13.79%) in two provinces and one municipality covered the Southwest and Central of China. The nucleic acid positivity rates of H7 were up to 4% in five provinces covered the Eastern and Central of China. The nucleic acid positivity rates of H9 were higher (13.07%-2.07%) in eleven PMA covered the Southern, Eastern, Central, Southwest and Northwest of China. The nucleic acid positivity rate of influenza A, H5, H7 and H9 showed the same seasonality. The highest nucleic acid positivity rates of influenza A, H5, H7, H9 subtypes were detected in December and January and lowest from May to September. Significant higher nucleic acid positivity rate of influenza A, H5, H7 and H9 were detected in samples collected from live poultry markets (LPM) (30.42%, 5.59%, 4.26%, 17.78%) and poultry slaughterhouses (22.96%, 4.2%, 2.08%, 12.63%). Environmental samples that were collected from sewage and chopping boards had significantly higher nucleic acid positivity rates for influenza A (36.58% and 33.1%), H5 (10.22% and 7.29%), H7(4.24% and 5.69%)and H9(21.62% and 18.75%). Multiple subtypes of AIVs including nine hemagglutinin (HA) and seven neuraminidase (NA) subtypes were isolated form the environmental samples. The H5, H7, and H9 subtypes accounted for the majority of AIVs in environment. CONCLUSIONS: In this study, we found the avian influenza viruses characteristics of geographic distribution, seasonality, location, samples types, proved that multiple subtypes of AIVs continuously coexisted in the environment associated with poultry and wild bird, highlighted the need for environmental surveillance in China.

Gene expression profiles comparison between 2009 pandemic and seasonal H1N1 influenza viruses in A549 cells.

OBJECTIVE: To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A (H1N1) influenza viruses. METHODS: A549 cells were infected with A/California/07/09 (H1N1) and A/GuangdongBaoan/51/08 (H1N1) respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection (p.i.). Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. RESULTS: Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. CONCLUSIONS: The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition.

Interferon-induced Transmembrane Protein 3 Prevents Acute Influenza Pathogenesis in Mice.

OBJECTIVE: Interferon-induced transmembrane protein 3 (IFITM3) is an important member of the IFITM family. However, the molecular mechanisms underlying its antiviral action have not been completely elucidated. Recent studies on IFITM3, particularly those focused on innate antiviral defense mechanisms, have shown that IFITM3 affects the body's adaptive immune response. The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection in vivo. METHODS: We performed proteomics, flow cytometry, and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer (NK) cell numbers, their activation, and their immune function in the lungs of Ifitm3-/- and wild-type mice. RESULTS: Ifitm3-/- mice developed more severe inflammation and apoptotic responses compared to wild-type mice. Moreover, the NK cell activation was higher in the lungs of Ifitm3-/- mice during acute influenza infection. CONCLUSIONS: Based on our results, we speculate that the NK cells are more readily activated in the absence of IFITM3, increasing mortality in Ifitm3-/- mice.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A (H5N1) Virus.

OBJECTIVE: To recover broad-neutralizing monoclonal antibodies (BnAbs) from avian influenza A (H5N1) virus infection cases and investigate their genetic and functional features. METHODS: We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients. The genetic basis, biological functions, and epitopes of the obtained BnAbs were assessed and modeled. RESULTS: Two BnAbs, 2-12D5, and 3-37G7.1, were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset. Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity (ADCC) activity. Albeit derived from distinct Ab lineages, i.e., V H1-69-D2-15-J H4 (2-12D5) and V H1-2-D3-9-J H5 (3-32G7.1), the BnAbs were directed toward CR6261-like epitopes in the HA stem, and HA 2 I45 in the hydrophobic pocket was the critical residue for their binding. Signature motifs for binding with the HA stem, namely, IFY in V H1-69-encoded Abs and LXYFXW in D3-9-encoded Abs, were also observed in 2-12D5 and 3-32G7.1, respectively. CONCLUSIONS: Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus. The HA stem epitopes targeted by the BnAbs, and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

[Clinical investigation of the clinical management and therapeutic strategies to the first human case infected by influenza A/H5N1 in Guizhou Province].

OBJECTIVE: To explore the optimized clinical management and therapeutic strategies for the survived human case infected by influenza A (A/H5N1). METHODS: All the data of the first human case infected by A/H5N1 in Guizhou province was collected and analyzed. RESULTS: The first case infected by A/H5N1 in Guizhou Province was confirmed by laboratory findings with reverse-transcription polymerase chain reaction (RT-PCR) and A/H5N1 isolation. Patient was healthy in the past and exposed in the environment of living poultry. The initial symptoms was high fever without influenza-like presentation, but with extremity hyperspasmia and conscious disturbance sometimes. A productive cough with a large mount of pink foaming sputum then appeared. The clinical situation was rapidly deteriorated with dyspnea, acute respiratory distress syndrome and atrial fibrillation. Multiple infiltration in bilateral lungs was progressively developed with moderate bilateral pleural effusion. Invasive ventilation was intervened since ARDS on day 8 after sickness. Oseltamivir was kicked off since day 9 after sickness. However, the clinical condition was still exacerbated. High titering antibody of A/H5N1 vaccinated plasma was administrated on day 10 after sickness. The clinical condition (including oxygen saturation, respiratory symptoms, etc.) was improved rapidly. The weaning of ventilation was carried out in two days. Atrial fibrillation was back to normal. The patient was clinical recovery and was discharged from hospital on day 23 after sickness. CONCLUSIONS: The prognosis was poor if A/H5N1 infected human cases developed as acute respiratory distress syndrome with heart injury. However, it could be ameliorated if the plasma of A/H5N1 vaccinated neutralizing antibody was administrated in time or within two weeks after sickness.

Molecular evolution of influenza B virus during 2011-2017 in Chaoyang, Beijing, suggesting the free influenza vaccine policy.

Two influenza B virus lineages, B/Victoria and B/Yamagata, are co-circulating in human population. While the two lineages are serologically distinct and TIV only contain one lineage. It is important to investigate the epidemiological and evolutionary dynamics of two influenza B virus lineages in Beijing after the free influenza vaccine policy from 2007. Here, we collected the nasopharyngeal swabs of 12657 outpatients of influenza-like illness and subtyped by real-time RT-PCR during 2011-2017. The HA and NA genes of influenza B were fully sequenced. The prevalence is the highest in the 6-17 years old group among people infected with influenza B. Yamagata-lineage virus evolved to two inter-clade from 2011-2014 to 2014-2017. The amino acids substitutions of HA1 region were R279K in strains of 2011-2014 and L173Q, M252V in strains of 2014-2017. Substitutions L58P, I146V were observed in HA1 region of Victoria-lineage virus in 2011-2012 and I117V, N129D were showed in 2015-2017. Phylogenetic analysis of NA showed Yamagata-Victoria inter-lineage reassortant occurred in 2013-2014. Influenza B mainly infect the school-aged children in Beijing and the free influenza vaccine inoculation does not seem to block school-age children from infection with influenza B. The antigen characteristics of circulating influenza B were different to the recommended vaccine strains. We concluded that the Victoria-lineage vaccine strain should been changed and the free influenza vaccine should be revalued.

HIV prevalence in suspected Ebola cases during the 2014-2016 Ebola epidemic in Sierra Leone.

BACKGROUND: The 2014-2016 Ebola virus epidemic in West Africa was the largest outbreak of Ebola virus disease (EVD) in history. Clarifying the influence of other prevalent diseases such as human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) will help improve treatment and supportive care of patients with EVD. CASE PRESENTATION: We examined HIV and hepatitis C virus (HCV) antibody prevalence among suspected EVD cases from the Sierra Leone-China Friendship Biological Safety Laboratory during the epidemic in Sierra Leone. HIV and HCV antibodies were tested in 678 EVD-negative samples by enzyme-linked immunosorbent assay. A high HIV prevalence (17.6%) and low HCV prevalence (0.22%) were observed among the suspected cases. Notably, we found decreased HIV positive rates among the suspected cases over the course of the epidemic. This suggests a potentially beneficial effect of an improved public health system after assistance from the World Health Organization and other international aid organizations. CONCLUSIONS: This EVD epidemic had a considerable impact on the public health system and influenced the prevalence of HIV found among suspected cases in Sierra Leone, but also provided an opportunity to establish a better surveillance network for infectious diseases.