"Smart markets": harnessing the potential of new technologies for endemic and emerging infectious disease surveillance in traditional food markets.

Emerging and endemic zoonotic diseases continue to threaten human and animal health, our social fabric, and the global economy. Zoonoses frequently emerge from congregate interfaces where multiple animal species and humans coexist, including farms and markets. Traditional food markets are widespread across the globe and create an interface where domestic and wild animals interact among themselves and with humans, increasing the risk of pathogen spillover. Despite decades of evidence linking markets to disease outbreaks across the world, there remains a striking lack of pathogen surveillance programs that can relay timely, cost-effective, and actionable information to decision-makers to protect human and animal health. However, the strategic incorporation of environmental surveillance systems in markets coupled with novel pathogen detection strategies can create an early warning system capable of alerting us to the risk of outbreaks before they happen. Here, we explore the concept of "smart" markets that utilize continuous surveillance systems to monitor the emergence of zoonotic pathogens with spillover potential.IMPORTANCEFast detection and rapid intervention are crucial to mitigate risks of pathogen emergence, spillover and spread-every second counts. However, comprehensive, active, longitudinal surveillance systems at high-risk interfaces that provide real-time data for action remain lacking. This paper proposes "smart market" systems harnessing cutting-edge tools and a range of sampling techniques, including wastewater and air collection, multiplex assays, and metagenomic sequencing. Coupled with robust response pathways, these systems could better enable Early Warning and bolster prevention efforts.

Detection and phylogenetic analysis of contemporary H14N2 Avian influenza A virus in domestic ducks in Southeast Asia (Cambodia).

Avian influenza virus (AIV) in Asia is a complex system with numerous subtypes and a highly porous wild birds-poultry interface. Certain AIV subtypes, such as H14, are underrepresented in current surveillance efforts, leaving gaps in our understanding of their ecology and evolution. The detection of rare subtype H14 in domestic ducks in Southeast Asia comprises a geographic region and domestic bird population previously unassociated with this subtype. These H14 viruses have a complex evolutionary history involving gene reassortment events. They share sequence similarity to AIVs endemic in Cambodian ducks, and Eurasian low pathogenicity and high pathogenicity H5Nx AIVs. The detection of these H14 viruses in Southeast Asian domestic poultry further advances our knowledge of the ecology and evolution of this subtype and reinforces the need for continued, longitudinal, active surveillance in domestic and wild birds. Additionally, in vivo and in vitro risk assessment should encompass rare AIV subtypes, as they have the potential to establish in poultry systems.

Detection and phylogenetic analysis of highly pathogenic A/H5N1 avian influenza clade 2.3.4.4b virus in Chile, 2022.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses continue to pose a significant threat to animal and human health worldwide. In late 2022, the first confirmed case of HPAI A/H5N1 infection in wild birds in Chile near the Chilean-Peruvian border was reported. Active surveillance by our group in the adyacent Lluta river estuary revealed an increase in A/H5N1 prevalence coinciding with the arrival of migratory birds from the Northern Hemisphere. Genomic analysis of A/H5N1-positive samples demonstrated a close genetic relationship to strains detected in Peru during the same period, which originated from A/H5N1 viruses causing outbreaks in North America. Notably, we identified genetic mutations that did not correlate with known enhanced transmission or binding traits to mammalian receptors. In summary, this study provides valuable genomic insights into the A/H5N1 Clade 2.3.4.4b viruses in wild birds in Chile, emphasizing the need for enhanced surveillance and response strategies to mitigate the threat posed by these highly pathogenic avian influenza viruses in South America.

Evolution of highly pathogenic H5N1 influenza A virus in the central nervous system of ferrets.

Central nervous system (CNS) disease is the most common extra-respiratory tract complication of influenza A virus infections in humans. Remarkably, zoonotic highly pathogenic avian influenza (HPAI) H5N1 virus infections are more often associated with CNS disease than infections with seasonal influenza viruses. Evolution of avian influenza viruses has been extensively studied in the context of respiratory infections, but evolutionary processes in CNS infections remain poorly understood. We have previously observed that the ability of HPAI A/Indonesia/5/2005 (H5N1) virus to replicate in and spread throughout the CNS varies widely between individual ferrets. Based on these observations, we sought to understand the impact of entrance into and replication within the CNS on the evolutionary dynamics of virus populations. First, we identified and characterized three substitutions-PB1 E177G and A652T and NP I119M - detected in the CNS of a ferret infected with influenza A/Indonesia/5/2005 (H5N1) virus that developed a severe meningo-encephalitis. We found that some of these substitutions, individually or collectively, resulted in increased polymerase activity in vitro. Nevertheless, in vivo, the virus bearing the CNS-associated mutations retained its capacity to infect the CNS but showed reduced dispersion to other anatomical sites. Analyses of viral diversity in the nasal turbinate and olfactory bulb revealed the lack of a genetic bottleneck acting on virus populations accessing the CNS via this route. Furthermore, virus populations bearing the CNS-associated mutations showed signs of positive selection in the brainstem. These features of dispersion to the CNS are consistent with the action of selective processes, underlining the potential for H5N1 viruses to adapt to the CNS.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

Detection of Clade 2.3.4.4b Avian Influenza A(H5N8) Virus in Cambodia, 2021.

In late 2021, highly pathogenic avian influenza A(H5N8) clade 2.3.4.4b viruses were detected in domestic ducks in poultry markets in Cambodia. Surveillance, biosafety, and biosecurity efforts should be bolstered along the poultry value chain to limit spread and infection risk at the animal-human interface.

A Look inside the Replication Dynamics of SARS-CoV-2 in Blyth's Horseshoe Bat (Rhinolophus lepidus) Kidney Cells.

Bats are considered the natural reservoir of numerous emerging viruses such as severe acute respiratory syndrome coronaviruses (SARS-CoVs). There is a need for immortalized bat cell lines to culture and investigate the pathogenicity, replication kinetics, and evolution of emerging coronaviruses. We illustrate the susceptibility and permissiveness of a spontaneously immortalized kidney cell line (Rhileki) from Blyth's horseshoe bat (R. lepidus) to SARS-CoV-2 virus, including clinical isolates, suggesting a possible virus-host relationship. We were able to observe limited SARS-CoV-2 replication in Rhileki cells compared with simian VeroE6 cells. Slower viral replication in Rhileki cells was indicated by higher ct values (RT-PCR) at later time points of the viral culture and smaller foci (foci forming assay) compared with those of VeroE6 cells. With this study we demonstrate that SARS-CoV-2 replication is not restricted to R. sinicus and could include more Rhinolophus species. The establishment of a continuous Rhinolophus lepidus kidney cell line allows further characterization of SARS-CoV-2 replication in Rhinolophus bat cells, as well as isolation attempts of other bat-borne viruses. IMPORTANCE The current COVID-19 pandemic demonstrates the significance of bats as reservoirs for severe viral diseases. However, as bats are difficult to establish as animal models, bat cell lines can be an important proxy for the investigation of bat-virus interactions and the isolation of bat-borne viruses. This study demonstrates the susceptibility and permissiveness of a continuous kidney bat cell line to SARS-CoV-2. This does not implicate the bat species Rhinolophus lepidus, where these cells originate from, as a potential reservoir, but emphasizes the usefulness of this cell line for further characterization of SARS-CoV-2. This can lead to a better understanding of emerging viruses that could cause significant disease in humans and domestic animals.

Genetic and Antigenic Characterization of an Influenza A(H3N2) Outbreak in Cambodia and the Greater Mekong Subregion during the COVID-19 Pandemic, 2020.

Introduction of non-pharmaceutical interventions to control COVID-19 in early 2020 coincided with a global decrease in active influenza circulation. However, between July and November 2020, an influenza A(H3N2) epidemic occurred in Cambodia and in other neighboring countries in the Greater Mekong Subregion in Southeast Asia. We characterized the genetic and antigenic evolution of A(H3N2) in Cambodia and found that the 2020 epidemic comprised genetically and antigenically similar viruses of Clade3C2a1b/131K/94N, but they were distinct from the WHO recommended influenza A(H3N2) vaccine virus components for 2020-2021 Northern Hemisphere season. Phylogenetic analysis revealed multiple virus migration events between Cambodia and bordering countries, with Laos PDR and Vietnam also reporting similar A(H3N2) epidemics immediately following the Cambodia outbreak: however, there was limited circulation of these viruses elsewhere globally. In February 2021, a virus from the Cambodian outbreak was recommended by WHO as the prototype virus for inclusion in the 2021-2022 Northern Hemisphere influenza vaccine. IMPORTANCE The 2019 coronavirus disease (COVID-19) pandemic has significantly altered the circulation patterns of respiratory diseases worldwide and disrupted continued surveillance in many countries. Introduction of control measures in early 2020 against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection has resulted in a remarkable reduction in the circulation of many respiratory diseases. Influenza activity has remained at historically low levels globally since March 2020, even when increased influenza testing was performed in some countries. Maintenance of the influenza surveillance system in Cambodia in 2020 allowed for the detection and response to an influenza A(H3N2) outbreak in late 2020, resulting in the inclusion of this virus in the 2021-2022 Northern Hemisphere influenza vaccine.

Human Infection with Avian Influenza A(H9N2) Virus, Cambodia, February 2021.

In February 2021, routine sentinel surveillance for influenza-like illness in Cambodia detected a human avian influenza A(H9N2) virus infection. Investigations identified no recent H9N2 virus infections in 43 close contacts. One chicken sample from the infected child's house was positive for H9N2 virus and genetically similar to the human virus.

Zinc-Embedded Polyamide Fabrics Inactivate SARS-CoV-2 and Influenza A Virus.

Influenza A viruses (IAV) and SARS-CoV-2 can spread via liquid droplets and aerosols. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of these viruses. However, IAV and SARS-CoV-2 are stable for hours on various materials, which makes frequent and correct disposal of these PPE important. Metal ions embedded into PPE may inactivate respiratory viruses, but confounding factors such as adsorption of viruses make measuring and optimizing the inactivation characteristics difficult. Here, we used polyamide 6.6 (PA66) fibers containing embedded zinc ions and systematically investigated if these fibers can adsorb and inactivate SARS-CoV-2 and IAV H1N1 when woven into a fabric. We found that our PA66-based fabric decreased the IAV H1N1 and SARS-CoV-2 titer by approximately 100-fold. Moreover, we found that the zinc content and the virus inactivating property of the fabric remained stable over 50 standardized washes. Overall, these results provide insights into the development of reusable PPE that offer protection against RNA virus spread.

Zinc-embedded fabrics inactivate SARS-CoV-2 and influenza A virus.

Infections with respiratory viruses can spread via liquid droplets and aerosols, and cause diseases such as influenza and COVID-19. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of respiratory droplets containing these viruses. However, influenza A viruses and coronaviruses are stable for hours on various materials, which makes frequent and correct disposal of these PPE important. Metal ions embedded into PPE may inactivate respiratory viruses, but confounding factors such as absorption of viruses make measuring and optimizing the inactivation characteristics difficult. Here we used polyamide 6.6 (PA66) fibers that had zinc ions embedded during the polymerisation process and systematically investigated if these fibers can absorb and inactivate pandemic SARS-CoV-2 and influenza A virus H1N1. We find that these viruses are readily absorbed by PA66 fabrics and inactivated by zinc ions embedded into this fabric. The inactivation rate (pfu·gram-1·min-1) exceeds the number of active virus particles expelled by a cough and supports a wide range of viral loads. Moreover, we found that the zinc content and the virus inactivating property of the fabric remain stable over 50 standardized washes. Overall, these results provide new insight into the development of "pathogen-free" PPE and better protection against RNA virus spread.

Enhanced Enterovirus D68 Replication in Neuroblastoma Cells Is Associated with a Cell Culture-Adaptive Amino Acid Substitution in VP1.

Since its emergence in the United States in 2014, enterovirus D68 (EV-D68) has been and is associated with severe respiratory diseases and acute flaccid myelitis. Even though EV-D68 has been shown to replicate in different neuronal cells in vitro, it is currently poorly understood which viral factors contribute to the ability to replicate efficiently in cells of the central nervous system and whether this feature is a clade-specific feature. Here, we determined the replication kinetics of clinical EV-D68 isolates from (sub)clades A, B1, B2, B3, and D1 in human neuroblastoma cells (SK-N-SH). Subsequently, we compared sequences to identify viral factors associated with increased viral replication. All clinical isolates replicated in SK-N-SH cells, although there was a large difference in efficiency. Efficient replication of clinical isolates was associated with an amino acid substitution at position 271 of VP1 (E271K), which was acquired during virus propagation in vitro Recognition of heparan sulfate in addition to sialic acids was associated with increased attachment, infection, and replication. Removal of heparan sulfate resulted in a decrease in attachment, internalization, and replication of viruses with E271K. Taken together, our study suggests that the replication kinetics of EV-D68 isolates in SK-N-SH cells is not a clade-specific feature. However, recognition of heparan sulfate as an additional receptor had a large effect on phenotypic characteristics in vitro. These observations emphasize the need to compare sequences from virus stocks with clinical isolates in order to retrieve phenotypic characteristics from original virus isolates.IMPORTANCE Enterovirus D68 (EV-D68) causes mild to severe respiratory disease and is associated with acute flaccid myelitis since 2014. Currently, the understanding of the ability of EV-D68 to replicate in the central nervous system (CNS), and whether it is associated with a specific clade of EV-D68 viruses or specific viral factors, is lacking. Comparing different EV-D68 clades did not reveal clade-specific phenotypic characteristics. However, we did show that viruses which acquired a cell culture-adapted amino acid substitution in VP1 (E271K) recognized heparan sulfate as an additional receptor. Recognition of heparan sulfate resulted in an increase in attachment, infection, and replication in neuroblastoma cells compared with viruses without this specific amino acid substitution. The ability of EV-D68 viruses to acquire cell culture-adaptive substitutions which have a large effect in experimental settings emphasizes the need to sequence virus stocks.

A High-Fat Diet Increases Influenza A Virus-Associated Cardiovascular Damage.

BACKGROUND: Influenza A virus (IAV) causes a wide range of extrarespiratory complications. However, the role of host factors in these complications of influenza virus infection remains to be defined. METHODS: Here, we sought to use transcriptional profiling, virology, histology, and echocardiograms to investigate the role of a high-fat diet in IAV-associated cardiac damage. RESULTS: Transcriptional profiling showed that, compared to their low-fat counterparts (LF mice), mice fed a high-fat diet (HF mice) had impairments in inflammatory signaling in the lung and heart after IAV infection. This was associated with increased viral titers in the heart, increased left ventricular mass, and thickening of the left ventricular wall in IAV-infected HF mice compared to both IAV-infected LF mice and uninfected HF mice. Retrospective analysis of clinical data revealed that cardiac complications were more common in patients with excess weight, an association which was significant in 2 out of 4 studies. CONCLUSIONS: Together, these data provide the first evidence that a high-fat diet may be a risk factor for the development of IAV-associated cardiovascular damage and emphasizes the need for further clinical research in this area.

Zika Virus Infection Induces Elevation of Tissue Factor Production and Apoptosis on Human Umbilical Vein Endothelial Cells.

Zika virus (ZIKV) infection is typically characterized by a mild disease presenting with fever, maculopapular rash, headache, fatigue, myalgia, and arthralgia. A recent animal study found that ZIKV-infected pregnant Ifnar -/-mice developed vascular damage in the placenta and reduced amount of fetal capillaries. Moreover, ZIKV infection causes segmental thrombosis in the umbilical cord of pregnant rhesus macaques. Furthermore, several case reports suggest that ZIKV infection cause coagulation disorders. These results suggest that ZIKV could cause an alteration in the host hemostatic response, however, the mechanism has not been investigated thus far. This paper aims to determine whether ZIKV infection on HUVECs induces apoptosis and elevation of tissue factor (TF) that leads to activation of secondary hemostasis. We infected HUVECs with two ZIKV strains and performed virus titration, immunostaining, and flow cytometry to confirm and quantify infection. We measured TF concentrations with flow cytometry and performed thrombin generation test (TGT) as a functional assay to assess secondary hemostasis. Furthermore, we determined the amount of cell death using flow cytometry. We also performed enzyme-linked immunosorbent assay (ELISA) to determine interleukin (IL)-6 and IL-8 production and conducted blocking experiments to associate these cytokines with TF expression. Both ZIKV strains infected and replicated to high titers in HUVECs. We found that infection induced elevation of TF expressions. We also showed that increased TF expression led to shortened TGT time. Moreover, the data revealed that infection induced apoptosis. In addition, there was a significant increase of IL-6 and IL-8 production in infected cells. Here we provide in vitro evidence that infection of HUVECs with ZIKV induces apoptosis and elevation of TF expression that leads to activation of secondary hemostasis.

Viral Factors Important for Efficient Replication of Influenza A Viruses in Cells of the Central Nervous System.

Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections. Remarkably, zoonotic H5N1 virus infections are more frequently associated with CNS disease than seasonal or pandemic influenza viruses. Little is known about the interaction between influenza A viruses and cells of the CNS; therefore, it is currently unknown which viral factors are important for efficient replication. Here, we determined the replication kinetics of a seasonal, pandemic, zoonotic, and lab-adapted influenza A virus in human neuron-like (SK-N-SH) and astrocyte-like (U87-MG) cells and primary mouse cortex neurons. In general, highly pathogenic avian influenza (HPAI) H5N1 virus replicated most efficiently in all cells, which was associated with efficient attachment and infection. Seasonal H3N2 and to a lesser extent pandemic H1N1 virus replicated in a trypsin-dependent manner in SK-N-SH but not in U87-MG cells. In the absence of trypsin, only HPAI H5N1 and WSN viruses replicated. Removal of the multibasic cleavage site (MBCS) from HPAI H5N1 virus attenuated, but did not abrogate, replication. Taken together, our results showed that the MBCS and, to a lesser extent, the ability to attach are important determinants for efficient replication of HPAI H5N1 virus in cells of the CNS. This suggests that both an alternative hemagglutinin (HA) cleavage mechanism and preference for α-2,3-linked sialic acids allowing efficient attachment contribute to the ability of influenza A viruses to replicate efficiently in cells of the CNS. This study further improves our knowledge on potential viral factors important for the neurotropic potential of influenza A viruses.IMPORTANCE Central nervous system (CNS) disease is one of the most common extrarespiratory tract complications of influenza A virus infections, and the frequency and severity differ between seasonal, pandemic, and zoonotic influenza viruses. However, little is known about the interaction of these viruses with cells of the CNS. Differences among seasonal, pandemic, and zoonotic influenza viruses in replication efficacy in CNS cells, in vitro, suggest that the presence of an alternative HA cleavage mechanism and ability to attach are important viral factors. Identifying these viral factors and detailed knowledge of the interaction between influenza virus and CNS cells are important to prevent and treat this potentially lethal CNS disease.

Mini viral RNAs act as innate immune agonists during influenza virus infection.

The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors1. However, it is generally accepted that a strong innate immune dysregulation known as 'cytokine storm' contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype2-4. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon expression5. Here, we show that short aberrant RNAs (mini viral RNAs (mvRNAs)), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind to and activate RIG-I and lead to the expression of interferon-ß. We find that erroneous polymerase activity, dysregulation of viral RNA replication or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells. By deep sequencing RNA samples from the lungs of ferrets infected with influenza viruses, we show that mvRNAs are generated during infection in vivo. We propose that mvRNAs act as the main agonists of RIG-I during influenza virus infection.

1918 H1N1 Influenza Virus Replicates and Induces Proinflammatory Cytokine Responses in Extrarespiratory Tissues of Ferrets.

Background: The 1918 Spanish H1N1 influenza pandemic was the most severe recorded influenza pandemic with an estimated 20-50 million deaths worldwide. Even though it is known that influenza viruses can cause extrarespiratory tract complications-which are often severe or even fatal-the potential contribution of extrarespiratory tissues to the pathogenesis of 1918 H1N1 virus infection has not been studied comprehensively. Methods: Here, we performed a time-course study in ferrets inoculated intranasally with 1918 H1N1 influenza virus, with special emphasis on the involvement of extrarespiratory tissues. Respiratory and extrarespiratory tissues were collected after inoculation for virological, histological, and immunological analysis. Results: Infectious virus was detected at high titers in respiratory tissues and, at lower titers in most extrarespiratory tissues. Evidence for active virus replication, as indicated by the detection of nucleoprotein by immunohistochemistry, was observed in the respiratory tract, peripheral and central nervous system, and liver. Proinflammatory cytokines were up-regulated in respiratory tissues, olfactory bulb, spinal cord, liver, heart, and pancreas. Conclusions: 1918 H1N1 virus spread to and induced cytokine responses in tissues outside the respiratory tract, which likely contributed to the severity of infection. Moreover, our data support the suggested link between 1918 H1N1 infection and central nervous system disease.

Phenotypic Differences between Asian and African Lineage Zika Viruses in Human Neural Progenitor Cells.

Recent Zika virus (ZIKV) infections have been associated with a range of neurological complications, in particular congenital microcephaly. Human neural progenitor cells (hNPCs) are thought to play an important role in the pathogenesis of microcephaly, and experimental ZIKV infection of hNPCs has been shown to induce cell death. However, the infection efficiency and rate of cell death have varied between studies, which might be related to intrinsic differences between African and Asian lineage ZIKV strains. Therefore, we determined the replication kinetics, including infection efficiency, burst size, and ability to induce cell death, of two Asian and two African ZIKV strains. African ZIKV strains replicated to higher titers in Vero cells, human glioblastoma (U87MG) cells, human neuroblastoma (SK-N-SH) cells, and hNPCs than Asian ZIKV strains. Furthermore, infection with Asian ZIKV strains did not result in significant cell death early after infection, whereas infection with African ZIKV strains resulted in high percentages of cell death in hNPCs. The differences between African and Asian lineage ZIKV strains highlight the importance of including relevant ZIKV strains to study the pathogenesis of congenital microcephaly and caution against extrapolation of experimental data obtained using historical African ZIKV strains to the current outbreak. Finally, the fact that Asian ZIKV strains infect only a minority of cells with a relatively low burst size together with the lack of early cell death induction might contribute to its ability to cause chronic infections within the central nervous system (CNS). IMPORTANCE The mechanism by which ZIKV causes a range of neurological complications, especially congenital microcephaly, is not well understood. The fact that congenital microcephaly is associated with Asian lineage ZIKV strains raises the question of why this was not discovered earlier. One possible explanation is that Asian and African ZIKV strains differ in their abilities to infect cells of the CNS and to cause neurodevelopmental problems. Here, we show that Asian ZIKV strains infect and induce cell death in human neural progenitor cells-which are important target cells in the development of congenital microcephaly-less efficiently than African ZIKV strains. These features of Asian ZIKV strains likely contribute to their ability to cause chronic infections, often observed in congenital microcephaly cases. It is therefore likely that phenotypic differences between ZIKV strains could be, at least in part, responsible for the ability of Asian ZIKV strains to cause congenital microcephaly.